Fastidious or rarely isolated gram-negative rods with a particular focus on Kingella kingae

SUMMARY

The bacterial species covered in this review are taxonomically diverse. Kingella kingae, a well-known infectious agent of pediatric bacteremia and osteomyelitis as well as septic arthritis in children aged 6–36 months, is the focus of this review. Recent advances in molecular diagnostic tools have allowed new aspects of pathogenesis and transmission to be investigated since the last review of K. kingae in this journal. Asymptomatic oropharyngeal colonization with noninvasive strains can be distinguished from invasive clones, so that a positive detection in the oropharynx can be assumed to be compatible with a clinical entity caused by K. kingae. Furthermore, novel and uncommon infections could be attributed to K. kingae. Clinical and microbiological aspects of the other Kingella spp. and of the genera Actinobacillus, Aggregatibacter, Capnocytophaga, Cardiobacterium, Chromobacterium, Dysgonomonas, Eikenella, Pasteurella, Simonsiella, Streptobacillus, and Suttonella are discussed separately. The identification of most genera by phenotypic methods is difficult. Direct microscopy and phenotypic key reactions, as well as commercially available systems, allow identification of the frequently encountered human species. If accurate identification of uncommon isolates is a concern, molecular methods, such as 16S ribosomal RNA gene PCR/sequencing, are necessary. Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry can also be helpful. Besides infections with only one infectious agent, all mentioned genera are often found in mixed infections. The accompanying bacteria and the clinical presentation might guide the antibiotic choice. In general, it can be said that empirical treatment of infections with the fastidious gram-negative rods based on the clinical picture sometimes fails due to antibiotic resistance. Susceptibility testing should be performed on all isolates according to the CLSI or EUCAST guidelines in order to adjust treatment if necessary.

INTRODUCTION

The aim of this review is an updated overview of clinical as well as microbiological aspects of fastidious or rarely isolated gram-negative rods with a particular focus on Kingella kingae; systemic fastidious zoonotic gram-negative rods, e.g., Brucella melitensis, Francisella tularensis, Haemophilus ducreyi, and Haemophilus influenzae are not included; in this context, we refer to previous review articles in this journal (1–4). The selection of challenging gram-negative rods included in this review is based on microbiological criteria as they present in the laboratory, i.e., there are no vaccines for humans and essentially no serological diagnostics. Therefore, Bordetella pertussis was not included; we refer to a previous review article in this journal (5). Rod-shaped Neisseria spp. that are often isolated together with other microorganisms in polymicrobial infected bites are often discussed with the oral cocci-shaped Neisseria spp. and are not included here. The species mentioned in this review are listed in Table 1. Medical students are taught by experienced teachers to focus first on what is common and not on what is rare. In their further training as assistant doctors, however, they must also familiarize themselves with uncommon diseases. Infections caused by the pathogens discussed in this review are rare in clinical practice. However, certain situations, such as wounds from cat or dog bites, probably indicate Pasteurella multocida as the pathogen because this association is very common (6). Another example is a gram-negative rod in a blood culture of a patient with endocarditis, where a bacterium of the HACEK group (which comprises Haemophilus sp. not influenzae, mainly Haemophilus parainfluenzae, Aggregatibacter spp., Cardiobacterium spp., mainly Cardiobacterium hominis, Eikenella corrodens, and Kingella spp., mainly Kingella kingae [7]) must be considered first, although these bacteria account for only 3% of all endocarditis (8).

TABLE 1.

Taxonomy of the bacterial species included in this review

Phylum	Class	Order	Family	Genus	Species
Pseudomonadota	β-proteobacteria	Neisseriales	Chromobacteriaceae	Chromobacterium	haemolyticum
					violaceum
			Neisseriaceae	Eikenella	corrodens
					exigua
					glucosivorans
					halliae
					longinqua
				Kingella	bonacorsii
					denitrificans
					kingae
					negevensis
					oralis
					potus
					pumchi
				Simonsiella	muelleri
	γ-Protobacteria	Cardiobacteriales	Cardiobacteriaceae	Cardiobacterium	hominis
					valvarum
				Suttonella	indologenes
		Pasteurellales	Pasteurellaceae	Actinobacillus	equuli
					hominis
					lignieresii
					pleuropneumoniae
					suis
					ureae
				Aggregatibacter	actinomycetemcomitans
					aphrophilus
					kilianii
					segnis
				Pasteurella	canis
					dagmatis
					multocida
					oralis
					stomatis
				[Pasteurella]a	aerogenes
					bettyae
					caballi
				Mannheimiab	glucosida
					haemolytica
				Rodentibacterb	pneumotropicus
Bacteroidota	Bacteroidia	Bacteroidales	Dysgonomonadaceae	Dysgonomonas	capnocytophagoides
					gadei
					hofstadii
					mossii
	Flavobacteriia	Flavobacteriales	Flavobacteriaceae	Capnocytophaga	bilenii
					canimorsus
					cynodegmi
					felis
					gingivalis
					granulosa
					haemolytica
					leadbetteri
					ochracea
					periodontitidis
					sputigena
					stomatis
Fusobacteriota	Fusobacteriia	Fusobacteriales	Leptotrichiaceae	Streptobacillus	felis
					moniliformis
					notomytis
				Pseudostreptobacillusc	hongkongensis

^a Uncertain allocation; reclassification possible in the future.

^b Formerly belonged to the genus Pasteurella.

^c Formerly belonged to the genus Streptobacillus.

The bacterial genera covered in this review are taxonomically diverse; they belong to the families Cardiobacteriaceae, Chromobacteriaceae, Dysgonomonadaceae, Flavobacteriaceae, Leptotrichiaceae, Neisseriaceae, and Pasteurellaceae and are mostly part of the microbiota of the nasopharynx and/or the oral cavity of animals and/or humans, with the only environmental genus being Chromobacterium. Some species are transmitted from animals to humans by bites and licking of wounds, e.g., Capnocytophaga canimorsus (9) and Pasteurella spp. (6); other species from humans to humans by droplets, e.g., Kingella spp. (10), or human bites, e.g., E. corrodens (11). These organisms may cause invasive infections and disseminate through the bloodstream to multiple body sites. We will focus on species that can occasionally be isolated in humans; species that have so far only been detected in animals will only be discussed in exceptional cases. The epidemiology and clinical significance of the genera Actinobacillus, Aggregatibacter, and Pasteurella of the family Pasteurellaceae, as well as the genera Cardiobacterium, Eikenella, and Kingella from the HACEK group, and other less frequently encountered genera are discussed separately.

Some bacteria of these genera are colonizers of the human or animal oral cavity; therefore, the evaluation of their presence in clinical specimens may be difficult. In specimens normally containing aerobic and anaerobic microbiota, e.g., from anatomical structures adjacent to the oral cavity, as well as from wounds, e.g., bite wounds, bacteria discussed in this review can be innocent bystanders or infectious agents. Even if typical pathogenic bacteria, such as Staphylococcus aureus or beta-hemolytic streptococci, are predominant, the additional presence of fastidious gram-negative rods can be relevant; the common empirical therapy with clindamycin for oral infections can select clindamycin-resistant E. corrodens (12). In such and similar situations, close cooperation between the clinical microbiology laboratory and the physician is necessary to discuss a repeat culture of the fastidious gram-negative rods for identification to the species level. If those bacteria are isolated as pure cultures from normally sterile body sites, they should be interpreted as infectious agents and reported with the susceptibility.

Identification of these genera is difficult by conventional phenotypic methods. Direct microscopy may help identify the corresponding bacteria, e.g., the typical morphology of spindle-shaped cells in the Gram stain of a positive blood culture, together with the clinical history of a dog bite, allows the presumptive identification of C. canimorsus. Commercially available systems (13, 14) allow identification of the frequently encountered human species, including the HACEK organisms causing infective endocarditis (15). However, phenotypic characteristics used for identification are subject to variation and dependent on individual interpretation and expertise. Correct identification to the species level often requires multiple substrates, which may not be available to routine laboratories (16) and are provided by automated systems (14). However, the VITEK 2 Neisseria-Haemophilus identification card (VITEK 2 ID-NH card, BioMérieux, Marcy l’Etoile, France) allows for the identification of most bacteria of the HACEK group (14). If accurate identification of uncommon isolates is a concern, molecular methods, such as 16S ribosomal RNA gene PCR/sequencing, may be necessary (17). Furthermore, molecular methods can identify new species in association with clinical entities. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) can be helpful and can be used to identify HACEK group members and other uncommon bacterial species, providing a precise and rapid (within minutes) identification of these and, thus, replacing the slow and laborious phenotypic procedures (18, 19). The species included in the databases of the two MALDI-TOF MS systems, i.e., VITEK MS system (BioMérieux, Marcy l’Etoile, France) and Bruker MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany), are mentioned in the corresponding paragraphs below. Finally, susceptibility testing according to Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) is mentioned. The susceptibilities of K. kingae are listed in Table 2, and the presumed susceptibilities of other fastidious gram-negative rods covered in this review are listed in part in Table 3. For the purely microbiologically detailed aspects of these bacteria, we refer you to the corresponding chapter in the Manual of Clinical Microbiology (20).

TABLE 2.

Kingella kingae antibiotic susceptibility patterns

Antibiotic class	Subclass	Drug	Susceptibility testing result
β-Lactams	Penicillins	Benzylpenicillin	Usually susceptiblea
		Ampicillin
		Amoxicillin-clavulanate	100% susceptible
	Isoxazolyl penicillins	Oxacillin	Non-susceptibleb
		Flucloxacillin
	Cephalosporins	First generation	100% susceptible
		Second generation
		Third generation
	Carbapenems	Meropenem
Macrolides		Erythromycin	Rarely resistantc
		Azithromycin
		Clarithromycin
Lincosamides		Clindamycin	Intrinsically resistant
Glycopeptides		Vancomycin	Intrinsically resistant
Aminoglycosides		Gentamicin	Rarely resistantc
Fluoroquinolones		Ciprofloxacin	Rarely resistantc,d
		Levofloxacin
Folate inhibitors		Trimethoprim	Intrinsically resistant
		Cotrimoxazole	Usually susceptiblee
Rifamycins		Rifampin	100% susceptible
Tetracyclines		Doxycycline	8% resistantd

^a β-lactamase is produced by 1%–2% of invasive isolates and 10% of carried strains.

^b Isolates exhibit elevated minimal inhibitory concentrations and are associated with clinical failure.

^c Less than 1% resistant.

^d Contraindicated in young children.

^e Resistance is frequently associated with β-lactamase production.

TABLE 3.

Presumed susceptibilities of gram-negative rods covered in this review^a

	Antibiotic class or representative antibiotic
	Aminopenicillins	Amoxicillin/clavulanic acid	First generation cephalosporins	Second generation cephalosporins	Third generation cephalosporins	Carbapenems	Macrolides	Clindamycin	Gentamicin	Fluoroquinolones	Tetracycline	Trimethoprim-sulfamethoxazole
Actinobacillus ureae	S	S	S	S	S		S		S		S
Aggregatibacter actinomycetemcomitans	R	S	S	S	S		S	R	S	S	R*	S
Aggregatibacter aphrophilus	R*	S	S	S	S		S		S	S	S	S
Capnocytophaga spp. oral	R*	S			S	S	R*	R*	R*	R*	R*
Capnocytophaga canimorsus/Capnocytophaga cynodegmi	R*	S			S	S
Cardiobacterium hominis/Cardiobacterium valvarum	S	S			S				S	S
Chromobacterium haemolyticum/Chromobacterium violaceum	R	R	R	R	R	S				S	S	S
Dysgonomonas capnocytophagoides		R*	R*	R*	R*	R*	S	S	R	R	S	S
Eikenella corrodens	R*	S			S	S	R	R		S	S
Pasteurella multocida/Pasteurella spp.	R*	S			S		S	R			S	S
Streptobacillus moniliformis	S	S	S	S	S		S	S	R*	R*	S	R

^a R, resistant in >80%; R*, resistant in >20%, but <80%; and S, susceptible in >80%.

KINGELLA SPECIES

The genus Kingella belongs to the β-subdivision of the proteobacteria, family Neisseriaceae, along with other important human pathogens such as Neisseria meningitidis and Neisseria gonorrhoeae. Kingella spp. inhabit the human buccal and pharyngeal epithelial surfaces and exhibit cocco-bacillary or bacillary morphology, aerobic or facultative anaerobic growth, positive oxidase activity, and negative catalase and indole reactions (21). Kingellae grow on blood agar and chocolate agar but do not develop on MacConkey or Kligler media.

The genus currently comprises Kingella kingae, Kingella denitrificans, Kingella oralis, Kingella potus, the recently described Kingella negevensis (22), and the two candidate species Kingella bonacorsii (23) and Kingella pumchi (24). The biochemical reactions that enable differentiation of the members of the genus are summarized in Table 4.

TABLE 4.

Biochemical characteristics of Kingella species^a

Test	Kingella kingae	Kingella negevensis	Kingella oralis	Kingella bonacorsii	Kingella denitrificans	Kingella potus	Kingella pumchi
Hemolysis	β	β	−	−	−	−	−
NO3 reduction	−	−	−	NA	+	−	−
NO2 reduction	−	−	NA	NA	+	−	−
DNase	−	−	−	NA	−	+	NA
Acid from glucose	+	+	−	−	+	−	+
Acid from maltose	+	−	−	−	−	−	NA
Pigment	−	−	−	−	−	Yellow	−

^a NA, not available.

The taxonomic study of the 20 genera comprising the Neisseriaceae family (including Kingella) has been complicated by the pervasive horizontal gene transfer and recombination between organisms, including the 16S rRNA gene, even among distantly related species. This dynamic process blurs species boundaries, and taxonomic trees built based on single gene sequencing have resulted in discrepancies in the allocation of members of the genus. Morreale et al. (25) have recently employed a core genome sequencing approach that better defines the relationship between kingellae and other members of the Neisseriaceae family and circumvents the difficulty. Surprisingly, the novel phylogenetic analysis revealed that Conchiformibius spp., the multicellular Alysiella spp., Simonsiella spp., and kingellae constitute a single genomic group named CASK clade, whereas K. potus is only remotely related to the other members of the genus and closer taxonomically to Neisseria bacilliformis (25).

The virulence of members of the Kingella genus shows wide variation; it ranges from K. kingae, which is the most common etiology of joint and bone infections in young children and causes endocarditis in pediatric and adult patients, K. denitrificans, which mostly infects immunocompromised individuals, K. oralis and K. bonacorsii, which are associated with periodontitis, whereas the clinical significance of K. negevensis remains to be established. K. pumchi and K. potus have been isolated only once, indicating that they are a rare etiology of human disease.

Kingella denitrificans

Kingella denitrificans is an obligate aerobic bacillus and the only member of the genus that reduces nitrates to nitrites. The organism is an uncommon opportunistic pathogen affecting particularly immunocompromised patients. K. denitrificans has been associated with pediatric and adult bacterial endocarditis (26, 27), pediatric vaginitis (28), bacteremia (29), retropharyngeal abscess (30), septicemic granulomatous disease in AIDS patients (31), peritonitis among patients with peritoneal dialysis-related indwelling catheters (32, 33), and keratitis (34).

Kingella oralis

Kingella oralis is a facultative anaerobe, agar-corroding rod with a mean size of 0.6–0.7 µm × 1–3 µm and rounded ends that appears in pairs or short chains (35). The organism is non-hemolytic, non-motile, and, like other species within the Neisseriaceae family, shows oxidase activity and a negative catalase reaction. It produces acid from glucose but not from maltose and does not reduce nitrite or nitrate. Being a mesophile, the organism can thrive in temperatures between 20°C and 45°C.

K. oralis is a common colonizer of the human oral cavity and can be isolated from the human dental plaque, mucosal surfaces, and saliva. It is particularly abundant in the subgingival biofilm in patients with chronic periodontitis, a bacterial-induced chronic inflammation affecting the tissues supporting the teeth, suggesting a potential role in pathogenesis (35). The disease is diet-driven, and patients exhibit an altered microbial composition of the oral microbiota. This dysbiosis results from an overgrowth of certain commensal species that metabolize the excess of dietary carbohydrates, producing acid. The acidic environment erodes the enamel, cementum, and dentin, initiating the progression to caries, endodontic, and periodontal diseases. The composition of the oral microbiota is extremely complex, and 730 different bacterial taxa coexist in the buccal community, of which many are not yet named and/or remain uncultured, and we only know their genomic signature. Not less than 18 of these species, including K. oralis, have been found statistically associated with caries formation and periodontal disease. However, the role of individual organisms in the pathogenesis of this polymicrobial medical condition is unclear and difficult to determine (36, 37).

Kingella bonacorsii

Kingella bonacorsii has been recently isolated and characterized in a French patient with stable periodontitis (23). The organism grows on blood-agar plates as 1 mm non-hemolytic colonies in a wide range of culture conditions ranging from aerobiosis to microaerophilic and anaerobic atmospheres. The Gram stain shows straight 0.4–0.7 µm by 1.3–2.6 µm rods with round tips. The organism exhibits positive oxidase, alkaline phosphatase, leucine arylamidase, C4 esterase, α-chymotrypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase reactions, negative catalase activity, and does not produce acid from sugars. K. bonacorsii is the taxonomically closest species to K. oralis, sharing 98.7% sequence identity of the 16S rRNA gene sequence. So far, no clinical disease other than periodontitis has been associated with the organism.

Kingella pumchi

This novel species has been isolated from the culture of a vertebral puncture of a Chinese patient (24). Contrary to other members of the genus, K. pumchi has optimal growth at pH 8.0, and its natural source remains to be determined. The organism is rod-shaped, 0.3–0.5 µm wide and 1.0–2.3 µm long, non-spore-forming, non-motile, and grows as 1.5–2.0 mm non-hemolytic, white, moist, and opaque colonies. It produces acid from glucose but not from maltose, fructose, or sucrose. It exhibits oxidase, lipase, esterase C4, leucine arylamidase, trypsin, and naphthol-AS-BI-phosphohydrolase activity, and negative catalase, urease, indole, ornithine decarboxylase, urease, alkaline phosphatase, β-galactosidase, proline arylamidase, and γ-glutamyl transferase reactions. In the phylogenetic tree based on the 16S rRNA gene, K. pumchi is located close to K. potus and N. bacilliformis (24), suggesting that its placement in the Kingella genus should be reassessed.

Kingella potus

This bacillus was initially isolated from an extensive soft tissue infection caused by the bite of a kinkajou (Potus flavus), a South and Central American arboreal mammal (21). The bacterium grows as α-hemolytic, yellow-pigmented 1.5–2 mm colonies. It is oxidase, casein, and DNase positive, catalase negative, and fails to produce acid from sugars. Alkaline phosphatase, β-glucuronidase, β-galactosidase, α-glucosidase, and indole activity are not detected. Analysis of the 16S rRNA gene showed that the novel bacterium represented a hitherto unknown Neisseriaceae and its closest relatives were other Kingella spp. with which it shows a low sequence similarity (94.8%–95.9%). Employing whole-genome sequence analysis, Morreale et al. (25) confirmed that K. potus is more closely related to the catalase-variable N. bacilliformis than to other Kingella spp. These genomic data suggest that K. potus should be transferred to a different genus within the Neisseriaceae family.

Kingella negevensis

In a prospective longitudinal study carried out in the 2004–2009 period among children residing in southern Israel aimed to detect K. kingae carriers, a β-hemolytic gram-negative coccobacillus was isolated from several oropharyngeal specimens (38). Except for its growth as small colonies, formation of long chains (whereas K. kingae appears as pairs or short chains), early autolysis, and negative production of acid from maltose, all other phenotypic features resembled K. kingae and, thus, was originally considered a K. kingae small colony variant (Fig. 1). Further analysis demonstrated that it belongs to a novel species and was named K. negevensis (Negev is the desert region of southern Israel where the organism was first isolated) (22). Based on culture on blood-agar-vancomycin (BAV) medium, K. negevensis colonization rate was approximately 1/10th of that of K. kingae, and the bacterium was not detected in children younger than 6 months (39). Similar to K. kingae, K. negevensis is carried on the oropharyngeal epithelium and does not colonize the nasopharynx (38).

Fig 1.

Growth of K. negevensis on a blood-agar plate, showing small colonies surrounded by β-hemolysis.

The organism has also been isolated from the pharynx of an asymptomatic Swiss child (39, 40), from a polymicrobial ocular infection in an American drug abuser (41), from a vaginal specimen (“eburonensis” strain) drawn from a Swiss patient with bacterial vaginosis (a condition associated with imbalance of the vaginal microbiota) (42), and, probably, as early as 2004 from an unknown USA source (Kingella sp., Genebank M03-232, accession number AY644511). In addition, the bacterium has been recently detected by nucleic acid amplification tests (NAATs) in the synovial fluid of a French child with culture-negative arthritis (43).

Similarly to K. kingae, K. negevensis is coated by a polysaccharide capsule, secretes an antiphagocytic exopolysaccharide, elaborates Knh-like trimeric autotransporter, type IV pili, and a potent and wide-spectrum RTX toxin (42–44). These findings imply that K. negevensis, like other species within the Neisseriaceae family, can colonize the upper respiratory and genital tracts, has a wide geographic dispersal, and could represent an invasive human pathogen.

The β-hemolysis induced by K. negevensis RTXA toxin facilitates its identification in oropharyngeal specimens plated on selective BAV medium (45). Available data suggest that the entire K. negevensis RTX production machinery is encoded at a single locus that contains only one copy of the rtxA gene (46). However, it should be pointed out that while over 600 K. kingae isolates from healthy carriers and patients with a variety of invasive infections have been analyzed, our knowledge of K. negevensis genomic content is based on the study of only 12 strains, all derived from colonized Israeli (n = 11) and Swiss (n = 1) children.

Although K. negevensis shares many virulence factors with K. kingae, no K. negevensis organisms have been isolated yet from the blood, and the organism was detected only once from the synovial fluid; thus, solid evidence of its status as a human pathogen is still lacking. It should be noticed that because K. negevensis RTXA encoding rtxA and rtxB genes are highly homologous to those of K. kingae, they are amplified with the K. kingae rtx operon primers and, thus, it is possible that K. negevensis infections are being erroneously attributed to K. kingae (42, 43).

All K. negevensis strains studied to date expressed a polysaccharide capsule identical to K. kingae’s capsule b (44). The species exhibits genomic variability, and five different pulsed-field gel electrophoresis (PFGE) clones have been identified among 24 isolates, of which one could not be typed (39). Similarly to K. kingae, K. negevensis PFGE clone T produces a TEM1 β-lactamase, but, unexpectedly, the encoding bla_TEM1 gene has a double chromosomal and plasmid location (47). Members of this clone have a 33 bp repetition of a fraction of the rtxA gene sequence (rtxA 17 allele).

K. negevensis can be easily differentiated from K. kingae based on the lack of acid production from maltose, formation of long chains, and widespread autolysis on 48-h-old cultures (Fig. 2). El Houmami et al. (43) and Opota et al. (42) independently developed a real-time PCR assay that amplifies K. kingae’s cpn60 (groEl) gene. Because the K. negevensis cpn60 gene sequence has substantial differences from that of K. kingae, no amplification occurs (42). Therefore, a genuine K. kingae will show amplification of the rtxA and cpn60 targets, while K. negevensis will only show amplification of rtxA. Unexpectedly, sequencing of the cpn60 gene of rtxA-negative/cpn60-positive isolates revealed close sequence similarity of the homologous gene of Simonsiella muelleri, a close member of the Neisseriaceae family (43). The differential diagnosis of K. kingae and K. negevensis based on this pair of molecular tests is summarized in Table 5.

Fig 2.

Left: Gram stain of K. negevensis after 48-h incubation, showing long chains of coccobacilli and early lysis. Right: typical Gram stain of K. kingae exhibiting coccobacilli in pairs and short chains and no bacterial lysis.

TABLE 5.

Presumptive bacterial identification of K. kingae and K. negevensis by rtxA and cpn60 PCR tests

Gene target		Interpretation
K. kingae rtxA	K. kingae cpn60
+	+	Kingella kingae
+	−	Kingella negevensis
−	+	Simonsiella muelleri
−	−	Unknown bacterium

Kingella kingae

Since K. kingae was last reviewed in CMR in 2015 (10), the popularization of the use of NAATs for its detection has confirmed the role of the organism as a common invasive bacterial pathogen in early childhood and resulted in remarkable progress in our understanding of the bacterium and its diseases. The present section summarizes novel studies published between 2015 and 2025, emphasizing the advances made over the last two decades in microbiology, epidemiology, clinical aspects, diagnosis, and therapy of the organism. For additional information, the reader is referred to the 2015 publication (10).

Pathogenesis

Virulence factors

Type IV pili and associated proteins

To establish a foothold on the oropharyngeal mucosa and avoid being washed out, K. kingae possesses long type IV pili and the pilus-associated PilC1 and PilC2 proteins that anchor the bacterium to the host’s epithelial cells. This adherence to the oropharyngeal surface is the first step in the colonization of the upper respiratory tract and the pathogenesis of invasive K. kingae infection. The pili are assembled by polymerization of the major PilA1 subunit by the PilF ATPase at the inner membrane. The fibers extend through the periplasm and are secreted through an outer membrane PilQ secretin ring, protruding through the outer membrane and can be retracted by the PilT ATPase. In addition to adherence, type IV pili fill other roles, including twitching motility and natural DNA transformation (48). Kingella kingae PilC1 and PilC2 are adhesive multifunctional proteins that promote bacterial adherence, twitching motility, DNA transformation, and pilus biogenesis, and inducing pilC1 and pilC2 gene deletions abolishes all these functions. PilC1 protein contains a metal ion-dependent adhesion site that plays a crucial role in the adhesive activity (49).

Knh trimeric autotransporter

K. kingae genome contains a large open reading frame similar to the meningococcal NhhA trimeric autotransporter adhesin, named Kingella NhhA homolog (Knh) (50). Although the Knh element makes a stronger attachment of the bacterium to the host epithelial cell possible, close contact with the epithelial receptor is physically blocked by the presence of the polysaccharide capsule. The adhesion of K. kingae to the epithelial cells requires a complex and well-orchestrated process involving pili, the Knh element, and the capsule. The type IV pili are responsible for the initial attachment. Retraction of the pili pulls the organism closer to the host cell by displacing the capsule, enabling the intimate contact of Knh with the host cell and resulting in a sturdy adherence to the epithelial surface (50).

RtxA toxin

K. kingae’s RTXA toxin belongs to the large family of RTX (repeat-in-toxins) elaborated by many gram-negative organisms (51). These bacterial components penetrate and permeabilize host cell membranes, making a critical contribution to the pathogenesis of infection. The toxin is produced by all K. kingae (and K. negevensis) isolates studied so far and is absent from the genomes of the commensal species K. denitrificans, K. oralis, and K. potus (38, 42–46, 52).

The manufacture of K. kingae’s RTXA toxin and its secretion are encoded by the five genes rtxA, rtxB, rtxC, rtxD, and tolC, which are located in a single cluster in the chromosome (46, 52). Insertion sequences flank this gene cluster, which has a decreased G + C content compared to the rest of K. kingae’s genome, suggesting that the rtxCrtxAtolC locus has been acquired by horizontal gene transfer from a donor bacterial species (52). This possibility is backed by the fact that K. kingae’s rtxA, rtxB, and rtxC gene sequences share >70% identity with their counterparts in Moraxella bovis, whereas the proteins encoded by K. kingae’s rtxD and tolC show 81% and 64% identity, respectively, with their N. meningitidis homologs (52). The analysis of K. kingae and K. negevensis genomic content suggests that the co-option of a preexisting type-I secretion system (TISS) encoded by rtxBrtxD enabled the full reconstitution of a complete toxin production and secretion system (46).

K. kingae’s rtxA gene cluster consists of two loci. Locus A contains the rtxB and rtxD genes that encode a TISS, and the rtxC gene, which encodes the RtxC acyltransferase enzyme that modifies and activates the toxin before its secretion. Locus B harbors a second and identical copy of rtxC, followed by rtxA and tolC genes (46). This genomic arrangement has been designated genotype I. One-third of the 255 clinical K. kingae isolates studied possess a second copy of the rtxA and tolC genes located in locus A, a configuration named genotype II (46). Genotype II is clonal and almost entirely restricted to multilocus sequence types (MLST) ST-6 and ST-14. It is exclusively associated with invasive capsule types a and b and found in strains causing clinical infections. Genotype II isolates do not generally produce more toxin in vitro than genotype I isolates (46). It is unknown whether genotype I, per se, increases invasiveness or is merely a marker for other associated virulence factors.

Most of the membrane-bound RTX A toxin is localized extracellularly, while five of seven transmembrane α-helices within the N-terminal half of the RTXA molecule are irreversibly embedded in the membrane (46). These α-helices, four of which are arranged as two back-to-back pairs, are required for forming an ion-conducting membrane pore, while the C-terminal half of RTXA is essential for the toxin’s interaction with the host’s cell surface and its irreversible insertion into the membrane via acyl chains covalently linked to the molecule (46).

Following enzymatic activation, the RTXA toxin is secreted through an outer membrane protein encoded by the rtxB, rtxD, and tolC genes (46). RTXA can also be secreted via outer membrane vesicles that are phagocyted by many eukaryotic cells, exerting a cytotoxic effect and enhancing K. kingae’s virulence (53). After release, the RTXA recognizes sialic acid residues located on the surface of the host’s cell and linked to gangliosides and glycoproteins (54). The RTXA toxin inserts into the cell membranes and induces the formation of large channels in host cell membranes, resulting in calcium influx and potassium efflux. This chain of events leads to a programmed death in a variety of human cells involved in the pathogenesis of K. kingae infections, such as erythrocytes—causing β-hemolysis (Fig. 3)—as well as epithelial, polymorphonuclear cells, and synoviocytes (52–55).

Fig 3.

K. kingae growing on a blood-agar plate surrounded by a large area of β-hemolysis.

In an experimental model of infection, 7-day-old rats inoculated with a wild-type K. kingae strain isolated from a patient with arthritis developed a severe systemic infection and rapid death, while animals inoculated with an RTX toxin-deficient mutant of the same strain did not show signs of disease (56). Inoculation of 21-day-old rats with the wild-type strain did not result in noticeable disease, demonstrating an age-dependent susceptibility similar to that found in the human host (56).

Analysis of a collection of 103 K. kingae isolates derived from different countries and associated clinical syndromes revealed remarkable polymorphism of the rtxA gene, suggesting that, similar to other crucial virulence factors, it is subjected to selective pressure by the host’s immune system. The study identified 18 different alleles indicating that the gene evolved to escape the host’s immune defenses (57).

Capsule

All K. kingae strains elaborate one of four chemically different polysaccharide capsules. The polysaccharide capsules, named a, b, c, and d, are synthesized by four distinct synthases encoded by the genomic loci csa, csb, csc, and csd, respectively. The molecular structure of the capsules has been determined as follows: type a: [3)-β-GalpNAc-(1→5)-β-Kdop(2→] polysaccharide capsule; type b: [6)-α-GlcpNAc-(1→5)-β-(8-OAc)Kdop-(2→]; type c: [3)-β-Ribf -(1→2)-β-Ribf-(1→2)-β-Ribf -(1→4)-β-Kdop-(2→]; and type d: [P-(O→3)[β- Galp-(1→4)]-β-GlcpNAc-(1→3)-α-GlcpNAc-1-] (50, 58). The distribution of the different capsule types was studied in 417 Israeli K. kingae strains, comprising 239 isolates from asymptomatic pharyngeal carriers and 178 derived from patients with a variety of invasive infections, including bacteremia, osteoarthritis, and endocarditis. While capsule type a was found in 80 (44.9%), and capsule b in 91 (51.1%) of the 178 invasive strains, capsules c and d were detected in only 4 (2.2%) and 2 (1.7%), respectively, while the capsule type could not be determined in one invasive isolate (58). The number and percentages of capsules a, b, c, and d among carried organisms were 117 (49.0%), 46 (19.2%), 29 (12.1%), and 47 (19.7%), respectively (58). In summary, capsule types a and b represented 171 of the 177 (96.6%) typable capsules of the invasive isolates, suggesting that they confer enhanced virulence or are linked with other virulence factors. Capsules c and d were mostly found among organisms colonizing the oropharynx and were detected in 76 (31.8%) of the 239 asymptomatically carried strains (58). In a small French study, the capsular type was determined in oropharyngeal specimens obtained from healthy children by a multiplex PCR assay that amplified the csa, csb, csc, and csd encoding genes. Four capsule type a, four capsule type b, and one capsule type a in combination with capsule type c were identified, confirming the predominance of capsule types a and b in the species (59). In an international collaborative study analyzing 150 strains derived from 10 countries in North America (USA, n = 28 and Canada, n = 12), Europe (France, n = 35, Spain, n = 45, Iceland, n = 12, Norway, n = 3, Switzerland, n = 3, and Russia, n = 1), and Oceania (Australia, n = 1, and New Zealand, n = 10), 76 of the 126 (60.3%) isolates for which an invasive clinical source was known elaborated capsule type a and 45 (35.7%) capsule type b (60). Overall, the capsule types of 121 of the 126 (96.0%) invasive strains were either a or b, fully confirming previous results (58–60). In a French independent study comprising 105 children with positive K. kingae NAATs or culture-positive osteoarthritis, capsule a was identified in 71 (67.6%), capsule type b in 33 (31.4%), and capsule type c in 1 (0.9%) sample, while no type d capsule was detected (61). In a rat model, the capsule prevented deposition of IgG, IgM, C4b, and C3b, thus impeding opsonization and complement-mediated killing and ensuring bacterial survival in the bloodstream (62). Additional research demonstrated that the polysaccharide capsule prevents neutrophil binding, activation, and production of reactive oxygen species (63, 64).

Similar to other pediatric pathogens such as pneumococci and H. influenzae, the polysaccharide capsules play a crucial role in the pathogenesis of invasive infections by conferring evasion of the host’s immune response. Human pathogens take advantage of the delayed maturation of the T-cell independent arm of the adaptive immune system that reacts to polysaccharides until the age of 2–4 years (65). This phenomenon explains the enhanced susceptibility to colonization and infection by encapsulated bacteria in general, and K. kingae, in particular, in early childhood. The fact that only two capsular types cause the vast majority of invasive K. kingae diseases may have significant implications for designing a future capsule-based conjugate vaccine for pediatric use.

Exopolysaccharide

Bacterial extracellular polysaccharides comprise a major component of the oral biofilm (66). Species colonizing human body surfaces elaborate a sticky “slime” consisting of jam-packed bacteria and polysaccharides that ensure a sturdy attachment to the host’s mucosal layers. The biofilm protects the organism from desiccation and makes the bacteria inaccessible to the host’s immune response and antimicrobial drugs. The establishment, growth, and remodeling of biofilm are dynamic processes that result in the periodic release of trapped bacteria, enabling the colonization of new body sites and transmission to new hosts.

To date, two chemically different exopolysaccharides have been described in K. kingae. A linear galactan homopolymer with the structure →3)- β -(1→6)-Galf-(1→ that inhibits biofilm formation by a wide range of oropharyngeal bacteria competing for the same niche (67) and a second polymer with the structure →5)- β-Galf-(1→ (68). The exopolysaccharides enable K. kingae’s survival by promoting resistance to phagocytosis and killing by neutrophils and preventing serum opsonin deposition and complement-mediated lysis and, thus, blocking the innate immunity response (62, 63). Anchoring of the galactan molecule on the bacterial cell surface is mediated through an atypical lipopolysaccharide (LPS) O-antigen. This LPS is synthesized by two glycosyltransferases encoded by the pamD and pamE genes (69).

Oropharyngeal colonization, age, and dynamics

Previous studies conducted in southern Israel, using culture on the BAV selective medium designed to facilitate K. kingae recognition in oropharyngeal specimens, found that the prevalence of the bacterium starts after the age of 6 months, indicating waning of maternally derived immunity to the bacterium and increasing social interaction (70, 71). The colonization rate increases to 12% at 12 months of age, remains stable during the second year of life, and gradually decreases as children grow older. To validate these initial results, Anderson de la Llana et al. (72) conducted a prospective study in which oropharyngeal samples were obtained from 744 Swiss children aged 7–48 months to investigate the age-and gender-related K. kingae carriage, and the seasonal colonization rate and its correlation with the incidence of clinical disease. Instead of cultures, a real-time NAAT targeting the rtx operon was employed. The study results were remarkably similar in terms of the age-related colonization prevalence to the data previously obtained in the Israeli population (10). Although the prevalence of K. kingae colonization was highest and remained stable between 12 and 36 months of age, the age-related curve of clinical disease was markedly skewed to the left, and 34 of 46 (73.9%) diseased children were aged 24 months or younger (72). These results indicate that the acquisition of resistance to invasive infection precedes the development of immunity to pharyngeal colonization (70). The carriage rate of the organism remained relatively unchanged throughout the entire year, but the attack rate of clinical disease peaked significantly in the fall, suggesting that cofactors and, most probably, seasonal viral infections facilitate dissemination of K. kingae outside the upper respiratory tract (73–75). The carriage rate in older children and adults is low (around 1%–2%) and transient (76–78). However, carriage rate substantially increases, approaching that of the young pediatric population, among parents of children aged 6–48 months and among personnel working in daycare centers (DCCs) affected by K. kingae disease outbreaks (78).

Transmission

K. kingae is readily transmitted by close contact among susceptible young children in the family setting, among playmates, and attendees to DCC facilities (38, 77, 79). The colonized buccal and oropharyngeal mucosae are the source of droplet transmission, and contaminated secretions facilitate the spread of the organism during viral upper respiratory infections (80).

The intrafamilial transmission was demonstrated in a prospective Swiss study in which a real-time PCR assay was employed (77). In total, 55% of children with K. kingae osteoarticular infections and 40% of asymptomatic carriers had siblings with a positive molecular test result (77).

Strain virulence and tissue tropism

Similarly to other Neisseriaceae, K. kingae is naturally transformable, and horizontal gene transfer is a foremost force driving genetic diversity. Employing large international strain populations derived from healthy carriers and patients with a variety of invasive infections, 74 PFGE clones, 18 rtxA alleles, 11 por alleles, and 76 MLST STs have been identified in the species (57, 81–83). Genotyping of K. kingae isolates by MLST and PFGE has been a crucial tool in the investigation of the child-to-child transmission of the bacterium and its spread in the community, the epidemiological research of clusters of invasive disease in DCCs, and the investigation of virulence differences among strains (84, 85). Despite the horizontal gene transfer, an almost perfect correlation has been found between the different genotyping methods, and individual strains are characterized by stable and unique combinations of PFGE fingerprints, MLST typing of housekeeping genes, and rtxA and por gene alleles (10). It is postulated that prevalent K. kingae strains contain mixtures of beneficial genomic elements and, thus, tend to resist the disrupting effect of recombination. These successful strains have been subjected to positive selection and, therefore, have persisted for many decades and spread globally (10).

Epidemiological studies have demonstrated that not all K. kingae organisms were created equal; while many strains are asymptomatically carried in the oropharynx but are a rare etiology of invasive K. kingae disease, a few others cause the vast majority of sporadic infections worldwide as well as clusters of invasive infections in DCCs (10, 85, 86). For instance, in a study in which 181 Israeli strains, isolated between 1991 and 2012 from patients with bacteremia, endocarditis, and osteoarthritis were analyzed, a total of 32 different PFGE clones were identified, of which only five, namely K, H, N, B, and P, were responsible for 132 (73%) of all infections (85).

In recent years, evidence has been accumulated indicating that some K. kingae strains show a proclivity to invade specific body systems and tissues. In the aforementioned Israeli study, the possible association of certain PFGE genotypes with specific clinical syndromes was also investigated (85). Clone K, which belongs to MLST sequence-type complex (STc) -23/25, showed a significant positive association with occult bacteremia, clone N (which correlates with STc-35) with pediatric arthritis and osteomyelitis, and clone P (STc-23/25) with bacterial endocarditis (85). Although tenosynovitis is an exceptional manifestation of K. kingae disease, three of the five children involved in a French daycare outbreak had a tendon sheath infection (87). Recent publications show that strains belonging to the highly virulent STc-23/25 have tropism toward the cardiac tissue and the central nervous system (83, 88, 89). However, it should be emphasized that the tissue tropism of K. kingae strains is only relative, and many invasive organisms can colonize a diversity of tissues and body sites (86, 89).

A recent study has shown significant genomic differences between K. kingae strains isolated from asymptomatic pharyngeal carriers, bacteremic children with no focal disease and skeletal system infections, and adult and pediatric patients with bacterial endocarditis, suggesting the presence of syndrome-specific genomic determinants (83). The presence or absence of single genes could not discriminate between strains carried asymptomatically on the oropharynx and those detected in invasive infections. However, it should be noted that all invasive strains had initially colonized the oropharynx, and thus, isolates of oropharyngeal origin represent a mixture of strains with diverse grades of virulence. This could have blurred genomic differences between mere colonizers without invasive capabilities and truly virulent organisms. Forty-three genes were significantly more frequent in invasive isolates compared to mere oropharyngeal colonizers, and a few genes showed a significant differential distribution among isolates from patients with bacteremia, osteoarthritis, or endocarditis (83). These results imply that, similar to other Neisseriaceae, K. kingae’s virulence and tissue tropism depend on combinations of multiple genomic determinants widely disseminated throughout the genome.

Viral infections, a contributing factor to invasive K. kingae disease

Shortly after the role of K. kingae as an invasive pathogen of early childhood was recognized, symptoms of preceding or concomitant upper respiratory viral infections were noticed among the diseased children (10, 74). In most cases, the clinical manifestations were non-specific, such as coryza or cough, and in a few children, a respiratory virus was detected with no associated clinical signs. Rhinoviruses, coronavirus OC43, parainfluenza, enteroviruses, or adenoviruses were the most common agents detected, and a temporal association was observed between the circulation of rhinoviruses in the community and the occurrence of K. kingae infections. Buccal ulcers caused by primary herpes simplex gingivostomatitis, varicella zoster infections, herpangina, and hand-foot-and-mouth disease were also diagnosed in association with sporadic cases and DCC outbreaks of K. kingae disease (10, 74, 78, 90, 91). In a French study, the seasonality of 322 invasive K. kingae infections was correlated with the detection of respiratory viruses in the community over 7 years (73). High viral activity and increased K. kingae osteoarticular infection attack rate occurred in the fall, and low activity occurred during the summer months. Weekly distribution of K. kingae disease significantly correlated with rhinovirus activity but not with respiratory syncytial virus, influenza virus, and metapneumovirus (73). These observations strongly support the concept that damage to the oropharyngeal mucosa induced by a seasonal viral disease facilitates the penetration into the bloodstream of K. kingae organisms already residing in the buccal cavity and the pharynx, followed by dissemination of the bacterium to joints, bones, or the endocardium, for which the organism shows a particular tropism.

Epidemiology

Global epidemiology of invasive infections

In the past, K. kingae infections were mainly reported from the industrialized countries, whereas notifications from the Developing World were scarce (10). This paucity of reports does not prove that the organism’s prevalence is restricted to the Western World. Instead, it is plausibly caused by the inaccessibility in resource-limited countries of the expensive automated blood culture systems and NAAT equipment required to detect this fastidious microorganism. However, the gradual adoption of molecular diagnostic methods in recent years and the increasing familiarity of microbiology laboratories with K. kingae’s phenotypic characteristics have resulted in a growing number of case reports from additional European countries, as well as South America, Africa, and Oceania (92–100). The patients’ age distribution, clinical manifestations, and laboratory features in these reports were entirely similar to those described in the USA, Western Europe, and Israel, indicating that the bacterium has a universal distribution and a distinctive presentation.

Outbreaks of invasive K. kingae disease in daycare centers

Although cases of K. kingae infection are usually sporadic, clusters of invasive disease have been reported in DCCs in the USA (n = 5), Canada (n = 1), Spain (n = 2), France (n = 11), Luxembourg (n = 2), and Israel (n = 6) (86). Multiple cases of invasive disease characterized these events within 1 month, involving, on average, 18% of the DCC attendees, and frequently following local outbreaks of viral respiratory disease. The median age of the 72 affected attendees was 14 months, ranging from 6 to 32 months (86). Skeletal system infections were diagnosed in 66 (92%) children—mostly septic arthritis and osteomyelitis. Three children (4%) presented with bacteremia with no focal infection, and three others (4%) had severe and complicated endocarditis, of which two died. Genomic analysis of the available causative strains identified the highly invasive STC-23/25, ST-14, or STC-6 in 12 of 13 (92.3%) outbreaks (86).

The epidemiological investigation of the clusters revealed a high prevalence of the invasive strains in the oropharynx of asymptomatic classmates, implying that the causative organisms combined, in addition to high virulence, enhanced colonization fitness and transmissibility. In 15 of the 27 DCC outbreaks, prophylactic ampicillin and/or rifampin for 2–4 days was administered to all children attending the affected classrooms, and no additional disease cases were detected (86). In 10 DCCs for which the information was available, the outbreak ended without pharmacological intervention, suggesting the acquisition of herd immunity induced by the prolonged carriage of the causative strain, extinction of the viral trigger, or both (86). The question of the need to prescribe antibiotics to asymptomatic children attending DCCs where K. kingae infections are detected remains open, but it will probably be difficult, if not impossible, to withhold antibiotic prophylaxis from children attending DCCs in which a case of life-threatening endocarditis has occurred (86).

Diagnosis

Culture

Bacteremia and endocarditis

Following colonization of the oropharyngeal epithelium, virulent K. kingae strains may penetrate the mucosal layer and invade the bloodstream, from which they translocate to bones, joints, or the endocardium (Fig. 4). Despite the increasing use of NAATs for detecting skeletal system infections, the isolation of the organism remains crucial for diagnosing K. kingae bacteremia and endocarditis.

Fig 4.

Gram stain of a positive blood culture vial of a 22-month-old child with K. kingae bacteremia. RBCs, red blood cells.

Bacteremia with no focal infection (also known as occult bacteremia) is probably the second most common manifestation of K. kingae disease and has been detected in 56 of 143 (39.2%) consecutive Israeli patients (10). In other countries, the fraction of bacteremic children is much lower, reflecting a more restricted use of blood cultures in well-appearing children presenting with low-grade fever and non-impressive inflammatory markers (10).

Since the last CMR review of K. kingae (10), novel blood culture systems such as the bioMérieux BacT/Alert Virtuo, Thermo Fisher VersaTrek, and Becton Dickinson Bactec FX have been introduced into clinical practice. The published experience with the performance of these systems and culture media for the recovery of K. kingae remains limited. In a prospective volume-controlled study, the performance of paired 559 pediatric BACTEC Peds Plus and the adult BACTEC Plus Aerobic blood culture vials for the isolation of common pediatric pathogens was compared (101). The study results showed no difference in terms of sensitivity and time-to-detection for all bacterial species, except for the two K. kingae isolates, which were solely recovered from the pediatric bottles (101).

Employing simulated blood cultures, vials of the three systems (BacT/Alert Vituo FAN Plus, Thermo Fisher VersaTrek Redox, and Becton Dickinson Bactec FX Bactec Plus) were spiked with a variety of bacteria, and their sensitivity was compared (102). Unfortunately, the blood culture vials were inoculated with a single K. kingae clinical isolate at the sole concentration of 5 colony-forming units (CFUs) per milliliter (102). The Virtuo BacT/Alert standard aerobic medium detected the bacterium in only 5 of 12 (42%) vials, while the pediatric aerobic medium detected the organism in all 9 vials. The VersaTrek and Bactec standard aerobic and pediatric aerobic media detected K. kingae in all inoculated vials, and the average time-to-positivity was comparable (32.3 h in the Virtuo BacT/Alert vials, 29.9 h in the VersaTrek vials, and 35.5 h in the Bactec vials). Despite the fact that K. kingae is a facultative anaerobic bacterium, the anaerobic media failed to detect the organism in all 12 Virtuo BacT/Alert and VersaTrek vials, and in 11 of 12 Bactec vials (102). Naturally, these experimental results should be validated with actual pediatric patients in large, prospective clinical studies in which vials are inoculated with blood, synovial fluid aspirates, and bone exudates.

Bone and joint exudates

Following the first description of K. kingae in 1960, the bacterium was considered a rare etiology of human infections (10). Twenty years later, it was serendipitously discovered that inoculating synovial fluid aspirates of young children with suspected infectious arthritis into blood culture vials enhanced the isolation of the organism, opening a new era in the study of K. kingae and its diseases (10). Diluting the tiny volume of joint fluid into a large volume of nutrient broth reduces the concentration of detrimental factors, enabling the recovery of fastidious K. kingae organisms.

Molecular diagnosis

Traditionally, the management of pediatric osteoarthritis has been hampered by the 2–4-day delay in isolating the causative organism, identifying it by traditional phenotypic methods and determining its antibiotic susceptibility testing. Because of the potential severity and long-term sequelae of septic arthritis and osteomyelitis in young patients, the initial therapy of these infections aimed to cover the highly virulent S. aureus and other gram-positive cocci, and H. influenzae type b, which were considered the most common etiology. Unfortunately, the traditional culture approach failed to detect the causative pathogen in one-third to two-thirds of patients (103).

Nucleic acid amplification tests

Over the past two decades, the diagnosis and management of pediatric skeletal system infections have improved drastically thanks to the development and widespread use of molecular methods. NAATs expedite the identification of the etiology of the disease and enable the detection of difficult-to-culture organisms.

The initial NAAT method consisted of extracting DNA from clinical specimens, followed by incubation with “universal primers” that anneal to conserved sections of the 16S rRNA gene—which is present in all bacteria—resulting in the amplification of the intervening species-specific sequences (104, 105). The amplicon was sequenced, and the results were aligned to a curated pathogen database to enable precise identification, or the amplification products were hybridized with pathogen-specific probes.

The 16S rRNA amplification test enabled the early microbiological diagnosis of joint and bone infections compared to the traditional culture isolation and identification procedures, reduced the fraction of frustrating culture-negative infections, and enabled the early administration of pathogen-tailored antimicrobial therapy, de-escalating the use of broad-spectrum antibiotics (105–107). Given that NAATs detect both viable and nonviable organisms, molecular tests are less affected than cultures by the administration of antibiotics, enabling the bacteriologic diagnosis in children already receiving antimicrobial therapy (106). However, amplification of the universal 16S rRNA gene when performed on blood specimens does not improve the bacteriological diagnosis over the yield of traditional blood cultures (108).

Because of its fastidious nature, K. kingae was considered a natural candidate for NAAT diagnosis from the onset. Routine use of the molecular tests significantly improved the detection of the organism by a fourfold factor compared to the BAV culture and firmly established the status of K. kingae as the prime etiology of osteoarthritis in children aged 6–48 months (10, 104, 105). The huge impact of the introduction of the 16S rRNA-based assay into clinical practice can be fully appreciated in a Spanish study in which the bacteriological diagnosis of children with suspected septic arthritis was compared in two consecutive periods (107). Between 2002 and 2008—prior to using the NAAT—the traditional cultures identified a pathogen in 11 of 35 (31.4%) patients and no K. kingae. In the 2009–2023 period, following the implementation of the broad-spectrum PCR method, the diagnostic yield increased to 29 of 46 (63.0%) infections, of which 14 (48.3%) were caused by K. kingae (107). However, in an in vitro study, the universal gene amplification assay exhibited a detection threshold of 300 CFU/mL (109), suggesting that, despite its enhanced sensitivity, the 16S rRNA target may fail to detect the low concentration of K. kingae organisms usually present in synovial fluid aspirates (from 11 to 300 CFUs/mL, median 15 CFUs/mL) (110).

The initial 16S rRNA-based NAATs have been superseded by novel real-time polymerase chain reaction assays that amplify K. kingae-specific genes. The genes that are targeted by the current molecular test are the rtxA and rtxB genes (109, 111) and the groEL gene (also known as cpn60) that encodes the chaperonin 60 protein (112–114). The assays that amplify the rtxA and rtxB genes of the rtx operon have a sensitivity of 30 CFU/mL but do not differentiate between K. kingae and K. negevensis (43). Whereas the groEL-based assays also showed suboptimal sensitivity, a novel test targeting the mdh gene was able to detect 18 variants of the gene’s sequence and was positive in seven K. kingae infections that were missed by groEL and rtx locus NAATs, suggesting that it should be considered the desired diagnostic test (115).

In a retrospective study conducted between 1997 and 2016 at a Geneva medical center, Samara et al. (116) reported that prior to the 2007 introduction of a molecular test that independently amplified the rtxA and rtxB genes, 60 children aged 6-48 months were hospitalized with an osteoarticular infection, and the combined use of conventional cultures and the universal 16S rRNA NAAT detected K. kingae in only one (1.7%). Between 2007 and 2016, K. kingae was detected in bone or joint exudates by a NAAT targeting the rtxA operon in 79 of 154 (51.3%) patients (P = 0.001) (116). In 25 additional children (16.2%), a K. kingae etiology was presumed on the basis of a positive molecular test result performed on an oropharyngeal specimen, reducing the fraction of cases of uncertain etiology from 55.0% to 24.5% (116). A similar experience was reported in Portugal by Gouveia et al. (117) where introduction of the species-specific NAAT increased the bacteriological diagnosis of pediatric suppurative arthritis from 49/133 (36.8%) to 52/106 (49.0%), P = 0.031, at the expense of the improved detection of K. kingae, which was found in 21 of the 30 (70.0%) children in which the molecular test was performed. Employing a K. kingae-specific molecular assay targeting K. kingae’s rtxA and rtxB genes independently, the study identified the organism in the osteoarticular samples of 64 of 134 (47.8%) children aged 6–48 months, and the bacterium was also detected in the oropharynx of an additional 12 children. K. kingae was not found in any of 13 infants aged less than 6 months and was detected in only 2 of 70 (2.9%) children older than 4 years, confirming the age-related susceptibility to K. kingae skeletal system infections (117). In a Canadian study, 50 synovial fluid specimens obtained from children with arthritis were submitted to the laboratory for analysis. The cultures yielded S. aureus in eight patients and Streptococcus pyogenes in two. Residual fluid was available for 27 culture-negative aspirates, and a rtx operon-based NAAT detected K. kingae in seven (25.9%), demonstrating that the use of K. kingae-specific NAATs substantially reduces the fraction of culture-negative osteoarthritis in early childhood (111). The most convincing argument for the use of species-specific NAATs for diagnosing K. kingae osteoarthritis was provided in a large retrospective study involving 217 Swiss patients aged 16 days to 15.7 years (118). In addition to cultures, the authors employed a real-time PCR assay targeting the rtx operon on oropharyngeal specimens, blood, articular aspirates, and intraoperative samples. K. kingae was the most common causative organism and was detected in 66 of the 138 (47.8%) microbiologically confirmed infections and in 76 of 85 (89.4%) children between the ages of 6 and 48 months. S. aureus was the second most frequent etiology, representing 4 of 10 bacteriologically proven cases below the age of 6 months, 2 of 85 (2.4%) in children between the ages of 6 and 48 months, and in 43 of 55 (78.2%) older children and adolescents (118). It should be pointed out that no pathogen was identified in 37 of 217 (17.1%) children. Although these patients could have had a non-infectious condition such as reactive arthritis or a rheumatologic disease, the possibility that they had joint or bone infections caused by microorganisms that are undetectable by our current diagnostic methods should also be entertained.

Multiplex NAATs

The diagnostic culture approach formulates the open question: what is the etiology of the patient’s disease? species-specific NAATs try to answer the close question: does THIS particular agent cause the infection? Therefore, requests for a NAAT that targets single pathogens are based on epidemiological and clinical considerations. The obvious drawback of using molecular tests that target a single species is that infections are caused by a wide array of pathogens that show overlapping symptoms and signs. To overcome this limitation and eliminate the guesswork of diagnosing infectious diseases, multiplex syndromic PCR tests have been developed in which several sets of primers are used to target the most plausible etiologic agents of particular medical conditions, such as septic arthritis, gastroenteritis, or central nervous system infections. Multiplex PCR assays include several pairs of primers in the reaction mixture, each targeting a syndrome-relevant individual etiology. This strategy enables the simultaneous and cost-effective analysis of multiple potential pathogens in a single biological specimen, saving time, laboratory resources, and precious patient samples (119). However, multiplex PCR tests may display inferior amplification efficiency compared to single PCR reactions. This diminished sensitivity occurs because multiple primer dimer sets in the same reaction can compete for components such as dNTPs and enzymes. Multiplex PCR also requires a single set of amplification conditions, including annealing temperatures, primer concentrations, and cycle parameters, for all the included targets, making optimizing individual sequences difficult (119).

BioFire

A commercial multiplex panel (BioFire Joint Infection) has been developed by bioMérieux (Marcy-l'étoile, France) for the simultaneous and rapid detection in synovial fluid samples of 31 bacterial and fungal pathogens (including K. kingae) commonly associated with osteoarthritis. In addition, the multiplex test targets eight relevant antibiotic-resistant genomic markers. Most studies assessing the test’s capabilities have enrolled adult patients, generally with infections of bone and joint implants (120). The long list of potential pathogens and resistance-encoding genes targeted by the BioFire Joint Infection system reflects the wide range of opportunistic and uncommon bacteria and yeasts responsible for these difficult-to-diagnose infections and their unpredictable antibiotic susceptibility patterns. Therefore, data on the system’s performance for detecting the agents of pediatric osteoarthritis are still limited.

In a multicenter US study in which 1,527 patients were enrolled, the performance of the BioFire Joint Infection was compared with that of conventional synovial fluid cultures (121). K. kingae was detected by the multiplex panel and missed by the culture in 3 of the 87 (3.5%) patients younger than 18 years (121). In a large international study, K. kingae was detected in only 3 of 1,544 joint fluid specimens, of which 23 were obtained from children aged less than 4 years (122). In a similar study comprising 399 synovial fluid samples, of which only 26 (1.7%) were obtained from patients younger than 18 years, K. kingae was detected in a single child (3.8%) and confirmed by a species-specific NAAT, while the culture result was negative (123). In a Colombian research study in which the BioFire Joint Infection multiplex was employed to diagnose pediatric osteoarthritis, 15 enrolled patients were aged 6 months to 5 years, and K. kingae was detected in one child missed by the culture (124). Recently, the performance of the BioFire Joint Infection panel was assessed for the diagnosis of 50 pediatric Spanish patients with suspected osteoarthritis and compared to traditional methods (125). Overall, K. kingae was detected in 11 patients, of which the blood culture isolated the organism in a single child and the synovial culture in six. In contrast, the BioFire Joint Infection test was positive in all 11 infections.

Home-brew multiplex test

In an attempt to improve the diagnostic performance of multiplex panels, a home-made arthritis syndromic panel was designed to target only clinically relevant pediatric pathogens, i.e., K. kingae, S. aureus, Streptococcus pneumoniae, S. pyogenes, and S. agalactiae (126). Synovial fluid aspirates obtained from children with presumptive joint infections were cultured and simultaneously tested with the home-brew multiplex NAAT (126). K. kingae was detected in 37 (66.1%) of the 71 (78.9%) children who met the inclusion criteria. However, despite the restricted number of genomic targets, the multiplex test missed five K. kingae infections diagnosed by culture, indicating a less-than-optimal sensitivity and the need to improve the test amplification technicalities.

Metagenomic next-generation sequencing

Despite the substantial advances in diagnosing pediatric joint and bone infections using NAATs, the Clinical Microbiology Laboratory still fails to identify a pathogen in many patients (118). Metagenomic next-generation sequencing (mNGS) is a novel cell-free DNA technology that promises to further advance the bacteriological diagnosis of joint and bone infections (127). Patient samples (usually blood, synovial fluid, or other normally sterile body fluids) are collected using a strict aseptic technique. Cell-free DNA is separated by centrifugation, and mNGS libraries are made and sequenced. DNA sequences of human origin are removed, and any remaining sequence is considered to represent the causative pathogen. The organism is identified by comparing the sequence with a curated database. The detection and identification can, then, be performed without prior knowledge of the suspected etiologic agent. This unbiased strategy is particularly helpful for establishing a microbiological diagnosis in immunocompromised patients and infections in which cultures and NAATs have been unsuccessful (127, 128). The mNGS tests, also known as “liquid biopsies,” are increasingly being used to establish the elusive etiology of periprosthetic joint infections in adults (129). However, because no mNGS-based test has yet received FDA endorsement for use in children, reports on the performance of this novel technology for pediatric skeletal system infections in general and the specific detection of K. kingae, in particular, are scarce (128, 130). Ramchandar et al. (130) performed an mNGS test on joint and bone samples of 42 children with suspected skeletal system infections and compared the results with those obtained by cultures and NAATs. Overall, a bacterial pathogen was identified by mNGS in 26 (61.9%) children vs 24 (57.1%) detected by the comparators. Three bacteremic patients were also positive for the same bacterium by cultures of the infected site and detected by mNGS. Metagenomic next-generation sequencing detected K. kingae DNA in six synovial fluid aspirates, of which five were also positive by PCR (130).

In a multicenter retrospective study, mNGS performed in plasma with the commercially available Karius test (Redwood City, CA, USA) detected K. kingae in 10 young children (median age: 16.5 months, range: 10–23 months) with spinal infections (131). All patients’ blood cultures were negative, while the standard-of-care PCR assay performed in a vertebral bone biopsy yielded K. kingae in a single child. The detection of K. kingae by mNGS established the diagnosis without biopsy in eight patients (in one child, a biopsy was performed before the mNGS test was submitted) and narrowed the antimicrobial coverage in nine without the traditional reliance on empiric MRSA therapy (131). Because no spinal biopsies were performed in most children and the poor performance of blood cultures for recovering K. kingae, only one patient had orthogonal validation of the diagnosis by other detection methods, raising the possibility of a false-positive microbial mNGS test result. However, the demographic, clinical, and laboratory presentation, excellent response to targeted antibiotic therapy, and lack of significant sequelae were entirely consistent with K. kingae spinal infections, supporting the accuracy of the Karius test result (131). This encouraging experience indicates that the novel pathogen-agnostic mNGS method made it possible to obtain a bacteriological diagnosis that otherwise would have remained unconfirmed. Although current mNGS testing is costly (1,000–2,500 USD per test) and requires expensive equipment and molecular biology and bioinformatics expertise, it has the potential to avoid spinal biopsies and expend the performance of laboratory tests of low yield, saving precious time, shortening hospital stays, and reducing overall costs (132–134).

Diagnosis of skeletal system infections by NAATs of oropharyngeal specimens

The microbiological diagnosis of childhood spondylodiscitis by the culture of spinal tissues is particularly problematic because of the reluctance of pediatricians to perform spinal biopsies under general anesthesia in young, non-cooperative patients. This invasive procedure is considered potentially risky, and an experimental study has convincingly demonstrated that even the minimal damage induced by a needle puncture may result in progressive degeneration of the intervertebral disk (134). In a recent study comprising children with spondylodiscitis, blood cultures were obtained from 62 of the 124 patients, with no positive results (135). A spinal biopsy was performed in only two children, and no pathogens were isolated (135).

To circumvent the difficulty, a surrogate non-invasive strategy has been proposed and implemented (136–138). Because colonization of the upper respiratory epithelium is a sine qua non requisite for the development of a K. kingae invasive infection, detecting the organism in oropharyngeal specimens by a sensitive NAAT has been used as a surrogate for the diagnosis of K. kingae osteomyelitis and septic arthritis (138, 139). The method consists of obtaining an oropharyngeal specimen and performing a sensitive K. kingae-specific NAAT assay. A positive molecular result in a young child, coupled with a benign clinical presentation, supports the role of K. kingae as the probable etiology of the infection. This diagnostic approach has the drawback that the prevalence of the bacterium in the oropharynx of children in the relevant 6–48 months age group is around 10% and substantially higher among those attending daycare center facilities, thus reducing the predictive value of a positive result (10). However, because the colonized oropharyngeal cavity is the source of the organism’s spread and invasion of other body sites, the assay’s negative predictive value is high, and failure to detect K. kingae’s DNA sequences virtually excludes the bacterium as the agent of the disease (10).

The strong correlation between the prevalence of the bacterium on the oropharynx and the occurrence of a K. kingae osteoarticular infection was demonstrated in a study conducted in parallel in Montreal, Canada, and Geneva, Switzerland (111). A total of 65 children aged 6–48 months complaining of joint pain or decreased limb movement without antecedent trauma who presented in good general condition, with or without fever, were enrolled. For each case, four age-matched controls admitted for trauma to the same emergency department were studied. A molecular test amplifying the rtxA operon detected K. kingae in the oropharyngeal specimens of 46 of the 65 cases (71%, 95% CI 59%–80%) and in 31 of the 34 (95% CI 77%–97%) with positive culture or PCR of the joint or bone sample, but in only 17 of the 286 controls (6%, 95% CI 4%–9%). The odds ratio for the association between K. kingae’s oropharyngeal colonization and the risk for a skeletal system infection was 38.3 (95% CI 18.5–79.1) (111). This non-invasive diagnostic approach is especially being used for young children aged 6–48 months presenting with suspected infections involving small or difficult-to-access bones or joints and spondylodiscitis (111, 136–139). In a binational prospective study involving young Italian and Swiss children with spondylodiscitis, the K. kingae-specific real-time PCR assay performed on blood specimens was positive in 4 of 29 (13.8%) children, whereas the same test performed on oropharyngeal samples was positive in 28 of 32 (87.5%) children (137). These encouraging results indicate that the PCR test performed on oropharyngeal swabs represents a convenient and valuable tool for the indirect diagnosis of invasive K. kingae infections such as spondylodiscitis and those involving small bones and joints (137).

Laboratory features of K. kingae infections

In a retrospective study conducted in Geneva, Switzerland, which enrolled 247 children with a variety of skeletal system infections caused by K. kingae, the laboratory workup results were gathered and analyzed (140). The patients’ mean age was 18.5 ± 10 months; only four were older than 48 months, and none were less than 6 months. The mean temperature at admission was 37.4°C ± 0.9°C; the mean leukocyte count was 12,700 ± 4,180/mm³; a left shift was present in only one patient; the mean platelet count was 419,000 ± 123,000/mm³, the mean C-reactive protein (CRP) level was 26.6 ± 27.8 mg/L, and the mean erythrocyte sedimentation rate (ESR) was 35.0 ± 18.9 mm/h (140).

The mild clinical and laboratory features of K. kingae bone and joint infections mimic non-infectious conditions such as transient synovitis, and the true nature of the disease is frequently not suspected. The familiar Kocher-Caird diagnostic algorithm for septic arthritis (141) is notoriously misleading, and the vast majority of patients with hip and knee arthritis would be missed by this predictive score, emphasizing the need for performing NAATs on oropharyngeal swabs and joint fluid in young children, even in the absence of fever, leukocytosis, and elevated inflammation markers (10, 142–144).

The mild characteristics of K. kingae skeletal system infections exhibit a sharp contrast with the symptomatology and inflammation markers of staphylococcal septic arthritis and osteomyelitis. A recent large study compared 100 K. kingae skeletal system infections with 116 caused by methicillin-susceptible S. aureus (145). In the multivariate analysis, age less than 4 years, CRP concentration <32.5 mg/L and a platelet count >400,000/mm³ were highly predictive of a K. kingae etiology, whereas the ESR and the white blood cell count were of limited predictive value.

Novel and uncommon infections

Traditionally, K. kingae has been associated with bacteremia, endocarditis, acute septic arthritis, and osteomyelitis, and uncommonly with lower respiratory tract disease, meningitis, and ocular infections (10). In the last two decades, the spectrum of clinical infections caused by the organism has been expanded by the description of novel syndromes affecting both immunocompetent and immunocompromised patients.

Chorioamnionitis

The organism was isolated from a patient with a rheumatologic disorder, resulting in septic abortion (146).

Pyomyositis

An infection involving the biceps crural muscle has been reported in a 21-month-old girl and a 14-month-old girl with a fluid collection in the anterior muscular compartment of the arm (80, 147).

Tenosynovitis and other infections of the hand and wrist

Like other pathogens infecting the skeletal system, K. kingae preferably invades the long bones and large joints of the extremities and, particularly, those of the legs (10). However, the bacterium also infects unusual locations, such as small bones and joints. Cochard et al. reviewed their experience with K. kingae hand and wrist infections in a Swiss pediatric hospital (148). Of 249 invasive K. kingae infections, 33 (13.5%) affected the hand or wrist. The patients’ age (mean ± SD) was 16.9 ± 9.4 months, with a range of 7–44 months; 25 (75.8%) children were afebrile, and in only one-half of the patients in which the CRP concentration was measured, the value was >20 mg/L, which is the value that predicts infection by the Caird criteria (141). The final diagnoses, confirmed by MRI, identified arthritis, tenosynovitis, osteomyelitis, cellulitis, and myositis, and in 19 of the 33 (57.6%) children, the disease simultaneously involved many anatomical structures. The introduction of a K. kingae-specific NAAT for blood, aspirates, and biopsy material in 2007 was followed by the use of the test for oropharyngeal specimens in 2009, resulting in a substantial increase in the detection of K. kingae, which was identified in 13.3% of the patients (148). Of the 33 children, 15 (45.5%) underwent surgical treatment in addition to antibiotics, and 18 (55.5%) received antibiotics alone, and full mobility and grasping were restored in all patients (148).

A similar experience was reported by El Houmami et al. (149), who analyzed 10 patients with flexor or extensor involvement of the tendon sheaths of the hands or feet, from France, Switzerland, and Israel, and by Gouveia et al. (150), who reported tenosynovitis in 8 of 58 (13.8%) Portuguese children aged 12–22 months. All children recovered and regained full-range motion, and the authors concluded that the administration of antibiotics could probably suffice for uncomplicated K. kingae tenosynovitis, whereas invasive interventions such as tendon sheath irrigation or debridement can be avoided.

Endovascular infection

An endovascular K. kingae infection without evidence of endocarditis was diagnosed in an elderly woman with myelodysplastic syndrome (151). The disease affected the transition of the external iliac artery with the common femoral artery and was complicated by culture-negative arthritis of the homolateral knee (151).

Transphyseal osteomyelitis

Primary epiphyseal and apophyseal subacute osteomyelitis are uncommon bone infections that can seriously damage the growing cartilage of the long bones, especially those of the lower extremities. These diseases particularly affect young children and are characterized by mild symptoms and low inflammation (152). Ceroni et al. (153) reported that K. kingae was detected by a species-specific NAAT in 8 of 14 children, while methicillin-susceptible S. aureus was cultured in one, and no pathogen could be isolated in the remaining five patients.

Subacute osteomyelitis

Subacute hematogenous osteomyelitis is an infectious process characterized by a >2 weeks course, mild or no systemic symptoms, slight bone pain, unimpressive laboratory test results, negative blood cultures, but positive radiological findings. This atypical presentation of a bone infection has been attributed to increased host resistance, a low-virulence organism, and/or prior antibiotic exposure (154). In a retrospective study conducted among 23 children younger than 48 months with subacute osteomyelitis, a K. kingae-specific real-time NAAT targeting the rtx operon was performed on an oropharyngeal specimen and/or a bone aspirate. The test was positive in 20 (95.2%), strongly suggesting that the organism is currently the prime etiology of subacute osteomyelitis among preschoolers. In two older children in whom the K. kingae molecular test was performed, the result was also positive, while S. aureus was the most common organism detected over the age of 4 years (154).

Skeletal system infections in adults

K. kingae has also been detected in septic arthritis of native and prosthetic joints in a few adult immunocompetent and immunocompromised patients (10, 155, 156).

Endocarditis

Endocardial infections represent an uncommon manifestation of K. kingae infections and were only diagnosed in 2 of 150 (1.3%) consecutive Israeli patients in the 1988–2015 period (10). Kingella kingae endocarditis also affects older children, adolescents, and adult patients, and only three-quarters of all pediatric patients with endocarditis are younger than 3 years compared to >96% of those with skeletal system infections or bacteremia. In a 2024 review of all 63 published patients, 45 (71.4%) were children, and 18 were adults (157). Predisposing conditions such as congenital cardiac malformations were found in 21 of the 45 (46.7%) children, and rheumatic valvular disease in two (4.4%). However, the remaining children had previously normal valves and were otherwise healthy, whereas among adults, endocarditis is usually associated with underlying chronic diseases or immunosuppressive conditions (157).

Children with endocarditis present in a poor general condition, have high fever, leukocytosis, and substantial increase in CRP levels and other acute-phase reactants (27). Whereas other pediatric K. kingae infections are characterized by an unimpressive presentation, a benign clinical course, and prompt and satisfactory response to antimicrobial therapy, leaving no long-term sequelae, despite the exquisite antibiotic susceptibility of the organism, K. kingae endocarditis is characterized by frequent life-threatening complications and a ≅10% mortality rate (158). Emergency surgical interventions are frequently needed due to heart failure unresponsive to medical therapy, severe valve malfunction, uncontrolled sepsis, the occurrence of septic emboli—particularly to the central nervous system—or the need to resect a large vegetation (158). In many patients, repair or replacement of the damaged cardiac valve would be later required (27, 157).

The epidemiological and clinical features of pediatric K. kingae endocarditis significantly differ from those caused by streptococcal species and S. aureus. Patients with K. kingae infection are much younger (mean ± SD: 16.0 ± 10.3 months vs 91.4 ± 74.1 months), their body temperature is higher (median of 40.0°C vs 39.0°C), hepatosplenomegaly is more common, and have higher leukocyte counts, lymphocytosis, and prevalence of buccal aphthae (27).

The profound contrast between K. kingae’s mild behavior in bacteremia or skeletal system infections and the severity of endocarditis suggests that strains causing endocarditis could belong to a subset of particularly invasive organisms with marked tropism to cardiac tissues. To test this hypothesis, a study was conducted in which 125 international K. kingae isolates derived from 23 healthy carriers and 102 patients with clinical infections, including bacteremia (n = 23), osteoarthritis (n = 61), and endocarditis (n = 18), were analyzed by whole-genome sequencing (83). Overall, 13 different MLST STs were identified among the 18 endocarditis-associated isolates, of which 8 (44.4%) belonged to STc-23/25. It should be pointed out that members of STc-23/25 were also recovered in the past from two children with endocarditis in France (83).

Disregarding their MLST ST grouping, endocarditis-associated K. kingae strains showed a statistically significant overrepresentation of six genes (PAS sensor domain-containing protein, elongation factor Tu, Eco57I restriction endonuclease, isoprenylcysteine carboxyl methyltransferase, ATP phosphoribosyltransferase, and a hypothetical protein of unknown function) compared to the other isolates (83). Remarkably, the gene encoding the iron-regulated protein FrpC was uniformly absent in all 18 endocarditis-associated strains but was present in 27 (32.1%) of the other 84 invasive strains (P < 0.05) (83). These preliminary results indicate that an in-depth study of K. kingae genes showing a differential distribution among the clinical infections should be conducted to elucidate their role in pathogenesis. However, it can also be argued that the severe clinical course of patients with K. kingae endocarditis does not exclusively result from the biological characteristics of the strain but also from the peculiar microenvironment of cardiac valve vegetations, which is poorly reachable by polymorphonuclear leucocytes, antibodies, and antibiotics (83).

MALDI-TOF identification of K. kingae

In recent years, the matrix-assisted laser desorption ionization-time-of-flight mass spectrometry has revolutionized how microorganisms are identified in the Clinical Microbiology Laboratory (159). The MALDI-TOF method is replacing the traditional slow and laborious phenotypic tests, enabling precise, reproducible, and rapid results that can be obtained by less-trained and experienced personnel (160). The published information on using novel technology to identify K. kingae is limited, probably because of the difficulties in detecting the organism in culture, and nowadays, most infections are diagnosed by molecular methods (112–118). In a pioneering study, Couturier et al. (160) assessed the Bruker instrument (Bruker Daltonics, Germany) and database capability to identify a HACEK group of bacteria collection, including five K. kingae isolates. All five were correctly identified, exhibiting identification scores ≥ 0.19. The Vitek MS system (bioMérieux, Marcy l'Etoile, France), the commercial competitor, enabled the precise identification of a K. kingae organism isolated from a patient with keratitis, but misidentified K. negevensis as K. kingae (42).

The MALDI-TOF spectra used for bacterial identification have been proposed for on-site and low-cost strain subtyping to assist in epidemiological investigations and surveillance. However, the approach’s usefulness depends on the bacterial lineages (161), and the limited data available indicate that the MALDI–TOF method is unreliable for discriminating between K. kingae strains (J. Schrenzel, personal communication).

Antibiotic susceptibility

Because K. kingae was initially considered an exceptional etiology of human disease, species-specific interpretive criteria for determining antimicrobial susceptibility were lacking for over five decades. Published literature on the bacterium’s antibiotic susceptibility was scarce, and antibiotic susceptibility results were generated employing different culture media and bacteriological methods. Thus, standardized disk diffusion and MIC data for K. kingae were unavailable.

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) initially recommended using general pharmacokinetic-pharmacodynamic-based breakpoints (162), whereas the last American Clinical and Laboratory Standards Institute (CLSI) guidelines (document M45, published in 2015) included K. kingae in the HACEK group of organisms and recommends collective single methods and interpretive criteria for all taxa in the group (163). This guideline ignores the fact that the HACEK acronym comprises heterogeneous and taxonomically unrelated bacteria joined together by a gram-negative staining, commensality in the oropharyngeal niche, fastidious growth, and being the uncommon etiology of endocarditis, disregarding their wide biological differences. In 2017, the EUCAST sponsored a large study to produce MIC–growth–inhibition zone diameter correlations. The study comprised 159 international K. kingae strains isolated between the 1960s and 2015. The strain collection was derived from healthy pharyngeal carriers (n = 47) and patients with a variety of clinical infections, including bacteremia (n = 28), osteoarthritis (n = 70), and endocarditis (n = 9) (164). Standardized EUCAST methods were employed for determining growth inhibition zones around antibiotic-containing disks and minimal inhibitory concentration distributions of 18 antimicrobial drugs, including penicillins, cephalosporins, carbapenems, fluoroquinolones, tetracyclines, rifampin, and trimethoprim-sulfamethoxazole (cotrimoxazole) (164). Cation-adjusted Mueller-Hinton agar plates and broth with 5% defibrinated horse blood and 20 mg/L-NAD (MH-F agar) were used and incubated at 35°C in a 5% CO₂-enriched atmosphere. The resulting antibiotic susceptibility results—inhibition zone diameters and MIC values—have been incorporated into the freely available EUCAST Breakpoint tables for the interpretation of MICs and zone diameters document, version 14.0 (1 January 2024), in which a table has been devoted to K. kingae on pages 94–95.

With rare exceptions, K. kingae is susceptible to aminoglycosides, macrolides, fluoroquinolones, and trimethoprim-sulfamethoxazole (frequently associated with β-lactamase production) (165), tetracyclines, and chloramphenicol (166–168).

All K. kingae isolates are intrinsically resistant to trimethoprim and glycopeptides and clindamycin. Traditionally, determining K. kingae’s susceptibility to clindamycin, an antimicrobial drug widely used for the treatment of pediatric joint and bone infections, was particularly problematic because of the blurred limits of the growth inhibition zones around the antibiotic disk, and 89 (61.4%) of 145 isolates were assumed to be susceptible (168). Employing the disk diffusion and microdilution methods, Matuschek et al. (164) found that clindamycin MIC values ranged from 0.5 to 2 mg/L, and thus, the EUCAST decided to consider the entire K. kingae population as intrinsically resistant to the drug. These data imply that the usual antibiotic choices that cover MRSA are inadequate for treating K. kingae infections (164–168). K. kingae also exhibits relatively high MICs to oxacillin (MIC₅₀: 3 mg/L; MIC₉₀: 6 mg/L), and treatment failure has been reported in a child with a spinal infection treated with flucloxacillin (169), indicating that isoxazolyl penicillins should not be used to treat invasive K. kingae infections (170, 171).

Beta-lactamase production

In a large international study on the prevalence and geographic distribution of β-lactamase production in K. kingae, it was found that the trait is uncommon and limited to a few geographic locations, such as southern Israel, Minneapolis (USA), New York, Reykjavik (Iceland), and Marseille (France) (81, 172, 173). Among the Israeli isolates, β-lactamase was significantly more prevalent among organisms carried on the oropharynx of asymptomatic children (16 out of 429, 15.4%) than among invasive strains (2 out of 190, 1.1%), P < 0.001, suggesting that the enzyme-producing strains have a colonization advantage because young children are subjected to repeat antibiotic exposure (174). Beta-lactamase production is clonal and restricted in Israeli isolates to the MLST ST-33, ST-34, and ST-38—all belonging to the STc-34; ST-3 and ST-49—both in STc-3; and ST-40 (in STc-29), and all the isolates of the aforementioned STs elaborate the enzyme (81). Strikingly, all the Minneapolis (n = 8) and Reykjavik (n = 4) β-lactamase-producing strains belonged to the highly invasive ST-6 in the STc-6, shared the closely related rtx alleles 8 or 9, and the por 1 allele. The genomic similarities among organisms isolated in these two distant locations do not seem to result from random convergent evolution. It has been speculated that they could be the result of the Scandinavian roots of many of the Minnesota inhabitants and the transatlantic crossing and contact between the populations of the two regions (81).

The β-lactamase enzyme is encoded by a bla_TEM-1 gene, which is usually contained in a plasmid, but it has also been chromosomally integrated in carried strains in France and Israel (47, 81). Bidet et al. (175) have shown that the plasmid bla_TEM-1 gene is located in the French KWG1 strain in an integrative and conjugative element (ICE) inserted in Met-tRNA and comprising genes that confer resistance to sulfonamides, tetracyclines, and streptomycin. Because K. kingae is intrinsically non-susceptible to trimethoprim, the ICE encoding sulfamide resistance explains the frequent linkage of trimethoprim-sulfamethoxazole resistance with β-lactamase production (165).

K. kingae’s β-lactamase has a weak hydrolytic activity, and the penicillin MICs of the enzyme producers range from 0.016 to 2 mg/L compared to 0.008–0.03 mg/L in non-producing strains (164). It does not hydrolyze cephalosporin drugs such as cefazolin, and its activity is fully inhibited by clavulanic acid (164, 165, 176). β-lactamase production should be suspected by a zone of growth inhibition diameter of 13–24 mm around the one unit disk benzylpenicillin and confirmed with the traditional colorimetric nitrocefin (Cefinase) disk (164). K. kingae’s antibiotic susceptibility patterns are summarized in Table 2.

Therapy

Traditionally, the treatment strategy for pediatric osteoarticular infections consisted of the prompt administration of broad antibiotic coverage, including drugs active against S. aureus, pending culture and antimicrobial susceptibility results. The initial antibiotic therapy was administered via the intravenous route until the patient’s local and general clinical conditions improved, fever subsided, and CRP levels decreased. The treatment was then switched to the oral route to complete 10–15 days for septic arthritis and 3–4 weeks for osteomyelitis, whereas spondylodiscitis was usually treated for a longer period (10).

The indications for surgical intervention included obtaining exudate or tissue samples for a microbiologic diagnosis, drainage of pus collections and abscesses to achieve infectious source control, and preservation of maximal function (177). This aggressive approach derives from the potentially severe complications of joint and bone infections caused by traditional pyogenic bacteria, particularly S. aureus, which was traditionally considered the primary etiology of these infections, regardless of age group. Staphylococcal septic arthritis and osteomyelitis, especially those caused by PVL-positive strains, follow a severe clinical course, a slower clinical response to therapy, and may result in severe morbidity and even mortality, as well as long-term functional sequelae (178, 179). However, a clear trend toward shorter antibiotic courses and a less invasive therapeutic approach for osteoarticular infections has been gradually developing, mostly in European countries (180–182). This trend has been considerably strengthened by recognizing K. kingae as the etiology of the vast majority of skeletal system infections in preschool children, which are characterized by a mild clinical course, remarkable antibiotic susceptibility, and favorable outcome, leaving no orthopedic disabilities (183). Therefore, it has been suggested that the current recommendations for treating K. kingae joint and bone infections are excessive and could be tailored to the low-virulence features of the organism (184). In addition, alternative non-invasive approaches have been advocated to dispense with the need for surgical procedures such as arthrotomy, bone drilling, and biopsies (136) and, probably in the near future, by detecting circulating mcfDNA in the plasma (131). These culture-independent diagnostic modalities suit K. kingae exceptionally well, not only because of the fastidious nature of the bacterium but also because it frequently invades small joints and bones and difficult-to-reach intervertebral discs.

Pioneering studies published over the last few years could represent the first steps toward a less invasive approach for managing uncomplicated pediatric joint and bone infections. A prospective 1-year investigation was carried out in a Spanish medical center in which 25 children aged 18–54 months, who presented in good general health and oral tolerance and who could be closely followed as outpatients, received exclusively oral antibiotics (185). These patients were compared with 228 hospitalized children who were administered sequential parenteral and oral antibiotics treated in other hospitals participating in the Spanish Network of Osteoarticular Infections (RioPed) registry (185). The two patient groups were comparable in age, gender, fever, ESR, and CRP concentration on admission. The exclusive oral group comprised 15 children with osteomyelitis, 7 with septic arthritis, 2 with osteoarthritis, and 1 with spondylodiscitis. As expected, this group had a lower percentage of S. aureus (8% vs 26%, P = 0.06) and a higher proportion of K. kingae (24% vs 9%, P = 0.017) than the sequential antibiotic therapy group. There were complications (24%) and follow-up sequelae (6%) in the sequential therapy group and none in the oral therapy group (185).

In a nationwide Spanish study, 893 hospitalized children who were initially treated with intravenous therapy were compared to 64 children who were administered oral antibiotics alone. Patients allocated to receive just oral antibiotics met all of the following criteria: good general condition, no underlying disease, age 6 months–3 years, appropriate oral tolerance, CRP ≤ 80 mg/L, ESR/CRP ratio ≥ 0.67, no skin injury, no recent surgery, no cervical spondylodiscitis, and no local complications at onset (186). Patients in the oral therapy group were younger (mean age 33.9 vs 20.3 months [P = 0.001]), had a significantly lower percentage of S. aureus (23.3% vs 3.1%, P < 0.001), a higher proportion of K. kingae (12.1% vs 28.1%, P = 0.001), higher ESR/CRP ratio (1.4 interquartile range [IQR] 0.6–3.6 vs 3.3 IQR 1.7–5.7, P < 0.001) and showed a lower rate of fever (63% vs 48.8%, P = 0.024). Neither acute complications nor long-term sequelae were found in the oral therapy group (186).

Oral vs intravenous empirical antibiotics were compared in a randomized, controlled, non-inferiority study involving Danish patients aged 3 months to 17 years (187). Patients with severe infections—the presence of septic shock, need for acute surgery, or substantial soft tissue involvement, prosthetic material, significant comorbidity, previous bone and/or joint infection, or antibiotic administration for longer than 24 h before enrollment—were removed from the study (187). Overall, 101 patients were randomized to receive oral therapy and 91 to receive intravenous treatment. Thirteen patients (five in the oral therapy group and eight in the parenteral therapy group) had a K. kingae infection. Need for surgery after randomization and adverse effects did not show statistically significant differences between the two groups, and no orthopedic disabilities were later detected in either group (187).

The experience of treating K. kingae osteomyelitis and septic arthritis exclusively with oral antibiotics is still limited. A small retrospective Spanish study comprising 12 children with culture- or NAAT-confirmed K. kingae skeletal system infections has shown that selected cases defined by age less than 4 years, good general condition, and possibility of follow-up as outpatients can be successfully treated with oral therapy alone, hospital stays can be shortened, costs reduced, and the need to insert a central venous line and its complications can be avoided (188).

ACTINOBACILLUS

Microbiological aspects

The genus Actinobacillus in the family Pasteurellaceae consists of facultatively anaerobic, nonmotile, gram-negative rods and comprises animal (Actinobacillus equuli, Actinobacillus lignieresii, Actinobacillus pleuropneumoniae, and Actinobacillus suis) and human (Actinobacillus hominis and Actinobacillus ureae) species, but no longer includes Actinobacillus actinomycetemcomitans, which was renamed Aggregatibacter actinomycetemcomitans and is therefore still known to dentists as the non-systematic abbreviation AAC (http://www.the-icsp.org/taxonomic-subcommittees [189]).

Actinobacillus spp. are medium-sized, gram-negative rod-shaped or coccoid bacteria with a tendency to bipolar staining; their arrangement is single, in pairs, and, rarely, in short chains. They require enriched media and grow best in a 5%–10% CO₂ atmosphere. Some Actinobacillus spp. are included in both commercial MALDI-TOF MS databases. A. ureae is included in the VITEK 2 ID-NH card database. In view of the phenotypical closeness, molecular methods (e.g., 16S ribosomal RNA gene or rpoB gene PCR/sequencing) may be needed for species identification of Actinobacillus, Aggregatibacter, Capnocytophaga, and Pasteurella spp. (17, 19, 190). EUCAST has defined non-species related PK/PD MIC breakpoints (191).

Clinical aspects, including epidemiology

Actinobacillus lignieresii (primary habitat in the oral cavities of sheep and cattle), A. equuli (in the oral cavities of horses and pigs), and A. suis (in the oral cavities of pigs) can be transmitted to humans by animal contact (192). A. hominis and A. ureae are pathogenic only for humans; their natural habitat is unknown (192, 193). Virulence factors are widespread among members of this genus and other species of the family Pasteurellaceae, particularly toxins belonging to the pore-forming RTX family. RTX toxins have repeats in the structural toxin peptide and exhibit cytotoxic and often hemolytic activity. The toxic activities of RTX toxins in host cells may lead to necrosis and apoptosis. A. equuli harbors the hemolytic RTX toxin aqxA and A. suis the cytotoxic RTX toxin ApxIA and ApxIIA (194).

A. lignieresii causes actinobacillosis, a granulomatous disease in cattle and sheep in which, similar to actinomycosis, sulfur granules form in tissues (195). A few human soft tissue infections after a cow or sheep bite or other animal contacts have been reported (6). A. equuli and A. suis have caused a variety of diseases in horses and pigs; human infections are generally due to horse or pig bites (6) or contact, e.g., a case of meningitis in a child after a previous visit to a farm (196). Recently, fatal sepsis and mitral valve endocarditis have been described (197, 198).

A. hominis is known to cause respiratory tract infections and bacteremia but can also occur as a commensal, albeit rarely. The most important predisposing factors are underlying cardiovascular, chronic lung disease, or liver disease in debilitated patients (193). A case of osteomyelitis without an underlying disease was reported in a 35-year-old woman (199). A. ureae can cause serious infections in patients with underlying conditions, including skull fracture, neurosurgical procedures, alcoholism, or immune compromise. In most cases, it caused meningitis or meningoencephalitis, bacteremia, endocarditis, and respiratory infections, while bone marrow infections, peritonitis, urogenital infections, and middle ear infections have also been reported (200). A patient with rheumatoid arthritis who received anti-TNF-α therapy developed septic arthritis and abscess formation with A. ureae (201).

Susceptibility studies of Actinobacillus spp. are extant for a few isolates of the human-pathogenic species that are susceptible to many antimicrobials, including penicillin (193, 201). A. ureae is susceptible to penicillin, ampicillin, and cephalosporins; β-lactamase production has not been reported. The organism is also susceptible to tetracycline, macrolides, and aminoglycosides (202). In meningitis, third-generation cephalosporins are indicated until the diagnosis of H. influenzae is ruled out; surgical repair of a dural leak may also be necessary in some patients (202). Empirical ceftriaxone for 2 weeks has also been successful in meningitis caused by A. equuli/suis (196).

AGGREGATIBACTER

Microbiological aspects

Multilocus sequence analysis of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus, and Haemophilus segnis (189) showed that these bacteria represent a monophyletic group. Actinobacillus actinomycetemcomitans, H. aphrophilus, H. paraphrophilus, and H. segnis have therefore been transferred into the new genus, Aggregatibacter, in the family Pasteurellaceae, with the species Aggregatibacter aphrophilus comprising the former H. aphrophilus and H. paraphrophilus, i.e., including V factor-dependent and -independent isolates (189). Aggregatibacter kilianii was proposed for human clinical isolates of Aggregatibacter as a new species (203).

Aggregatibacter spp. are coccoid to rod-shaped gram-negative bacteria, occasionally exhibiting filamentous forms. Aggregatibacter spp. require enriched media and grow best in a 5%–10% CO₂ atmosphere. Some A. aphrophilus strains and A. segnis require V-factor and therefore do not grow on blood agar but on chocolate agar medium (204). A. actinomycetemcomitans colonies initially show a central dot and a slightly irregular edge and, on further incubation, develop a star-like configuration resembling “crossed cigars” and pit the agar. Colonies of other Aggregatibacter spp. are granular or smooth, grayish white to yellowish, and opaque. For species identification, a battery of tests (requirements for V- and X-factors, biochemical tests, and colony morphology) is necessary to avoid confusion with Haemophilus spp. Aggregatibacter spp. exhibit negative reactions for indole, ornithine decarboxylase, and urease and are not dependent on X-factor, whereas Haemophilus spp. are positive for at least one of these three reactions or cannot synthesize heme components from delta-amino-levulinic acid. Automated systems may present difficulties in identifying these bacteria to the species level (14, 205). A. segnis is best identified by a specific NAAT (205). Aggregatibacter spp.—except for A. kilianii—are included in both MALDI-TOF MS databases (18, 19), as well as in the VITEK 2 ID-NH card database (14). A decisive phenotypic test for the identification of A. kilianii sp. nov. is alanine-phenylalanine-proline arylamidase as tested by VIKEK 2 ID-NH card, which is positive for A. kilianii and negative for the three other Aggregatibacter spp. (203). In view of the phenotypical closeness of the species, molecular methods (e.g., 16S ribosomal RNA gene or rpoB gene PCR/sequencing) may be needed for species identification of Actinobacillus, Aggregatibacter, Capnocytophaga, and Pasteurella spp. (17, 19, 190). A commercial NAAT-based hybridization assay for the detection of A. actinomycetemcomitans and other periodontal pathogenic bacteria (e.g., E. corrodens and Capnocytophaga spp.) is available (206). EUCAST has defined non-species related PK/PD MIC breakpoints (191). CLSI published guidelines for broth microdilution susceptibility testing of the HACEK group (163).

Clinical aspects, including epidemiology

The habitat of Aggregatibacter spp. is the human oral cavity; the species of the genus are occasionally recovered from other body sites; the primary habitat of A. actinomycetemcomitans is the tooth (189). The main virulence factors of A. actinomycetemcomitans are the leukotoxin A (LtxA) of the RTX family and the cytolethal distending toxin; its lipopolysaccharides (LPS) and cytokine-binding factors contribute to its pathogenicity. Serotyping into serotypes a to f and non-serotypeable A. actinomycetemcomitans is based on the structural differences in the O-antigen part of LPS (207); an additional serotype g has been characterized (208). A. actinomycetemcomitans is one of the major agents of juvenile and adult periodontitis (207) and may occur together with Actinomyces spp. in actinomycotic sulfur granules (189). Patients with aggressive periodontitis harbor serotypes b and c of A. actinomycetemcomitans, with serotype c being the most prevalent serotype in diseased and healthy subjects, at least in Brazil (209). Serotypes a, b, and c are globally dominant, but the distribution may vary depending on ethnicity and geography. The correlation between different serotypes and different periodontal conditions remains unclear (210). However, a particular clone of A. actinomycetemcomitans belonging to the serotype b cluster, JP2 genotype, is associated with an increased risk of developing periodontitis because a 530 deletion within the promoter region of the leukotoxin gene is responsible for a higher level of leukotoxin than in other strains (211). A recent study suggests that infection with the periodontal pathogens A. actinomycetemcomitans and Porphyromonas gingivalis impairs the therapeutic response in rheumatoid arthritis (212). Several periodontal pathogens, including P. gingivalis and A. actinomycetemcomitans, are associated with atherosclerosis-related diseases (213).

Aggregatibacter belongs to the HACEK group of gram-negative bacteria and, as such, is an etiology of bacterial endocarditis. Only a few layers of crevicular epithelial cells separate the gingival location from the host’s parenteral space, and incidental translocation of the bacterium to the bloodstream may occur during tooth brushing, injury, and chewing of granular substances, which can be accelerated by the presence of periodontitis (214). Prosthetic valve endocarditis (PVE) is common in A. actinomycetemcomitans (15) but pacemaker endocarditis is rare (215). Other non-oral infectious diseases include osteomyelitis, arthritis, synovitis, brain abscess, meningitis, thyroid abscess, urinary tract infection, soft tissue infections, and pneumonia (214–217). Pulmonary co-infection of A. actinomycetemcomitans and Actinomyces sp., forming a mediastinal mass, may mimic malignancy (218). Skin infection with soft tissue involvement may also be a co-infection of Actinomyces israelii/meyeri and A. actinomycetemcomitans, even if Actinomyces sp. cannot be cultured; actinomycete-like druses may be seen on histological examination (219). A review of infections in children showed mainly pulmonary or chest wall infections, including a pulmonary empyema and subphrenic abscess, as well as myositis and osteomyelitis; some infections can be mistaken for malignancy (217).

A. aphrophilus can lead to canniculitis and endophthalmitis, as well as systemic diseases, in particular bone and joint infections, spondylodiscitis with spinal epidural abscess, brain abscess, ventriculitis after transarticular C1–C2 screw fixation, liver abscess, empyema, and peritonitis (15, 16, 189, 219–224). In children, dental procedures, close contact with dogs, and congenital heart disease represent predisposing conditions for A. aphrophilus brain abscesses (225). A large multinational cohort study of HACEK endocarditis showed that A. aphrophilus and C. hominis are equally frequent but less frequent than H. parainfluenzae and A. actinomycetemcomitans and more frequent than E. corrodens and Kingella sp.; endocarditis has also been described in prosthetic heart valves (15). A single case of pacemaker endocarditis with A. aphrophilus is reported (226).

A. segnis, whose frequency may be underestimated due to obvious misdiagnoses, can rarely cause endocarditis (15, 205, 227). A. segnis may be an important cause of bacteremia and sometimes of other infections (16, 205). A. kilianii has been isolated from blood, wounds, abdominal abscess, conjunctivitis, and dacryocystitis (203). A case of cholecystitis has been described recently (228).

Aggregatibacter spp. are mainly susceptible to cephalosporins, tetracyclines, and aminoglycosides (15). Resistance to penicillins is common, but aminopenicillin combined with a β-lactamase inhibitor has been found to be effective. However, some isolates of Aggregatibacter spp. may be resistant to ampicillin due to mechanisms other than β-lactamase production (163). A larger study in Switzerland of 125 strains of A. actinomycetemcomitans showed no resistance to amoxicillin/clavulanic acid, but 88% were resistant to penicillin; clindamycin and tetracycline were resistant in 88% and 21%, respectively (229). However, in a large collection of oral strains from diverse geographical origins, no resistance to amoxicillin was detected in A. actinomycetemcomitans, even in strains previously reported as resistant to amoxicillin. The authors concluded that there is currently no convincing evidence to replace oral amoxicillin when antimicrobials are indicated for treatment of A. actinomycetemcomitans-associated periodontitis (230). The JP2 genotype appears to be more sensitive to penicillins than the non-JP2 genotypes; in addition, the JP2 genotype is susceptible to fusidic acid, which may be an option for topical therapy (231). For infective endocarditis, an intravenous course of at least 4 weeks with ceftriaxone is reasonable, which should be prolonged to 6 weeks for PVE strains. Ampicillin is an option when the appropriate susceptibility test is available, otherwise in combination with a beta-lactamase inhibitor. For patients who cannot tolerate ceftriaxone, other third-generation cephalosporins, or ampicillin, fluoroquinolone is a considerable option for adult patients, even when taken orally. Gentamicin is not recommended by the American Heart Association Committee because of its nephrotoxicity risk (232). Osteomyelitis is usually treated with an intravenous therapy followed by oral antibiotics for weeks (216). A. actinomycetemcomitans is usually susceptible to oral antibiotics such as fluoroquinolones, macrolides, and trimethoprim-sulfamethoxazole (231, 233), but tetracycline and clindamycin require susceptibility testing (229). For other non-oral infections, prolonged therapy is recommended depending on the extent of tissue involvement and the clinical response. For the pediatric infections with A. actinomycetemcomitans mentioned above, the mean treatment duration was 4.6 months (217).

A. aphrophilus is mostly susceptible to penicillin and amoxicillin, tetracycline, and azithromycin (231, 233); an in-house investigation of 18 strains of A. aphrophilus revealed susceptibility to trimethoprim-sulfamethoxazole and ciprofloxacin. These oral antibiotics are options for the treatment of non-systemic infections. In the case of brain abscesses, surgical intervention followed by prolonged treatment with antibiotics that penetrate the brain, i.e., ceftriaxone or carbapenems, is necessary to achieve a favorable outcome (220, 225). However, brain abscesses with A. aphrophilus are sometimes polymicrobial (234); metronidazole or a beta-lactam with anaerobic spectrum is indicated if anaerobic bacteria are suspected, even if the anaerobic cultures remain negative. The therapy for A. aphrophilus HACEK endocarditis is described above (232). As for other HACEK organisms, endocarditis is characterized by a relatively long interval between first symptoms and diagnosis (range, 2 weeks to 6 months), large vegetation sizes on native or artificial valves of the left side, and frequent embolization (15). This is also the case for rare A. segnis endocarditis (15, 205, 232). The therapy for other infections with A. segnis and A. kilianii is similar to that for infections with A. aphrophilus, as long as no specific regimens are published. Cephalosporins can be considered as susceptible, but susceptibility testing for penicillins, i.e., beta-lactamase production, macrolides, tetracycline, and clindamycin, is warranted.

CAPNOCYTOPHAGA

Microbiological aspects

The genus Capnocytophaga in the family Flavobacteriaceae at present consists of at least 10 species (Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica, Capnocytophaga leadbetteri, Capnocytophaga ochracea, and Capnocytophaga sputigena) of facultatively anaerobic, nonmotile, gram-negative rods, with gliding motility (235). Isolates from human dental plaques or from a human periapical abscess have recently been described as Capnocytophaga periodontitidis sp. nov. and Capnocytophaga bilenii sp. nov. (235, 236). Capnocytophaga genomospecies AHN8471 might be the same species as C. periodontitidis (235). Capnocytophaga endodontalis sp. nov. has been proposed for an isolate from a human periapical abscess (237). In addition to C. canimorsus and C. cynodegmi, Capnocytophaga canis and Capnocytophaga stomatis have been proposed as new species of dog- and cat-hosted isolates (238, 239). Capnocytophaga felis sp. nov. was recently described for four isolates from the oral cavities of healthy cats in Japan (240).

Capnocytophaga spp. are mainly fusiform, medium-to-long cells with tapered ends. In bacteremia with abundant C. canimorsus, the fusiform rods can be detected directly in the peripheral blood smear or in polymorphonuclear leukocytes as intracytoplasmic rods in the Pappenheim or May-Grünwald-Giemsa stain (241, 242). C. canimorsus has most often been isolated from blood cultures. However, it may not be detected by commonly used automated blood culture systems; therefore, clinicians should inform the laboratory about risk factors for C. canimorsus infection so that subcultures on enriched media can be performed in a blind manner (243). C. canimorsus is a very fastidious bacterium, and subcultures must be performed frequently. For keeping strains in a culture collection, the isolates should be frozen in a cryoprotective solution, e.g., skim milk, at −70°C. C. canimorsus can also be detected and identified by 16S ribosomal RNA gene PCR/sequencing (244).

Primary isolation of Capnocytophaga spp. requires 5%–10% CO₂ and enriched media; the composition of the blood agar base influences the ability to grow (243). Colonies of Capnocytophaga spp. on blood agar are very small after 24 h at 37°C and reach 2–4 mm in diameter after 2–4 days; they are convex or flat and often slightly yellow when scraped off agar, show regular or spreading edges with gliding motility, and adhere to the agar surface (245). C. canimorsus, C. cynodegmi, as well as the recently described C. canis, C. stomatis, and C. felis are catalase and oxidase positive. Phenotypic differentiation of species in the oxidase-negative group may be inconclusive due to the similarity of many biochemical reactions and the lack of suitable substrates even in automated systems (14). Most Capnocytophaga spp. are included in the database of the Bruker MALDI Biotyper (19) and some are in the database of the VITEK MS system. The VITEK 2 ID-NH card database includes Capnocytophaga spp. at the genus level (17). The new species C. periodontitidis is not yet in the databases, but the positive urease reaction separates it from other Capnocytophaga spp. (235); the more recent human oral and zoonotic Capnocytopha sp. are also not included in the databases. In view of the phenotypical closeness of the species, molecular methods (e.g., 16S ribosomal RNA or rpoB gene PCR/sequencing) may be needed for species identification of Actinobacillus, Aggregatibacter, Capnocytophaga, and Pasteurella spp. (17, 19, 190). EUCAST has defined non-species related PK/PD MIC breakpoints (191).

Clinical aspects, including epidemiology

The oxidase- and catalase-negative species C. bilenii, C. gingivalis, C. granulosa, C. haemolytica, C. leadbetteri, C. ochracea, C. periodontitis, and C. sputigena are members of the human oral microbiota (235, 236). The oxidase- and catalase-positive species C. canimorsus and C. cynodegmi reside in the oral cavities of healthy dogs (25% of dogs harbor C. canimorsus as determined by culture and 85%–100% as determined by PCR) and cats (15% of cats harbor C. canimorsus as determined by culture) (246). C. canis is also a commensal of dogs and cats; the proportion of C. canimorsus vs C. canis is lower in cats than in dogs (238). C. stomatis and C. felis are additional species of Capnocytophaga with zoonotic potential (239, 240). Capsular serotyping of C. canimorsus has been done by PCR and ELISAs; C. canimorsus has nine different serovars (A–I) of capsular polysaccharide (CPS), which are implicated in resistance to the innate immune system of the host. However, only three serovars A, B, and C cover 88% of human isolates, so the high prevalence of these three serovars compared to the low prevalence of these serovars among dog isolates (7.6%) confirms that these three serovars are more virulent for humans than other serovars and suggests that the low incidence of disease may be related to the low prevalence of the A, B, and C serovars in dogs (247). The capsular serovar B can also be found in C. canis and C. cynodegmi (248). C. canimorsus resists phagocytosis by macrophages and killing by complement and leukocytes; macrophages incubated with the bacterium failed to produce several proinflammatory cytokines and nitric oxide, suggesting that it can escape immune surveillance by failing to stimulate the innate immune response (249). The lipopolysaccharide of gram-negative bacteria, composed of lipid A, core, and O-antigen, is one of the most pro-inflammatory bacterial compounds, although the C. canimorsus LPS is 100-fold less endotoxic than Escherichia coli LPS. C. canimorsus has a unique penta-acylated lipid A that does not interact with the usual LPS receptors unless lipid A is attached to its core oligosaccharide. Overall, the properties of the lipid A-core may explain how this bacterium first escapes the innate immune system but nevertheless can cause shock at the septic stage (250). The presence of CPS is likely to be a common property of C. canimorsus; the capsule is related to the lipooligosaccharide (LOS) of the LPS and probably composed of the same polysaccharide units. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages (251). Except for LPS, the pathogenicity factors of human oral Capnocytophaga have not yet been investigated.

C. gingivalis, C. granulosa, C. haemolytica, C. leadbetteri, C. ochracea, and C. sputigena have been reported as agents of septicemia and other endogenous infections (endocarditis, endometritis, ophthalmic lesions, osteomyelitis, peritonitis, pneumonia, and soft tissue infections) in immunocompetent and immunosuppressed (mainly neutropenic) patients (16, 252–254), but also as a cause of chorioamnionitis and neonatal sepsis (255, 256). The cases of chorioamnionitis with preterm deliveries and neonatal infections caused by oral Capnocytophaga spp. not commonly found in the genital tract may reach the uterus via orogenital contact; in some cases, Capnocytophaga sp. could be isolated from a partner with periodontal disease (255). Meningitis with C. sputigena has been described following endonasal resection of a clival meningioma complicated by cerebrospinal fluid leak through the nose (257). A retrospective review of all patients with Capnocytophaga infection from January 2010 to August 2020 at three main hospitals of Mayo Clinic showed that human-oral-associated Capnocytophaga bacteremia occurs primarily in immunocompromised patients, particularly those with hematologic malignancy. Human-oral-associated Capnocytophaga infection without bacteremia is frequently isolated in polymicrobial infection (258). An unusual septicemia, including purpura in an asplenic patient after a dog bite, was due to C. ochracea (259). The human oral Capnocytophaga spp. can be opportunistic pathogens associated with periodontal diseases. C. periodontitidis has been isolated from the subgingival plaque of a periodontitis patient (235). The recently described C. bilenii was detected in a gingivitis subject (236) and C. endodontalis in a human periapical abscess (237).

Infections with C. canimorsus and C. cynodegmi are most commonly associated with dog or cat bites or contact. This also applies to C. canis and C. stomatis (260). The Mayo Clinic’s 10-year review confirmed that patients infected with C. canimorsus most often present with septicemia. However, not all patients had classical risk factors; of 16 patients with C. canimorsus bacteremia, only 2 patients had asplenia and alcohol use disorder, respectively. Additionally, only 68% of the C. canimorsus bacteremia patients were linked to animal bites or scratches (258). A rare case of a patient with chronic sinusitis and without animal wound has been reported. C. canimorsus probably entered the nasal cavities through contact with the patient’s dog, probably by licking the patient’s face. The chronic sinusitis probably resulted in mucous membrane disruption that served as a point of entry for the bacterium into the bloodstream (261). A multicenter study in France observed 44 cases of Capnocytophaga zoonotic infections over 10 years, i.e., C. canimorsus, n = 41, and C. cynodegmi, n = 3. The most frequent clinical presentations were sepsis (n = 37), skin and soft tissue infections (n = 12), meningitis (n = 8), osteoarticular infections (n = 6), and endocarditis (n = 2). Case fatality rate was 11%; mortality and meningitis rates were higher for alcoholic patients. Eighty-two percent of cases involved contact with dogs, mostly including bites (63%) (262). In a review of all cases with C. canimorsus infection before 2014 (median age 55 years, two-thirds male), prior splenectomy or hyposplenism and alcoholism were predisposing factors in around 20% of cases each. In 60% of cases, dog bites occurred before the onset of the disease; in 27%, scratches, licking, or other contact with dogs or cats also occurred (9). The overall case fatality was 26%. In fulminant cases, which have a poor prognosis, disseminated intravascular coagulation, acute renal failure, respiratory distress syndrome, and shock may develop; hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura are other possible sequelae (9). Lethal Waterhouse-Friderichsen syndrome is the extreme consequence (263). Arthritis, aortitis, mycotic aneurysm, blebitis, endophthalmitis, keratitis, meningitis with subsequent sensorineural hearing loss, and peritonitis have been reported as well (9, 264–272). A woman who had close contact with her dog developed target lesions on her upper and lower extremities and pneumonitis during the bacteremia with C. canimorsus (273). Endocarditis, myocarditis, or myocardial infarction following dog bites is rare but may even occur in immunocompetent individuals (241, 274, 275). C. cynodegmi was isolated less frequently, mainly from localized wound infections, most resulting from dog bites or cat scratches (276). However, it was isolated from the blood after a dog bite, with most cases showing a favorable outcome, but fatal cases have also been reported (258, 260, 262). Pneumonitis and cellulitis were described in a diabetic patient with C. cynodemi bacteremia after a dog bite (277). Peritonitis in a peritoneal dialysis patient has also been described (276). C. canis can also induce sepsis or even septic shock with and without risk factors; some of them occurred after a cat scratch (278–281). C. canis can also cause skin manifestations such as cellulitis or a target-looking rash (280, 282). C. stomatis has been found to be pathogenic to humans when isolated from infected wounds resulting from dog bites, and it can also cause bacteremia with cellulitis in immunosuppressed patients (260). Recently, endophthalmitis caused by C. felis was described in a healthy patient after a cat had bitten the upper and lower left eyelid and the nasal part of the conjunctiva of the left eye (283).

Oral Capnocytophaga spp. are susceptible to amoxicillin, amoxicillin-clavulanic acid, ciprofloxacin, moxifloxacin, doxycycline, and tetracycline but showed variable susceptibility to clindamycin and penicillin. Thus, some periodontal disease-related bacteria can be resistant to antibiotics commonly used in adjuvant periodontal therapy (284). However, mouth rinsing containing chlorhexidine can be recommended in immunocompromised patients with periodontal diseases (253). For other infections, oral Capnocytophaga spp. are usually susceptible to beta-lactam/beta-lactamase combinations, broad-spectrum cephalosporins, carbapenems, but susceptibility to other antibiotics such as aminoglycosides, aminopenicillins, clindamycin, erythromycin, fluoroquinolones, and tetracycline should be tested (253, 284). The beta-lactamase producer and resistance to macrolide, together with clindamycin resistance, are highly prevalent in human oral Capnocytophaga spp. (285). A multidrug-resistant C. gingivalis with resistance to third-generation cephalosporins, fluoroquinolones, gentamicin, macrolides, and clindamycin has been encountered in a patient with acute exacerbation of chronic obstructive pulmonary disease (286). A child with a brain abscess revealed a multiresistant C. ochracea; the strain was highly resistant to third and fourth-generation cephalosporins but susceptible to clindamycin and moderately susceptible to ciprofloxacin. After a craniotomy, the child was treated for 4 weeks with imipenem and metronidazole, and for 2 weeks with ciprofloxacin (252). Initial empirical treatment of a C. sputigena meningitis in a neurosurgical patient consisted of vancomycin and meropenem, but this led to exacerbation of the patient’s urticaria pigmentosa. Subsequent treatment with ceftriaxone also resulted in worsening of the rash. Treatment was empirically switched to chloramphenicol (15 mg/kg of body weight four times a day) and continued for 3 weeks with full recovery. Systemic chloramphenicol is uncommonly used in contemporary UK practice but remains an excellent antibiotic for penetrating the CNS and has excellent bioavailability (257). Maternal and neonatal disease due to oral Capnocytophaga spp. should be treated with third-generation cephalosporins or carbapenems or a combination of beta-lactam/beta-lactamase inhibitor, as beta-lactamase is often produced, and susceptibility to aminoglycosides is variable (255, 256). Patients with human-oral Capnocytophaga spp.-associated bacteremia had a higher frequency of beta-lactamase production than in C. canimorsus bacteremia in the Mayo Clinic 10 years survey. However, in non-bacteremic polymicrobial infections, the presence of Capnocytophaga sp. is unlikely to affect treatment and the choice of antibiotic therapies, so other antibiotics were used in addition to clindamycin and newer fluoroquinolones (258). Most strains of the zoonotic Capnocytophaga sp. are susceptible to beta-lactams, tetracycline, erythromycin, clindamycin, fluoroquinolones, and carbapenems but show resistance to aminoglycosides, trimethoprim-sulfamethoxazole, and metronidazole (9).

In the bacteremic group of the Mayo Clinic survey, a beta-lactam/beta-lactamase inhibitor combination, carbapenems, or third-generation cephalosporins were the common therapy against C. canimorus and C. cynodegmi infections (258). In the French survey, the most frequent initial therapy against C. canimorsus and C. cynodegmi infections was third-generation cephalosporins and amoxicillin/clavulanic acid, which is the currently recommended treatment for soft tissue infection after animal bites; some cases with beta-lactamase negative strains were treated with amoxicillin alone (262). Ciprofloxacin and clindamycin have been administered for skin, soft tissue, and osteoarticular infections. A bacteremic pneumonitis with C. canimorsus was initially treated with intravenous meropenem and clarithromycin, followed by oral clarithromycin for 2 weeks (273). The patient with C. canimorsus endocarditis was initially treated with amoxicillin and gentamicin as empirical therapy, which was later changed to intravenous benzylpenicillin to complete 4 weeks of treatment for native valve infective (274). An elderly patient with rheumatoid arthritis who developed septic arthritis of the shoulder with C. canimorsus was treated with intravenous amoxicillin/clavulanic acid and oral doxycycline for 2 days. The patient also underwent surgical joint drainage. A 7-week course of doxycycline was administered with good clinical results. A review showed that arthritis should be treated for at least 6 weeks, mostly combined with surgical treatment (272). A patient with peritonitis due to C. cynodegmi was cured with an empiric therapy of cefuroxime, gentamicin, and metronidazole; gentamicin, as well as erythromycin and clindamycin, was tested resistant (276). Metronidazole is often part of an empirical therapy as long as anaerobic bacteria are not excluded. Three cases of sepsis with C. canis were treated with beta-lactams, i.e., one patient with ceftriaxone followed by piperacillin/tazobactam, one patient complicated by cellulitis with amoxicillin/clavulanic acid, and one patient despite a wound of a cat bite with a newer carbapenem/beta-lactamase inhibitor combination (278, 280, 281). A bacteremia in a patient with multiple myeloma after direct contact with a cat due to C. stomatis was successfully treated with ampicillin/sulbactam (260). The C. felis endophthalmitis was first empirically treated with local moxifloxacin and gentamicin combined with intravenous ceftazidime and imipenem. During vitrectomy, vancomycin and ceftazidime were applied intravitreally, and therapy was then switched to oral clindamycin (283). Overall, the zoonotic Capnocytophaga bacteremia can be treated with third-generation cephalosporins, beta-lactam/beta-lactamase inhibitor combination, or carbapenems. For severe infections with C. canimorsus, newer therapies such as hemoadsorption or extracorporeal blood purification techniques are being investigated (287, 288).

CARDIOBACTERIUM

Microbiological aspects

The genus Cardiobacterium in the family Cardiobacteriaceae consists of facultatively anaerobic, nonmotile, gram-negative rods, with two species, Cardiobacterium hominis and Cardiobacterium valvarum (289). C. hominis stains irregularly and appears as straight, gram-negative rods in short chains, pairs, or rosettes, sometimes with bulbous ends; occasionally, filaments are formed. Cardiobacterium spp. are pleomorphic on blood agar. HACEK members do not need more than 5 days of incubation in modern blood culture systems (290). In cases of culture-negative endocarditis, microscopy of the resected valve or emboli, together with 16S ribosomal RNA gene PCR/sequencing, has led to the diagnosis (291).

Cardiobacterium spp. mostly requires 5%–10% CO₂ and humidity for initial growth on blood agar. Cardiobacterium spp. have most often been isolated from blood. Colonies of Cardiobacterium spp. attain a diameter of approximately 1 mm after 48 h at 37°C on blood agar; they are circular, smooth, opaque, and may pit the agar. A blood culture isolate with a positive indole reaction and a negative catalase is diagnostic for C. hominis. Some automated systems may misidentify C. valvarum (292). Both species are included in databases of the Bruker MALDI Biotyper (18, 19). The VITEK 2 ID-NH card database includes C. hominis (14). EUCAST has defined non-species related PK/PD MIC breakpoints (191). CLSI published guidelines for broth microdilution susceptibility testing of the HACEK group (163).

Clinical aspects, including epidemiology

The normal habitat of Cardiobacterium spp. is the human oral cavity and nasopharynx but possibly also the gastrointestinal and urogenital tracts (293). Disease caused by both Cardiobacterium spp. is mainly endocarditis (15, 289). Vertebral osteomyelitis, discitis, and epidural abscess as a complication of a C. hominis endocarditis have been described (294). Two cases of prosthetic valve endocarditis with C. hominis were identified after a colonoscopy (295). Bacteremia with C. hominis without endocarditis is rare, e.g., a case of neonatal sepsis (296). Other cases of overt bacteremia without endocardial involvement have been reported in a patient with adenocarcinoma of the right kidney, in a patient with metastatic adenocarcinoma from an unknown primary tumor, and in a patient with a pacemaker lead infected with C. hominis associated with vertebral osteomyelitis (295). An endocarditis with C. hominis has also been reported in a pediatric patient with congenital heart disease (297). On rare occasions, C. hominis has been isolated from other body sites, e.g., from the pericardial fluid in a child with pericarditis without endocarditis or an acute dacryocystitis with lacrimal abscess (298, 299). Most reported C. valvarum endocarditis cases were of odontogenic origin, related to periodontal diseases (300). An unusual case of C. valvarum causing aortic endograft infection and osteomyelitis, as well as an infection of arthritis without lesions, has been reported (301, 302). Pacemaker-related endocarditis with C. valvarum has also been reported (303).

C. hominis and C. valvarum are susceptible to penicillins with or without beta-lactamase inhibitor combinations, cephalosporins, quinolones, and gentamicin (289, 298). Beta-lactamase production is rare, and its effect can be neutralized by clavulanic acid (163, 304). The child with pericarditis with C. hominis was cured with the administration of 4 weeks of cefotaxime treatment (298). The patient with an aortic endograft infection and osteomyelitis with C. valvarum was successfully treated with ceftriaxone (301). The therapy for Cardiobacterium endocarditis is also the recommended standard therapy for HACEK endocarditis (232).

CHROMOBACTERIUM

Microbiological aspects

The genus Chromobacterium in the family Chromobacteriaceae contains several facultatively anaerobic, motile species, of which Chromobacterium violaceum and Chromobacterium haemolyticum are agents of human disease (305–307). C. violaceum is a medium-sized, gram-negative rod or coccus that is motile by one polar flagellum and one to four lateral flagella (305). C. haemolyticum forms regular gram-negative rods (306).

C. violaceum grows on routine media, even on most enteric ones, e.g., MacConkey agar, at 30°C–35°C, its optimal temperature (305). C. haemolyticum grows on the same media and is hemolytic (306). Colonies of C. violaceum measure 1–2 mm in diameter after 24 h of growth, are round and smooth, have an almond-like smell, and may be beta-hemolytic. Most strains produce a violet pigment called violacein, which is soluble in ethanol but not in water. Identification is easy if this pigment is produced, although the positive oxidase reaction will only be detected by a modified technique (308). C. haemolyticum does not form a pigment. C. violaceum is included in both MALDI-TOF MS databases and in the VITEK 2 ID-GNB card. C. haemolyticum is not in the MS databases and cannot be separated from C. violaceum by these systems (306).

Clinical aspects, including epidemiology

C. violaceum inhabits soil and water in tropical and subtropical climates between latitudes of 35° N and 35° S (South Africa, Southeast Asia, Australia, Southeastern United States, and rarely, South America) (309). C. violaceum, together with Vibrio spp., Aeromonas spp., and Shewanella spp., belongs to the gram-negative, oxidase-positive VACS organisms that occur naturally in aquatic environments, requiring estuarine or marine conditions; Aeromonas spp. are also found in fresh water (310). The natural habitat of C. haemolyticum is river and lake water, even in temperate regions (311). In addition to endotoxin, several other virulence factors have been described, e.g., adhesins, invasins, and cytolytic proteins (312). The high virulence of C. violaceum involves two type III secretion systems (T3SSs); the Chromobacterium pathogenicity island-1 encoded T3SS plays a crucial role in the interaction with host cells (313). The pigment violacein has been related to cytotoxic activity; however, non-pigmented strains with similar clinical severity have also been isolated. Asymptomatic colonization with C. violaceum in the gastrointestinal tract and respiratory tract has been documented (309).

Localized skin and soft tissue infections with C. violaceum usually develop through contaminated wounds, especially on the limbs, and can lead to septicemia and multiple organ abscesses. Important risk factors include neutrophil dysfunction in the case of a glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients with chronic granulomatous disease (CGD). C. violaceum remains a serious infection in immunocompetent patients, requiring intensive care management; comorbidities are common, including malignancy, diabetes, and excessive alcohol use (309). The association of C. violaceum infection with CGD occurs mainly in children and almost never in adults and manifests with skin and soft tissue infections. By 2021, there had been 200 cases of skin and soft tissue infections; most of them presented with acute onset, half of them had to be hospitalized with sepsis in the first week, and the fatality rate was about 20% (314). Reinfection and relapses with C. violaceum are well known. A recurrent C. violaceum bloodstream infection has been described in a G6PD-deficient 37-year-old man (WHO class I severe G6PD deficiency with 3.5% activity of the normal); his twin brother also had G6PD deficiency and died at age 3 from septic infection with C. violaceum after playing in the mud (315). Sepsis can be accompanied by extensive violaceous to hemorrhagic and pus-filled vesiculobullous eruptions (316). Facial necrotizing fasciitis has been described in a 14-year-old boy who presented first with sinusitis after swimming in a lake (317). Nosocomial pneumonia in two Japanese patients at an intensive care unit has been reported (318). A urinary tract infection (UTI) with C. violaceum was reported in a 41-year-old man with a neurogenic bladder due to spinal cord injury (319). Some infections with C. violaceum have been observed in Europe as well (320, 321). C. haemolyticum can cause bacteremia after trauma and exposure to water (311). A bacteremia with C. haemolyticum was discovered in a patient in Idaho, near Yellowstone National Park, who slipped on wet rocks and fell into a natural hot spring (322). Several cases of C. haemolyticum bacteremia have been reported from Japan; all patients had contact with river or city canal water or runoff water from a ditch. An elderly patient with alcoholic hepatitis developed meningitis and died, another developed necrotizing fasciitis of the upper part of the foot, and two others developed pneumonia (311, 323–325). Pediatric bacteremia with C. haemolyticum in a 11-year-old boy with a complicated history of congenital heart disease and a prosthetic mitral valve-associated endocarditis with K. kingae has also been reported from Cincinnati, OH, USA (326). Diarrhea with C. violaceum has been reported earlier, but a pediatric case from Thailand suggests that C. haemolyticum can cause mucous bloody diarrhea leading to proctocolitis (327).

C. violaceum is resistant to many antimicrobials, including beta-lactams and colistin, but is usually susceptible to imipenem, fluoroquinolones, tetracyclines, and trimethoprim-sulfamethoxazole (318). Resistance to ciprofloxacin, cefepime, and gentamicin has rarely been reported (310). For invasive C. violaceum infections, combination therapy with effective antimicrobials for at least 3 months should be considered to avoid relapses (328). In most cases of skin and soft tissue infections, the definitive treatment was a beta-lactam and fluoroquinolone combination (326). The UTI in the patient with neurogenic bladder was treated with ofloxacin (319). C. haemolyticum has a similar resistance pattern (306). Intravenous therapy with meropenem or fluoroquinolone is recommended for infections with C. haemolyticum (311, 323, 325). Only one fatal case of C. haemolyticum infection was noted under the therapy of meropenem, probably due to the alcoholic hepatitis comorbidities (324).

DYSGONOMONAS

Microbiological aspects

The genus Dysgonomonas in the family Dysgonomonadaceae consists of facultatively anaerobic, nonmotile, gram-negative rods (329, 330). Four species have been isolated in humans: Dysgonomonas capnocytophagoides and Dysgonomonas gadei (331); Dysgonomonas mossii (332); and Dysgonomonas hofstadii (333). A single isolate from a stool sample of a healthy man from the Democratic Republic of Congo was proposed as the type strain of Dysgonomonas massiliensis sp. nov. (334). Dysgonomonas spp. are small, gram-negative rods or cocci like Moraxella spp. (329).

Dysgonomonas spp. grow slowly on blood agar in a CO₂-enriched atmosphere (329). Colonies of Dysgonomonas spp. are entire, measure 1–2 mm in diameter after 24 h of growth, have a strawberry-like odor, and do not spread or adhere. Aerobically growing isolates of Leptotrichia buccalis may be confused with Dysgonomonas spp.; microscopic examination of morphology, different cellular fatty acid profiles, and determination of the production of lactic acid from glucose in Leptotrichia spp. (Dysgonomonas spp. produce propionic and succinic acid) (16) may assist in the identification of the species if 16S ribosomal RNA gene PCR/sequencing is not available (16, 331, 333). Dysgonomonas spp. are not yet included in the MALDI-TOF MS or in the VITEK 2 cards databases.

Clinical aspects, including epidemiology

Most D. capnocytophagoides strains have been isolated from the stools of immunocompromised patients, with a few strains isolated from other sources (335). In a systematic search of stool samples, D. capnocytophagoides was present in 1.1%–2.3% of the samples (336). The natural habitat of D. capnocytophagoides and the other Dysgonomonas spp. is unknown (332, 333). A recent analysis of a specific strain of D. mossii, i.e., strain Shenzhen WH 0221, showed many genes coding virulence factors, e.g., for capsules, lipopolysaccharides, and a cytolysin (337).

D. capnocytophagoides has been associated with human disease, mainly chronic diarrhea, but was considered a cause of diarrhea in less than half of the cases in which it was detected in stool (336). It also causes bacteremia (335, 338). Patients with D. capnocytophagoides bacteremia share several features, including malignancy, recent neutropenia, and presumed gastrointestinal source (339). In one case of a severely immunocompromised patient, the same D. capnocytophagoides was isolated from the blood and the stool specimen (338). D. capnocytophagoides was also isolated from the biliary drainage of a patient with obstructive cholangitis who had undergone a liver transplant 5 years earlier, together with Citrobacter freundii and Morganella morganii (336). In a patient with hepatocellular carcinoma, D. capnocytophagoides was isolated from the blood and liver abscess (340). In an 81-year-old woman, D. capnocytophagoides was isolated from the urine after anal surgery (341). D. mossii was isolated from the blood of a patient with cholangitis and from the intestinal juice of a patient with pancreatic carcinoma (342, 343). D. gadei was isolated from a human gallbladder (331) and from blood (344), D. hofstadii from a wound (333), and D. massiliensis from the stool of a healthy man (334).

D. capnocytophagoides strains are susceptible to tetracyclines, clindamycin, macrolides, and trimethoprim-sulfamethoxazole, while being resistant to cephalosporins, aminoglycosides, and fluoroquinolones, and exhibit variable susceptibility to other beta-lactam antibiotics and imipenem (336, 338). Some cases of bacteremia with D. capnocytophagoides were treated with doxycycline (339). In some case reports, it was discussed whether the clearing of the bacteria was due to the restitution of the immune function in immunosuppressed patients, since the corresponding isolates of D. capnocytophagoides were resistant against the antibiotics administered during the bacteremic phase of the disease (338, 344). A recent fatal case of peritonitis caused by D. capnocytophagoides showed a novel metallo-beta-lactamase (DYP-1) (345). The susceptibilities of the other Dysgonomonas spp. are similar to those of D. capnocytophagoides, so that the therapeutic options for systemic and local infections are similar but not yet standardized. In mixed infections, the accompanying bacteria and the clinical situations might trigger the antibiotic choice.

EIKENELLA

Microbiological aspects

The genus Eikenella in the family Neisseriaceae consists of facultatively anaerobic, nonmotile gram-negative rods. Eikenella corrodens is the most common species. New species isolated from human clinical materials have been described as Eikenella exigua sp. nov., Eikenella glucosivorans sp. nov., Eikenella halliae sp. nov., and Eikenella longinqua sp. nov. (346, 347). E. corrodens is a slender, straight, small, gram-negative rod with rounded ends. A specific NAAT is also commercially available (206).

With few exceptions, E. corrodens requires hemin for growth, unless 5%–10% CO₂ is present. E. corrodens grows well on commercial Columbia agar (243). Colonies of E. corrodens are 1–2 mm in diameter after 48 h of growth and may pit the agar. E. corrodens can be identified well by the VITEK 2 ID-NH card and by MALDI-TOF MS (14, 18, 19). The new Eikenella spp. are not yet in the databases. EUCAST has defined non-species related PK/PD MIC breakpoints (191). CLSI has published guidelines for broth microdilution susceptibility testing of the HACEK group (163).

Clinical aspects, including epidemiology

The natural habitat of E. corrodens is the oral cavity and possibly the gastrointestinal tracts of humans and some mammals, from which it can be transmitted via saliva, bites, and syringes with oral secretions. Endogenous infections prevail, however (11). Typing of E. corrodens by random priming PCR sequencing showed a change of clones over time and multiple clones in the oral cavity, especially in patients with aggressive periodontitis (348). E. corrodens can trigger a cascade of events that induce cell proliferation and expression of IL-8 and adhesion molecules in endothelial cells in periodontal tissue via mitogen-activated protein kinase, promoting chronic periodontitis without bacteria-cell contact (349).

E. corrodens is associated with juvenile and adult periodontitis but is also an agent of infections of the upper respiratory tract, abdomen, joints, bones, wounds (e.g., from a human bite), and, rarely, of other infections like dacryocystitis and thyroid abscess (16, 350, 351). A case of secondary hand infection with E. corrodens and S. aureus has been reported, which led to necrotizing fasciitis in an elderly woman with Behçet’s disease (352). E. corrodens is responsible for a range of head and neck infections in adults and children, often in combination with other members of the oropharyngeal microbiota, particularly streptococci and staphylococci. However, most affected patients are hosts with normal immunity (12, 353). Neck infections can be complicated by abscesses and are sometimes associated with underlying conditions such as diabetes or hypertension (354). Sometimes sinusitis or a surgical procedure in the nasal cavity can lead to brain abscesses with E. corrodens, although such risk factors or HACEK endocarditis with emboli do not always precede. Mixed infections are also found in brain abscesses (234, 354). Deep-seated infections such as pleuropulmonary and abdominal infections have been described in patients with comorbid conditions who have undergone invasive procedures that may serve as the pathogens’ route of entry. Pleuropulmonary infections such as empyema occur in patients with a history of aspiration pneumonia and alcoholism, and abdominal infections with liver or intraabdominal abscesses often result from previous surgery (355). E. corrodens can cause vertebral osteomyelitis, especially in the presence of risk factors such as diabetes mellitus. Abdominal pain can be the first clinical sign (356, 357). E. corrodens is one of the pathogens that cause HACEK endocarditis. Intravenous drug users are at risk of bacteremia and endocarditis (8). Bacteremia can be detected in systemic infections without endocarditis, e.g., in patients with pelvic inflammatory disease. Gynecological infections such as chorioamnionitis or tubo-ovarian abscesses caused by E. corrodens are mainly described in women using an intrauterine device (358). Infections caused by other Eikenella sp. are rare. E. exigua was found mainly in abscesses but not in endocarditis (346, 359). A single case of empyema caused by E. halliae has been described in an immunosuppressed woman who had undergone lung surgery (360). Single strains of E. glucosivorans isolated from a throat swab of a male patient and of E. longinqua isolated from blood without clinical information were used to describe the type strain (346, 347). Clinical entities of these bacteria could be described in the future.

E. corrodens is generally susceptible to amoxicillin/clavulanate, piperacillin/tazobactam, ceftriaxone, meropenem, and moxifloxacin but is resistant to clindamycin and macrolides (12, 361). An in-house investigation of 23 strains of E. corrodens showed susceptibility to tetracycline, ciprofloxacin, and levofloxacin. Beta-lactamase-positive strains have been reported, but the enzyme was inhibited by beta-lactamase inhibitors (163). Beta-lactamase production is well documented among members of the HACEK group and beta-lactamase-producing isolates resistant to ampicillin; some isolates may be resistant to ampicillin by mechanisms other than beta-lactamase production; routine beta-lactamase testing is recommended for all HACEK isolates (163). Clindamycin is a commonly used antibiotic for head and neck infections because it penetrates well into tissue and bone. However, E. corrodens is resistant to it and can be selected as a result, leading to treatment failure (12). A better choice for the presumptive treatment of these head and neck infections would be a combination of beta-lactam/beta-lactamase inhibitors, third-generation cephalosporins, carbapenems, or new quinolones for adults in combination with antibiotics against anaerobic bacteria, i.e., clindamycin or metronidazole (12, 353). In patients with a pyogenic infection caused by E. corrodens, timely and adequate surgical drainage of abscesses and debridement of non-viable tissue should be considered (11). The patient with a brain abscess caused by A. aphrophilus and E. corrodens underwent surgery to drain the abscess and was successfully treated with intravenous antibiotics (ceftriaxone, vancomycin, and metronidazole) (234). Deep-seated pleuropulmonary, abdominal infections have been successfully treated with piperacillin/tazobactam, carbapenems, or newer quinolones in combination with surgical intervention (355). A similar combination of surgery and antibiotic treatment is required for vertebral osteomyelitis (356, 357). The therapeutic options for systemic and local infections caused by other Eikenella spp. are similar to those for infections caused by E. corrodens. Treatment of endocarditis with Eikenella is also the standard treatment for HACEK endocarditis (232).

PASTEURELLA

Microbiological aspects

The taxonomy of the family Pasteurellaceae has undergone important changes (http://www.the-icsp.org/taxonomic-subcommittees; [362]); the family consists of over 100 named species distributed in over 25 genera, of which 4 include human pathogens: Actinobacillus, Aggregatibacter, Haemophilus, and Pasteurella. Many taxa of this family, Pasteurellaceae, are in the process of being or have recently been classified, e.g., bacteria of the Bisgaard taxon 5 and 7 have been classified as Caviibacterium pharyngocola gen. nov., sp. nov. and Conservatibacter flavescens gen. nov., sp. nov., respectively (363). The taxonomy of the genus Pasteurella has been in flux for some time ([362, 363], http://www.bacterio.net). Pasteurella multocida can be separated into P. multocida subsp. multocida, P. multocida subsp. septica, and P. multocida subsp. gallicida; Pasteurella canis, Pasteurella dagmatis, Pasteurella oralis, and Pasteurella stomatis are other species isolated from humans (362). Some related (Pasteurella) spp. found in human infections, i.e., (Pasteurella) aerogenes, (Pasteurella) bettyae, and (Pasteurella) caballi, may be allocated in the future to other genera (20). Some former (Pasteurella) spp. have already been reclassified as Mannheimia spp., e.g., Mannheimia glucosida and Mannheimia haemolytica, as well as Rodentibacter pneumotropicus (364). Pasteurella spp. are small, gram-negative rods or cocci that occur singly, in pairs, or in short chains. Bipolar staining is frequent. “Related” species are more rod-like. A NAAT for their detection is mainly applied in the veterinary field (365).

In contrast to some Haemophilus spp., Pasteurella spp. are hemin and CO₂ independent and therefore grow on media without blood (366). Colonies of Pasteurella spp. are 1–2 mm in diameter after 24 h of growth at 37°C and are opaque and grayish. Encapsulated strains tend to be mucoid. A slight greening underneath the colonies may be noted. Indole-positive isolates exhibit a mouse-like odor. The oxidase test has to be performed on blood agar; a weak reaction after 30 s is characteristic of many Pasteurella spp. (367). Nutritionally fastidious, nonmotile strains of Enterobacteriaceae, e.g., Escherichia coli, may be misidentified as Pasteurella spp. unless an oxidase test is performed. Pasteurella spp. are not in the database of the VITEK 2 ID-NH card but are in the database of the identification card for gram-negative bacilli (VITEK 2 ID-GNB card, BioMérieux) (13). P. multocida has a typical pattern of acidification of sugars and a positive indole reaction, and together with a history of cat bite, the diagnosis is feasible (368). In view of the phenotypic closeness of some taxa (http://www.bacterio.net), species identification in automated systems may be unsatisfactory (13, 369, 370). 16S ribosomal RNA gene and sodA gene PCR/sequencing have provided reliable species identification, the latter with even more discriminatory power (190). Most Pasteurella spp. are included in the database of the Bruker MALDI Biotyper, and some Pasteurella spp. are included in the database of the Biotype VITEK MS system (13, 19). The VITEK 2 ID-GNB card includes Pasteurella spp. (13). EUCAST has defined breakpoints for antimicrobial dilution and disk susceptibility testing of P. multocida; for other Pasteurella spp., non-species related PK/PD MIC breakpoints (191) are available. Approved guidelines for broth microdilution and disk susceptibility testing of Pasteurella spp. have been published by CLSI (163).

Clinical aspects, including epidemiology

Pasteurella spp. are widespread in healthy and diseased wild and domestic animals, including dogs and cats, inhabiting their nasopharynx and gingiva (366). Human isolates are transmitted predominantly from animals by contact (bites or licking or scratching of wounds). Of the related species, (P.) aerogenes occurs primarily in pigs (6) and (P.) caballi in pigs and equines (6, 371). The natural habitat of (P.) bettyae is uncertain, but it is detected in genitourinary samples from infected patients, suggesting the possibility of sexual transmission (372). The main habitats of Mannheimia (Pasteurella) glucosida and Mannheimia (Pasteurella haemolytica) are ruminants (190), while Rodentibacter (Pasteurella) pneumotropicus mainly lives in rodents (373). For typing of Pasteurella spp., PCR profiling, restriction endonuclease analysis, ribotyping, multilocus sequence typing, and pulsed-field gel electrophoresis have been employed (374). Virulence factors are capsules (five serotypes A, B, D, E, and F, of which A and D account for most human isolates), lipopolysaccharide, sialidases, hyaluronidase, surface adhesins, iron acquisition proteins (375), and the P. multocida toxin, a highly potent mitogen (376). P. multocida strains can be differentiated by multiplex PCR into eight lipopolysaccharide genotypes (L1–L8). P. multocida diseases and host predilection are often correlated with capsule and LPS structure; in a recent analysis, most human isolates were P. multocida ssp. septica with capsule type A and LPS type L1 and L3 (377). The putative virulence factors of P. multocida include mechanisms for iron acquisition, membrane lipopolysaccharide that confer serum resistance, a capsule that prevents phagocytosis, surface components that provide adhesion properties, enzymes that degrade extracellular matrix, such as hyaluronidase, neuraminidase, and proteases that facilitate colonization and/or dissemination (376).

In various overviews, P. multocida occurs much more frequently than other Pasteurella spp. Bacteremia and invasive infections occurred in about 20% of cases. Two-thirds of bacteremias occurred in adults over 65 years of age. More severe infections, including bacteremia, invasive and deep local infections, were more common in lower limb infections and in individuals with underlying medical conditions. Bloodstream infections were more often associated with contact with cats, while invasive, deep local, and superficial infections were more often associated with dog bites. The subspecies most frequently encountered is subsp. multocida, which is also more frequent in respiratory infections and bacteremias than subsp. septica, which is most often associated with wound infections and central nervous system infections (6).

Polymicrobial infections are common (378–380). Human isolates of P. multocida are mostly found in wound or soft tissue infections. Less frequent are colonization or infection of the respiratory tract and (by hematogenous or contiguous route) systemic disease, epiglottitis, conjunctivitis, endophthalmitis, uterine abscess, intrapelvic abscess, and septicemia, with cirrhosis of the liver being a particular risk factor (376, 381–388). An existing cirrhosis can also decompensate due to bacteremia with P. multocida (389). Cirrhosis was present in about 10% of patients with infective endocarditis caused by Pasteurella spp. P. multocida accounted for over 70% (390). On the other hand, P. multocida bacteremia can also cause liver abscess (391). Cases of peritonitis with P. multocida occur in patients with liver cirrhosis, but most cases occur in patients with continuous ambulatory peritoneal dialysis who have contact with cats (392). A similar route can lead to urinary tract infections with P. multocida in anatomically susceptible individuals who have congenital neurological and urological abnormalities and occasionally come into contact with cats (393, 394).

Mycotic aneurysms caused by P. multocida affect not only the abdominal and thoracic aorta but also the cerebral vessels and the femoral artery, usually in the presence of underlying conditions. Mycotic Pasteurella aneurysm should be considered in immunocompromised cat or dog owners with recurrent bacteremia (395–398). P. multocida can cause meningitis with or without bacteremia, especially in newborns, but also in adults; the mortality rate for P. multocida neonatal meningitis has been reported at 20%. P. multocida meningitis is usually transmitted directly through animal bites and saliva (399). P. multocida bacteremia may also be associated with epidural abscess without meningitis (400).

In the respiratory tract, colonization may eventually lead to sinusitis or bronchitis as well as pneumonia and empyema, the latter two mostly in patients with prior respiratory disease (382). Chronic obstructive respiratory disease seems to be a risk factor for pneumonia caused by P. multocida (401, 402); a cavity formation secondary to P. multocida infection can be a complication in patients with chronic lung disease (403). Fatal outcome of COVID-19 and Pasteurella multocida pulmonary coinfections is well known (404).

Cases of septic arthritis caused by P. multocida after contact with cats or dogs can lead to septic shock and death in compromised individuals (405, 406). Osteomyelitis may be a complication of septic arthritis; most osteomyelitis cases are attributed to direct inoculation of the periosteum or spread from cellulitis. However, hematogenous spread of P. multocida leading to osteomyelitis in a remote anatomical site or spread from the oral cavity, sinuses, or respiratory tract is also possible. Vertebral osteomyelitis has also been reported (407). In recent years, cases of P. multocida infection from joint prostheses after cat bites or scratches have been reported (408–410).

In some of the cases mentioned above, the patients also suffered from diarrhea, but P. multocida was not specifically responsible for it. However, severe diarrhea has been described in a patient with cirrhosis and bacteremia with P. multocida, and a connection was made without a stool culture being able to prove it (411).

Cases of human infection with P. dagmatis, P. oralis, and P. stomatis (from dogs or cats) are less frequent than those of P. multocida (369, 370); however, P. canis is the most common species isolated from infected dog bite wounds (6). P. canis bacteremia is particularly common in patients with immunosuppression and can also lead to septic shock (412), septic arthritis (413, 414), and osteomyelitis (413, 415), which have also been described as polymicrobial (416). Endophthalmitis and other ocular infections occur after animal bites and scratches (417), respiratory infections, including exacerbation of COPD (418), breast implant infection (419), and peritonitis (420). Some earlier case reports on P. canis could be assigned to P. oralis, as described later; furthermore, the distinction was not possible in individual case reports (412, 416).

P. dagmatis has been found in systemic infections such as pneumonia, peritonitis, septicemia, and endocarditis. In addition, P. dagmatis can cause exacerbation of bronchiectasis symptoms in COPD patients (369). A prosthetic valve endocarditis caused by P. dagmatis has been reported following a cat bite (421). In some cases of pasteurellosis, animal contact could not be established (413). Double infections with two Pasteurella spp. have also been observed (369).

Of the related species, (P.) aerogenes has caused wound infections from bites and scratches by pigs and hamsters (6, 422), but also in vertebral osteomyelitis without a known history of animal contact, bites, or scratches (423). (P.) bettyae has been found in infections of newborns and in infections of the male and female genital tract (424). A recent report described an infection with (P.) bettyae in men who have sex with men in France. It could be speculated that (P.) bettyae could be added to the sexually transmitted diseases (425). (P.) caballi has been isolated from horse bites (6). Rodentibacter pneumotropicus is a rare agent of systemic infection in humans (373). Reported human cases of R. pneumotropicus infection following a bite or scratch by a pet include endocarditis, meningitis, cellulitis, osteomyelitis, sepsis, pneumonia, and peritonitis. An older woman with diabetes was diagnosed with septic arthritis after being bitten by a rat in the street (426). However, human bites can also cause localized infections with R. pneumotropicus, such as a hand phlegmon on her right forearm following a rare and unusual human bite during a brawl (427). There are reports of human wound infections with Mannheimia glucosida and Mannheimia haemolytica, both of which were previously assigned to the genus Pasteurella. One case involved a wound infection with M. glucosida after a bite on the right thumb by a 10-month-old lamb (428). Sepsis due to M. haemolytica is rare but can be fatal, as shown in a 7-month-old infant with persistent fever, sepsis, and pneumonitis without an obvious history of animal bites or contact (429). Endocarditis with M. haemolytica is rarely reported but has been known for decades (390, 430). Pyogenic cervical spondylitis is caused by M. haemolytica in men who had extensive/frequent dog contact (431). Finally, M. haemolytica was recovered from the tracheal secretion of a 9-month-old girl who had been admitted to hospital with sudden onset of dyspnea and unconsciousness, which had been considered as bacterial croup (432).

Pasteurella spp. are generally susceptible to penicillin, broad-spectrum cephalosporins, tetracyclines, fluoroquinolones, trimethoprim-sulfamethoxazole, and azithromycin but resistant to clindamycin (376, 382, 393, 433–435). Rare penicillin-resistant isolates of Pasteurella spp. have been encountered, but they are susceptible to beta-lactam/beta-lactamase inhibitor combinations (163). For isolates of Pasteurella spp. from bite wounds, routine susceptibility testing is usually not necessary. Multiple organisms are often present in these specimens, and empiric therapy toward these organisms is generally effective for P. multocida as well (163). The empirical treatment for animal bites is usually a penicillin antibiotic in combination with a beta-lactamase inhibitor, such as amoxicillin/clavulanic acid. An alternative is clindamycin plus a fluoroquinolone, although the combination of a third-generation cephalosporin and a fluoroquinolone would also be effective (389).

Patients with complicated local Pasteurella infections should undergo surgical intervention and receive antimicrobial therapy, mainly amoxicillin-clavulanic acid or in combination with gentamicin (379). However, novel multiresistance-mediating integrative and conjugative elements were detected in M. haemolytica and P. multocida that carry unusual antibiotic resistance genes for tetracycline, streptomycin, chloramphenicol, macrolides, and sulfonamides (435). Furthermore, empirical treatment may fail in the case of resistance. In a patient with COPD exacerbation and pneumonia, ceftriaxone and azithromycin were ineffective at initial diagnosis; the presence of P. multocida in sputum prompted a switch to intravenous doxycycline, and clinical improvement allowed patient discharge after 10 days of oral doxycycline therapy (402). An amoxicillin/clavulanic acid-resistant P. multocida was described in an implant-associated infection that was cured by targeted antimicrobial treatment and debridement, replacement of moving parts, and retention of the prosthesis. The isolate proved to be sensitive to cefotaxime, levofloxacin, ciprofloxacin, tetracycline, and trimethoprim-sulfamethoxazole (436).

The empirical therapy is usually based on the clinical entity, e.g., in the case of mycotic aneurysms, the American Heart Association recommends a 6-week to 6-month course of antimicrobial therapy followed by lifelong suppression therapy (396). In general, it can be said that empirical treatment based on the clinical picture sometimes fails due to antibiotic resistance. Susceptibility testing should be performed on all isolates of the genus Pasteurella or the related genera mentioned, according to the CLSI or EUCAST guidelines (163, 191), in order to adjust treatment if necessary.

SIMONSIELLA

Microbiological aspects

The genus Simonsiella in the family Neisseriaceae consists of several obligately aerobic species. Simonsiella spp. are common and abundant in dogs and cats (305). Simonsiella spp. are gram-negative, crescent-shaped rods (0.5–1.0 µm long) arranged in multicellular filaments (10–50 µm by 2–8 µm) and segmented into groups of mostly eight cells, resulting in a caterpillar-like appearance. Due to the rarity and unusual appearance of this bacterium, it can be mistaken for inorganic contaminants (e.g., crystals) or fungi in cytologic stains of oral swabs (437). The only species isolated from humans is Simonsiella muelleri. It grows aerobically on blood agar and is beta-hemolytic. Colonies of S. muelleri are 1–2 mm in diameter after 24 h, may exhibit gliding motility, and may produce a pale-yellow pigment (305). S. muelleri is not included in the MALDI-TOF MS or VITEK 2 card databases.

Clinical aspects, including epidemiology

The natural habitat of S. muelleri is the human oral cavity. In healthy human populations, it is present in 30%–40%, with possibly higher rates in children than in adults (305). S. muelleri has not been associated with disease. Simonsiella can be encountered in exfoliative cytology specimens of the upper gastrointestinal tract, erosions of the oral mucosa, and the respiratory tract (437). S. muelleri was isolated decades ago from a gastric aspirate taken from a neonate 15 min postpartum; the neonate showed a dental cyst and early eruption of teeth. The organism was resistant to clindamycin and susceptible to penicillin, ampicillin, cephalothin, tetracycline, and gentamicin (438).

STREPTOBACILLUS

Microbiological aspects

The genus Streptobacillus in the family Leptotrichiaceae consists of facultatively anaerobic, nonmotile, gram-negative rods; Streptobacillus moniliformis, Streptobacillus felis, Streptobacillus hongkongensis, and Streptobacillus notomytis have been isolated in humans (439–441). S. hongkongensis has been reclassified as Pseudostreptobacillus hongkongensis gen. nov., comb. nov. (442). Streptobacillus spp. are non-motile, non-encapsulated, non-spore-forming gram-negative rods with rounded or pointed ends or pleomorphic bacilli with coccobacillary, bacillary, and filamentous forms; they occur singly or form long, wavy chains (443). Depending on age and culture conditions, S. moniliformis may appear as straight, small to medium-size rods or as 100–150-µm-long tangled chains and filaments with bulbar swellings (20). In culture-negative cases, 16S ribosomal RNA gene PCR/sequencing has been employed (444). S. felis may form pleomorphic, fusiform to filamentous rods, 0.45 µm in width and 0.83 µm in length arranged in chains and clumps; sometimes irregular and lateral bulbar swellings are displayed (445). S. hongkongiensis shows pleomorphic bacilli, with coccobacillary, bacillary, and filamentous forms as described for the genus (446). The morphological description of S. notomytis is identical to that of S. felis (443, 445).

The growth of most strains of the genus Streptobacillus is non-hemolytic and dependent on 5%–10% CO₂ and on blood, serum, or ascitic fluid (445). S. moniliformis is best isolated from blood, joint fluid, or abscess material. For culture, media enriched with sheep, horse, rabbit blood (15% seems to be optimal), serum, or ascitic fluid and a 5%–10% CO₂ atmosphere at 37°C are required. Sodium polyanethol sulfonate, the anticoagulant contained in blood culture media, is inhibitory (447). S. moniliformis may show wild-type and L-phase colonies in the same culture. The former are 1–3 mm in diameter after 48–72 h of growth on blood agar and are round and smooth. L-phase colonies grow better on clear media, yielding the “fried egg” appearance, with irregular outlines and coarse lipid globules. In liquid media, growth occurs mainly in the form of “puff balls” at the bottom of the tube. The organism dies quickly unless subcultured (447). It is negative for catalase and oxidase, indole production, and nitrate reduction, like the other Streptobacillus spp. The growth of S. felis occurs after 2–5 days at 37°C in a capnophilic atmosphere of 10% CO₂ on sheep blood agar (SBA) or in trypticase soy broth (TSB) with 20% cattle or horse serum. Colonies are tiny, drop-like, shiny, and slightly convex, measuring 0.1–0.4 mm in diameter. Colonies are alpha-hemolytic on SBA. Conversion to L-phase or transitional phase variants may occur spontaneously during cultivation. In liquid media (e.g., TSB), with the addition of 20% serum, growth can be detected after 2–4 days with a typical “cotton ball” or “bread crumb”-like appearance (445). S. hongkongensis grows best on Columbia agar with 5% defibrinated sheep blood as small (0.5 mm) colonies after 48 h of incubation at 37°C in an anaerobic environment or aerobic environment with 5% CO₂ (446). S. notomytis grows similarly to S. felis but is not alpha-hemolytic (443). S. moniliformis is included in the database of the Bruker MALDI Biotyper; the MALDI-TOF patterns of the other three human Streptobacillus sp. differ (443). 16S rRNA gene PCR/sequencing was used to detect S. moniliformis in clinical samples and for laboratory confirmation of suspicious isolates. To differentiate S. moniliformis from other Streptobacillus sp., other genes such as the gene for gyrase subunit B (gyrB), recombinase subunit A (recA), and chaperonin (groEL) are used (448). Indirect serological investigations are so far mainly conducted for research purposes. In a patient with endocarditis, an ELISA could detect IgG and IgM; in the future, serologic analysis could be a useful tool for diagnosing S. moniliformis (449).

Clinical aspects, including epidemiology

Streptobacillus moniliformis and other Streptobacillus spp. occur naturally in the upper respiratory tract of up to 100% of wild and laboratory rats and other rodents (mice, gerbils, and squirrels) and are occasionally found in dogs and cats that prey on rodents. Transmission to humans occurs either from bites of those animals or consumption of food or water contaminated by rat urine. However, the natural animal reservoir for S. hongkongensis is not yet identified, but the oropharynx of humans may be the natural reservoir (443, 445, 447, 450). As with other gram-negative bacteria, lipopolysaccharide probably plays an important role in the pathogenesis. S. moniliformis can agglutinate blood in several animals and grow within mouse phagocytes in vitro (451). In experimental infections in mice, S. moniliformis can persist in the joint spaces despite the clearing from blood, liver, and spleen (447).

S. moniliformis infection, commonly called rat bite fever (RBF), is a systemic illness beginning with fever and chills, followed by migratory, sometimes even suppurative polyarthritis, and a maculopapular rash on the extremities. After exposure, the incubation period ranges from 3 days to over 3 weeks but typically is less than 7 days. Many patients report symptoms suggestive of an upper respiratory tract infection. The bite wound typically heals quickly, with minimal residual inflammation and no significant lymphadenopathy. Infections caused by rat bites in Asia are likely to be caused by Spirillum minus; in contrast to RBF, the bite site becomes indurated with associated regional lymphadenopathy; joint manifestations are rare (447). A review of S. moniliformis osteoarticular infections revealed that the clinical triad of RBF (fever, arthritis, and rash) was incomplete in more than half of the cases, mainly because rashes were rarely observed among adult patients. Second, the clinical presentation of RBF is quite non-specific, and exposure to rodents is not mentioned by patients on admission in one-third of cases (452). Hematogenous dissemination can cause arthritis and abscesses; a recent report describes cervical discitis/osteomyelitis with an epidural abscess requiring neurosurgical decompression (453). Nevertheless, local infections at the site of the bite are rarely described. One patient presented with a rat bite to the right hand, a thumb abscess with cellulitis, and septic monoarthritis without systemic features of RBF (454). The increasing popularity of rats as pets could explain why RBF occurs in people aged 0–19 years in the USA (455). S. moniliformis also causes Haverhill fever (HF), which refers to an outbreak of an epidemic disease resulting from contaminated milk in 1926. Patients with HF develop the same signs and symptoms as with RBF (439, 447). Rare complications include endocarditis, myo- or pericarditis, systemic vasculitis, polyarteritis nodosa, meningitis, hepatitis, nephritis, amnionitis, and pneumonia (447, 456). Prosthetic valve endocarditis caused by S. moniliformis is also possible (444). Untreated RBF has a mortality rate of approximately 10% (447). S. moniliformis endocarditis has also been described in a pregnant woman (457). Recently, periprosthetic infections have also been described (458, 459). A case of RBF with S. felis showed its zoonotic potential (440).

Streptobacillus hongkongensis, reclassified as Pseudostreptobacillus hongkongensis, has been isolated from patients with arthritis (442, 446). S. notomytis is reported as a cause of RBF several times, sometimes with polyarthritis (441, 460, 461). In a patient with S. notomytis infection, septic arthritis of both knee joints, meningitis, and hepatitis were observed, and complications of meningitis, spondylodiscitis, and epidural abscess were also observed, which are similar to the clinical symptoms of RBF caused by S. moniliformis (462).

S. moniliformis is susceptible to penicillin and tetracyclines, the mainstays of treatment, and to cephalosporins, carbapenems, aztreonam, clindamycin, erythromycin, and tetracycline; it shows intermediate susceptibility to aminoglycosides and fluoroquinolones and is resistant to colistin and trimethoprim-sulfamethoxazole (447). Patients with S. moniliformis endocarditis require dual therapy with high-dose penicillin G for 6 weeks in combination with an aminoglycoside for 2 weeks; the use of an aminoglycoside appears to enhance activity against the cell wall-deficient “L” forms of S. moniliformis (444, 447, 456). The patient with meningitis complicated by an epidural abscess required neurosurgical decompression and received a 6-week postoperative course of intravenous ceftriaxone, resulting in complete resolution of the abscess (453). The periprosthetic infections in the two patients described were treated over a period of 6 weeks in one patient, following a two-stage revision, and over a treatment period of 12 weeks in the other case, with debridement, antibiotics, and an implant maintenance procedure (458, 459).

The susceptibilities of the other Streptobacillus spp. are similar to those of S. moniliformis, which is why therapy usually involves a beta-lactam, i.e., a penicillin or ceftriaxone, and in case of allergy, a tetracycline. In the case of infection due to S. notomytis with meningitis, spondylitis, and abscess, the patient was treated with intravenous ceftriaxone and then switched to oral amoxicillin, with full recovery (462).

SUTTONELLA

Microbiological aspects

The genus in the family Cardiobacteriaceae (293) consists of facultatively anaerobic, nonmotile, gram-negative rods and so far contains only one human species, Suttonella indologenes (formerly Kingella indologenes). S. indologenes is a plump, irregularly staining gram-negative rod; occasionally, pairs, chains, or rosettes are formed. S. indologenes grows slowly on blood agar. Aerobic growth is enhanced by high humidity and CO₂, which makes the organism appear to be facultatively anaerobic (293). Colonies of S. indologenes may spread or pit the agar surface of the blood agar. S. indologenes is included in both MALDI-TOF MS databases.

Clinical aspects including epidemiology

Human isolates of S. indologenes are very rare: they were isolated from ocular sources, including a corneal abscess (16, 463) from the peritoneal dialysis fluid of a patient with peritonitis, who was performing a continuous ambulatory peritoneal dialysis, and from blood cultures in a case of endocarditis (464, 465). A patient with congenital heart disease and an implanted pulmonary valve developed vegetation in the pulmonary homograft, complicated by septic pulmonary embolism. Standard therapy with a 6-week intravenous ceftriaxone for HACEK endocarditis was supplemented with weeks of oral amoxicillin (466). The susceptibility of S. indologenes is similar to that of the HACEK organisms.

CONCLUDING REMARKS

Since the publication of the CMR review of K. kingae in 2015, the status of the bacterium as a common invasive pathogen in early childhood has been strengthened by the increasing use of sensitive species-specific NAATs. The organism is firmly recognized as the most common etiology of septic arthritis and osteomyelitis in children aged 6–48 months. The routine use of molecular tests has widened the spectrum of skeletal system infections caused by the organism, which now also comprises spondylodiscitis, subacute osteomyelitis, apophyseal and epiphyseal bone infections, and tenosynovitis. Research in the last few years has identified the novel and phenotypically similar K. negevensis, which was originally considered a K. kingae small colony variant. The asymptomatic colonization of the oropharyngeal surface by these species and the progression from asymptomatic K. kingae carriage to invasive infection is complex and depends on virulence factors such as polysaccharide capsule, antiphagocytic exopolysaccharides, type IV pili, and the RTX-toxin. Although K. negevensis shares many virulence factors with K. kingae, no K. negevensis organisms have been isolated from blood or normally sterile body fluids to date. It should be noted that the RTXA encoding rtxA and rtxB genes of K. negevensis are highly homologous to those of K. kingae, such that they are amplified with K. kingae rtx operon primers, and it is therefore possible that K. negevensis infections may still be misidentified as caused by K. kingae. The rtxA operon is absent from the genomes of the commensal species K. denitrificans, K. oralis, and K. potus. K. kingae strains that combine the wide-spectrum RTXA with the polysaccharide capsule types a or b are remarkably invasive, and many exhibit tropism for skeletal system and cardiac tissues. Successful strains have been subjected to positive selection, have readily disseminated among susceptible young children and daycare center attendees, have caused sporadic cases and outbreaks of disease, and have persisted and spread globally for many decades. In the future, a capsule-based conjugate vaccine targeting capsule types a and b could be beneficial for preventing invasive K. kingae disease in children. Since colonization of the upper respiratory epithelium is a sine qua non requisite for the development of invasive K. kingae infections, detection of the organism in oropharyngeal specimens by a sensitive NAAT is successfully used as a surrogate for diagnosing K. kingae skeletal system infections, especially those involving difficult-to-sample sites such as intervertebral discs, the pelvis, and the small pediatric joints and bones. A clear trend toward shorter courses of antibiotics has emerged for treating skeletal system infections in children, as the prime role of K. kingae and its mild clinical presentation, prompt response to antibiotics, and excellent prognosis have been recognized.

The taxonomy of the genera Actinobacillus, Aggregatibacter, Capnocytophaga, Cardiobacterium, Chromobacterium, Dysgonomonas, Eikenella, Pasteurella, Simonsiella, Streptobacillus, and Sutonella has changed repeatedly in recent years, but the clinical pictures have remained essentially the same. Thanks to improved diagnostic options, particularly with MALDI-TOF identification and molecular detection methods, many clinical microbiology laboratories nowadays can recognize the most important species and, thus, report new clinical entities. For some species, many pathogenic mechanisms are still unknown; for other species, they are increasingly understood, but no intervention measures are currently available, e.g., periodontitis caused by A. actinomycetemcomitans. New therapeutic approaches are being investigated for the highly dangerous C. canimorsus infections. Treatment of HACEK endocarditis has been standardized for a long time, but this is usually not the case for other infections with fastidious gram-negative rods. Many infections caused by animal bites are often polymicrobial, requiring the administration of broad-spectrum antimicrobial coverage. Standard antibiotic therapy for fastidious gram-negative rods with a beta-lactam/beta-lactam inhibitor or a cephalosporin is usually effective, but there is an increasing number of reports of broad-range resistance; so reliable antibiotic susceptibility testing according to CLSI or EUCAST guidelines is required.

Biographies

Reinhard Zbinden is Professor Emeritus of Medical Microbiology at the University of Zurich, Switzerland, and former head of the department of diagnostics and deputy director (Director: Prof. E. C. Böttger) and ad interim director (8/2018-7/2022) at the Institute of Medical Microbiology at the University of Zurich. Dr. Zbinden received his M.Sc. in Medical Microbiology in 1981 and his M.D. in 1984 from the University of Bern, Switzerland. His specialist training in virological diagnostics (Prof. L. Lindenmann, Zurich; Dr. Ivo Heinzer, Aarau, Switzerland) and in microbiological diagnostics (Prof. A. von Graevenitz, Zurich) led to the title of specialist in laboratory analysis FAMH Microbiology (Foederatio Analyticorum Medicinalium Helveticorum). He worked as a senior bacteriologist for 30 years. He has published over 180 articles and 8 invited book chapters in the fields of clinical microbiology, serology and diagnostics and had extensive teaching experience with medical, dental and biology students. Dr. Zbinden served on the Swiss Antibiogram Committee from 2011 to 2021 to implement EUCAST in Switzerland. Dr. Zbinden was president of the Swiss Society for Microbiology from 2010 to 2012 and received honorary membership in 2023. Dr. Zbinden has a long-standing research interest in the fields of serology, diagnostics, and conventional identification of gram-positive and gram-negative rods.

Pablo Yagupsky is Professor Emeritus of Pediatrics and Microbiology at the Ben-Gurion University of the Negev and the former Director of the Clinical Microbiology Laboratory of the Soroka University Medical Center (SUMC), Beer-Sheva, Israel. Dr. Yagupsky received his M.D. from the University of Buenos Aires, Argentina, in 1973, followed by a residency in Pediatrics at the SUMC and a fellowship in Medical and Public Health Microbiology at the University of Rochester, NY. For more than four decades, he worked as a senior attending pediatrician at the Pediatric Intensive Care Unit of the SUMC and the director of the hospital Clinical Microbiology Laboratory. Between 2007 and 2011, he has served as Vice-Dean and Director of the Joyce and Irving Medical School of the Ben-Gurion University of the Negev. He has published more than 200 research articles and 28 invited book chapters in the fields of Pediatrics, Clinical Microbiology, and Infectious Diseases, and has been the recipient of numerous awards for teaching excellence. Dr. Yagupsky has a long-standing research interest in the epidemiology, clinical presentation, and laboratory diagnosis of pediatric Kingella kingae infections, and the epidemiology and detection of human brucellosis in endemic countries.