Non-faecium non-faecalis enterococci: a review of clinical manifestations, virulence factors, and antimicrobial resistance

SUMMARY

Enterococci are a diverse group of Gram-positive bacteria that are typically found as commensals in humans, animals, and the environment. Occasionally, they may cause clinically relevant diseases such as endocarditis, septicemia, urinary tract infections, and wound infections. The majority of clinical infections in humans are caused by two species: Enterococcus faecium and Enterococcus faecalis. However, there is an increasing number of clinical infections caused by non-faecium non-faecalis (NFF) enterococci. Although NFF enterococcal species are often overlooked, studies have shown that they may harbor antimicrobial resistance (AMR) genes and virulence factors that are found in E. faecium and E. faecalis. In this review, we present an overview of the NFF enterococci with a particular focus on human clinical manifestations, epidemiology, virulence genes, and AMR genes.

INTRODUCTION

The Enterococcus genus comprises a group of Gram-positive bacteria that are frequently found as commensals in the human and animal gastrointestinal (GI) tract. Although the term “entérocoque” was introduced by Thiercelin (1, 2) in 1899, the genus was regarded as a subgroup of Streptococcus until 1984. Most enterococcal human disease is caused by two major species—Enterococcus faecium and Enterococcus faecalis. However, in recent years, there has been an increasing number of infections caused by non-faecium non-faecalis (NFF) enterococci. The NFF enterococci predominantly affect immunocompromised patients and may cause a range of clinical manifestations. Treatment of NFF enterococcal infections may be clinically challenging as some species are intrinsically resistant to glycopeptides and/or can acquire resistance mechanisms to multiple antibiotics through horizontal gene transfer (HGT). In addition, NFF enterococci may host multiple virulence factors. This review provides an overview of NFF enterococci, with a focus on their clinical manifestations, virulence factors, and antimicrobial resistance (AMR) genes.

PHENOTYPIC CHARACTERISTICS

Enterococci are typically spherical or ovoid in shape and are arranged in pairs or short chains. They are sometimes described as coccobacillary when grown on solid media but are typically ovoid when grown in broth (2, 3). β-hemolysis is more frequently associated with E. faecalis, particularly in non-sheep blood, while the other enterococci are typically α-hemolytic or non-hemolytic (4–6). On blood agar after 24 hours of incubation, enterococci appear as smooth white colonies 1–2 mm in diameter, although for some species the colonies may be smaller. Enterococci are non-spore-forming facultative anaerobes and obligatory fermentative chemoorganotrophs. Members of the genus have a temperature growth range from 10°C to 45°C, with an optimum growth temperature of 35°C (7). Enterococci are usually homofermentative, producing lactic acid as the product of glucose fermentation, without the production of gas. Although most species are non-motile, motility has been observed in Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus massiliensis, and “Enterococcus timonensis” (7).

POPULATION STRUCTURE AND EVOLUTION

The Enterococcus genus is a member of the Enterococcaceae family, which consists of six other genera: Bavariicoccus, Catellicoccus, Melissococcus, Pilibacter, Tetragenococcus, and Vagococcus. While typically associated with the GI tract of humans and production animals, enterococci are ubiquitous and can be found in the GI tract of birds, reptiles, insects, and rats, as well as soil, plants, and water (8–10).

As of 2022, 74 enterococcal species are recognized within the genus (Table S1), 62 of which have validly published species names (11).

While both E. faecium and E. faecalis are the most frequently isolated species in the human GI tract, E. faecium predominates in the gut of production animals such as pigs, poultry, and cattle (9, 10). However, NFF enterococci, such as E. gallinarum, Enterococcus cecorum, Enterococcus durans, and Enterococcus avium, are also found in production animals (12–14). In the environment, Enterococcus mundtii and E. casseliflavus are typically found in plants (15) (Fig. 1).

Fig 1.

Non-faecium non-faecalis enterococci and the host/environments from which they are most frequently isolated.

16S rRNA gene sequencing has shown that enterococci are more closely related to Vagococcus, Tetragenococcus, and Carnobacterium than they are to Streptococcus and Lactococcus (16). The Enterococcus genome ranges from 2.7 to 3.5 Mb and has a low GC content with an average of 37.9% (17–19). Based on whole-genome sequencing of 37 enterococci, which included 34 species, Zhong et al. (18) recently reported that the core genome of Enterococcus consisted of 605 genes. Using a maximum likelihood phylogenetic tree based on the 605 core genes, six phylogenetic groups were identified: the E. faecium group, the E. faecalis group, the Enterococcus dispar group, the E. casseliflavus group, the Enterococcus pallens group, and the Enterococcus canis group (Table 1).

TABLE 1.

Phylogenetic groups of the genus Enterococcus based on 605 core genes^a

	Branch name	Species in branch
Group 1	E. faecium branch	E. faecium, E. mundtii, E. durans, Enterococcus hirae, Enterococcus ratti, Enterococcus villorum, Enterococcus thailandicus, and Enterococcus phoeniculicola
Group 2	E. faecalis branch	E. faecalis, Enterococcus termitis, Enterococcus quebecensis, Enterococcus moraviensis, Enterococcus caccae, Enterococcus haemoperoxidus, and Enterococcus silesiacus
Group 3	E. dispar branch	E. dispar, Enterococcus canintestini, and Enterococcus asini
Group 4	E. casseliflavus branch	E. casseliflavus, E. gallinarum, Enterococcus aquimarinus, Enterococcus saccharolyticus, Enterococcus italicus, Enterococcus sulfureus, Enterococcus cecorum, and Enterococcus columbae
Group 5	E. pallens branch	E. pallens, Enterococcus hermanniensis, Enterococcus devriesei, Enterococcus gilvus, Enterococcus malodoratus, Enterococcus avium, and Enterococcus raffinosus
Group 6	E. canis branch	E. canis

^a Phylogenetic groups were based on a maximum likelihood tree from 605 core genes generated by Zhong et al. (18).

Evolutionary studies suggest that E. canis is the most ancient enterococcal species, while the enterococcal species found in mammals have evolved more recently. It is hypothesized that the mammalian enterococcal species expanded into the plant, bird, soil, and food environments (18).

EPIDEMIOLOGY OF NFF ENTEROCOCCI CAUSING BLOODSTREAM INFECTIONS

In humans, approximately 90% of enterococcal bloodstream infections are caused by E. faecium and E. faecalis (20, 21). The global prevalence of NFF enterococci associated with enterococcal bloodstream infections in humans is summarized in Fig. 2 (22–43).

Fig 2.

The prevalence of non-faecium non-faecalis Enterococcus infections among all enterococcal infections worldwide (16–37). Non-faecium non-faecalis Enterococcus infection data from all countries were collected from continuous national surveillance, except for Malawi (only from one health center). Four countries (Norway, Spain, New Zealand, and Australia) identified the isolates down to the species level. The remaining countries identified the isolates as enterococci not belonging to E. faecium or E. faecalis.

Except for Malawi in Africa (44), the NFF enterococcal infection data were obtained from continuous national surveillance programs. In some parts of the world, limited information is available with only one study cited from Africa and two studies each in Europe and Asia.

One of the largest ongoing national enterococcal bloodstream surveillance programs is performed in Australia by the Australian Group for Antimicrobial Resistance. Known as the Australian Enterococcus Surveillance Outcome Program (AESOP), the program collects continuous data on all enterococcal species from blood cultures from up to 30 laboratories servicing 48 hospitals from all Australian states and mainland territories. From 2013 to 2022, of the 11,681 unique episodes of enterococcal bacteriemia collected in the program, 5.6% were caused by NFF enterococci and include E. casseliflavus, E. gallinarum, and E. avium as the predominant species (Table 2) (21, 45–53).

TABLE 2.

Number of enterococcal isolates from human bloodstream infections collected as part of the AESOP from 2013 to 2022 (21, 45–53)^c

Species	Year										Total	30-day all-cause mortality rate (%)a
	2013	2014	2015	2016	2017	2018	2019	2020	2021	2022
E. avium	5	9	11	8	9	18	13	12	6	16	107	10.1
E. casseliflavus	16	19	16	25	19	21	22	19	20	21	198	6.7
E. cecorum	−	1	−	−	−	−	−	1	−	1	3	0
E. dispar	−	−	−	−	−	−	−	−	−	1	1	100
E. durans	3	1	1	−	5	3	3	2	5	4	27	16.6
E. faecalis	478	523	562	595	602	676	699	667	705	812	6,319	14.5
E. faecium	325	380	402	413	481	491	596	488	523	613	4,712	25.2
E. gallinarum	11	13	7	11	14	29	21	20	23	17	166	13.3
Enterococcus gilvus	1	−	1	−	−	−	−	−	−	2	4	50
Enterococcus hirae	5	2	4	3	2	6	3	8	7	5	45	12.9
E. mundtii	−	1	−	−	−	−	−	−	1	−	2	0
Enterococcus lactisb	−	−	−	−	−	−	−	−	−	29	29	3.44
Enterococcus raffinosus	3	3	7	3	4	3	3	12	9	13	60	13.9
Enterococcus saccharolyticus	−	−	−	−	1	−	−	−	−	−	1	100
Enterococcus thailandicus	−	−	−	−	−	−	1	−	−	−	1	NA
Enterococcus sp.	2	−	−	−	−	1	−	1	1	1	6	66.6
Total	849	952	1,011	1,058	1,137	1,248	1,361	1,230	1,300	1,535	11,681

^a 30-day all-cause mortality was calculated only from patients in which the outcome was known.

^b Prior to 2022, E. lactis was classified as E. faecium clade B.

^c −, no isolates detected; NA, data not available.

CLINICAL MANIFESTATIONS

Enterococci are considered opportunistic pathogens, as they are observed as commensal gut colonizers up to 20 times more frequently than being noted as the causal agents of symptomatic infections (17). Although enterococci are associated with endocarditis, septicemia, urinary tract infections, and wound infections, microbiological cultures are often polymicrobial, and enterococci are frequently dismissed as the causal agent of disease.

Of the NFF enterococci, E. casseliflavus and E. gallinarum are the most frequently reported, each causing 1%–3% of enterococcal infections (54). NFF enterococci can cause a diverse range of infections. The most frequent NFF enterococcal clinical manifestations are catheter-associated bloodstream infections followed by biliary tract infections and intra-abdominal infections (55). Other types of infections such as urinary tract infections, soft tissue infections, infective endocarditis, and respiratory infections tend to be less frequent. In recent years, there have been sporadic case reports of NFF enterococci causing meningitis, multifocal joint infections, cholangitis, and endophthalmitis (56–59).

NFF enterococcal infections are often polymicrobial, with 40%–60% of infections caused by more than one organism (20, 60). In Australia, 59.8% of NFF enterococcal bloodstream infections are polymicrobial (55). In polymicrobial infections, NFF enterococci are almost always associated with Gram-negative bacilli, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Klebsiella aerogenes (55, 60, 61). In rare cases, NFF enterococci are also isolated with E. faecium or E. faecalis.

When infections due to NFF enterococci occur, almost all patients (>95%) have underlying conditions (20, 61). The most frequently described conditions are malignancy, biliary disease, solid organ transplant, bone marrow transplant, and diabetes mellitus. Risk factors for bacteremia include biliary drainage catheters in place, history of prior hospital admission, leukopenia, and central venous catheters (20). Additionally, NFF enterococcal infections tend to be associated with the immunocompromised and the elderly (55).

Globally, the all-cause 60-day mortality incidence for NFF enterococcal infections ranges from 13% to 20% but can be significantly higher in immunocompromised patients (20, 60, 61). In bacteremia, mortality can be as low as 2.5% but as high as 42% in immunocompromised patients (20, 62). In Australia, the all-cause 30-day mortality incidence for enterococcal bloodstream infections is 14.2% (55).

NFF ENTEROCOCCI CAUSING INFECTIONS IN HUMANS

Several different species of NFF enterococci have been reported as the causative agent of infections in humans, and a summary of the infections caused by each species is listed in Table 3.

TABLE 3.

Non-faecium non-faecalis Enterococcus infections in humans

Species	Meningitis	Endophthalmitis	Periodontitis	Pneumonia	Endocarditis	Bloodstream infection	Biliary tract infection	UTI	Osteomyelitis	Peritonitis
E. avium	x		x		x	x	x	x	x	x
Enterococcus canintestini						x
E. casseliflavus	x	x			x	x
Enterococcus cecorum	x				x	x				x
E. dispar						x		x
E. durans	x			x	x	x		x
E. gallinarum	x	x			x	x	x	x	x
Enterococcus gilvus						x	x
Enterococcus hirae			x	x	x	x		x	x	x
Enterococcus lactis						x
E. mundtii		x				x		x
Enterococcus pseudoavium								x
E. pallens										x
Enterococcus raffinosus		x			x	x			x
Enterococcus saccharolyticus						x
Enterococcus thailandicus						x	x			x

E. avium

Originally known as group Q streptococci, E. avium was first recognized in 1984 and is a rare cause of human infections (3). The species is principally found in low abundance in the GI tract of poultry and mammals (including humans) (63). The first E. avium bloodstream infections were documented in 1992 and were related to ulcerative colitis (64). While bloodstream infections are the most frequently reported clinical manifestation, E. avium can be associated with a diverse range of infections, including surgical site infections (65, 66), abscesses (67–69), biliary tract infections (20), meningitis (70, 71), endocarditis (72, 73), osteomyelitis (74), periodontitis (75), sinusitis (76), and urinary tract infections (77). In Australia, 18% of NFF enterococcal bacteremia are caused by E. avium, 45.5% of which are polymicrobial (52).

Enterococcus canintestini

E. canintestini resides in the GI tract of healthy dogs (78). E. canintestini has been reported as a cause of infection in only one published case (20). Reported in Taiwan in 2004, the organism was isolated from the blood of a 72-year-old female patient with cervical cancer who was undergoing chemotherapy. The bacteremia was not polymicrobial. Although no additional human infections have been reported, the species is frequently detected in the vaginal swabs of mothers and the meconium of newborns (79).

E. casseliflavus

E. casseliflavus is the most frequently isolated NFF enterococcal species found in human infections. The organism resides in the GI tract of humans, cattle, birds, and pigs. Bacteremia is the most frequently reported clinical manifestation with the global prevalence of bloodstream infections ranging from 1.9% to 9% (45, 47, 80), and the mortality rate can be as high as 51.2% (60). E. casseliflavus has also been associated with endocarditis (81–84), eye infections (84–92), and meningitis (93–95). In Australia, 32.6% of NFF enterococcal bacteremia is caused by E. casseliflavus, with a 30-day all-cause mortality rate of 6.7% (52).

E. cecorum

E. cecorum was first isolated in 1983 from the GI tract of healthy chickens (96). While it mainly appears as a gut commensal in poultry, it can also be found in the GI tract of pigs, calves, ducks, cats, and dogs and may cause infections in these animals. In very rare circumstances, it may cause a diverse range of human infections and is responsible for 0.01% of NFF enterococcal bloodstream infections in Australia (52). Sporadic cases of human infections caused by E. cecorum have been reported over the last 30 years and include septicemia (97), peritonitis (98, 99), thoracic empyema (100), endocarditis (101, 102), meningitis (103), incisional hernia plate infections (104), and urinary tract infections (104). There is increasing concern over E. cecorum as an AMR reservoir, as detection of resistance to multiple classes of antibiotics is becoming more frequent in poultry isolates (105–113).

E. durans

Originally known as Streptococcus durans, E. durans was first isolated in 1937 and was reclassified to the Enterococcus genus in 1984 (114, 115). The species is predominantly found in the alimentary tract of foals, piglets, calves, and puppies and is a known cause of enteritis in these animals. It is frequently detected in a range of animal and dairy products (116–142). E. durans was first recognized as a cause of human infection in 1990 and was cultured from an infected wound(54) . It is now known to cause a range of infections, including bloodstream infections (21, 143), respiratory tract infections (144), endocarditis (62, 145–154), urinary tract infections (155), and brain infections (156, 157). In Australia, E. durans is responsible for 4% of NFF enterococcal bacteremia and has a 30-day-all-cause mortality rate of 16.6% (21).

E. gallinarum

Although E. gallinarum is predominantly a gut commensal of mammals and birds, it is one of the most prevalent NFF enterococcal species found in human disease. In humans, E. gallinarum has been associated with endocarditis (84, 158–163), bloodstream infections (60, 80, 164–172), brain infections (57, 173–175), cholangitis (58), arthritis (56), peritonitis (176), endocarditis (84, 158–163), ophthalmitis (84, 177), and urinary tract infections (84, 178–180). Globally, E. gallinarum accounts for 0.8%–3.5% of enterococcal bacteremia (47, 50), with a mortality rate of 26.7% (60). In Australia, E. gallinarum is the second most frequent cause of NFF enterococcal bacteremia (27.2%), with a 30-day-all-cause mortality rate of 13.3% (45, 46, 48–50, 52).

Enterococcus gilvus

Although E. gilvus is predominantly associated with dairy products, such as cheese and milk, the species can occasionally be found in poultry (134, 181, 182). The first human infection of E. gilvus was reported in 2002, in a bile sample from the biliary tract (183). E. gilvus is an extremely rare cause of infection in humans, with only two additional cases reported worldwide since its initial discovery, both being bloodstream infections in Australia (45, 47).

Enterococcus hirae

E. hirae was first detected in 1985 and was associated with growth defects in young chickens (184). There are sporadic case reports of the species causing a diverse range of human infections, including endocarditis (185–188), pneumonia (189), bloodstream infections (190–193), spondylitis (194), pyelonephritis (191, 195), gastritis (196), cystitis (197), periodontal infections (198, 199), and peritonitis (200). In Australia, E. hirae is responsible for 7% of NFF enterococcal bacteremia, with a 30-day-all-cause-mortality rate of 12.9% (45, 46, 48–50, 52, 55).

Enterococcus lactis

E. lactis was first isolated from raw milk and was previously considered to be rarely pathogenic (201). Studies on this bacterium primarily focus on its use in the food industry, particularly as a starter culture for cheese and milk products (202). Whilst E. lactis was not typically considered a cause of human infections, recent studies based on the core and pan-genome, multi-locus sequence typing, and digital DNA-DNA hybridization have found E. faecium clade B should be reclassified to E. lactis (203, 204). Based on the reclassification, E. lactis may be a more frequent cause of human infections than was previously thought. In the Australian AESOP 2022 report, 29 enterococci were identified as E. lactis based on the new classification, making up 1.9% of enterococcal bloodstream infections (53).

Additionally, based on DNA-DNA hybridization, Enterococcus xinjiangensis, which was first found in yogurt in 2016, has also recently been reclassified as E. lactis (201, 205).

E. mundtii

E. mundtii was initially isolated in cow teats and cow milk handlers (206). Having previously been misidentified as E. faecium and E. faecalis by commercial identification systems, E. mundtii was officially recognized as a species in 1984 (207). It has since been detected in numerous animal and environmental sources, including water (208, 209), poultry (137), horses (210), corn stovers (211, 212), dairy farms (213, 214), slaughterhouses (215, 216), fish (217), caterpillars (218), and other wildlife (219). Although E. mundtii rarely causes human infections, it has been found in thigh abscesses (207), sinusitis (207), endophthalmitis (220), bloodstream infections (170, 178), and urinary tract infections (221). Between 2013 and 2021, the AESOP has only reported two episodes of E. mundtii bacteremia in Australia (52).

Enterococcus raffinosus

E. raffinosus was first described in 1989 from patients with deep vein thrombosis, liver disease, and rheumatoid arthritis (222). Compared to other NFF enterococci, E. raffinosus is a relatively infrequent cause of human infections but has been found in cases of vertebral osteomyelitis (223), hematoma (224), vaginal infections (225), decubitus ulcers (226), endocarditis (227, 228), endophthalmitis (143, 229), sinusitis (143, 230), bloodstream infections (20, 61, 231, 232), and among hemodialysis patients (233). In Australia, E. raffinosus is responsible for 8.2% of NFF enterococcal bacteremia and has a 30-day-all-cause-mortality rate of 13.9% (21).

Enterococcus saccharolyticus

Originally grouped with Streptococcus bovis, E. saccharolyticus was first described as a species in 1984 (234). The organism is primarily isolated from animal and environmental sources, such as freshwater or sewage and is occasionally found as the causative agent of bovine mastitis (235). In rare circumstances, it is the cause of human infections and has been identified in cases of infected lymphocele (236) and bacteremia (49).

E. thailandicus

Initially isolated from fermented sausages in Thailand, E. thailandicus is an extremely rare cause of human disease (138). The species is infrequently isolated from the GI tract of animals, such as cows and beavers, and in environmental sources, such as farms and water bodies (216, 237–239). There is some debate as to whether E. thailandicus has been found in the human GI tract, as the species was previously grouped with E. sanguinicola, which has been detected in humans (238, 240, 241). To date, there have only been two recorded cases of E. thailandicus causing human infections, both of which were polymicrobial. The first case was a biliary tract infection reported in Australia (55) and the second case occurred in a patient in Belgium with an intra-abdominal infection (242).

Less common NFF enterococci causing human infections

While overwhelmingly detected in animals and in the environment, there are several less common NFF enterococci, which occasionally cause human infections. Species include E. dispar, which has caused urinary tract infections and septicemia (243–245), Enterococcus pseudoavium, which is responsible for 2.6% of enterococcal urinary tract infections in India (243), and E. pallens, which sporadically causes bacterial peritonitis (183, 246).

Additionally, there are NFF enterococci species isolated in humans, which are not known to cause infection: Enterococcus villorum, Enterococcus rivorum, and Enterococcus phoeniculicola have been found in the vagina of pregnant women and meconium of newborn infants (79, 247–249); Enterococcus caccae, “E. massiliensis,” and “Enterococcus mediterraneensis” have been found in the fecal samples of healthy individuals (250–252).

VIRULENCE FACTORS

As the majority of enterococcal infections are caused by E. faecalis and E. faecium, most studies involving virulence factors have focused on these two species. When the NFF enterococci species are isolated, the studies are often limited due to insufficient sample size. Additionally, the species are rarely studied in vitro, so it is unknown if these virulence genes are responsible for the same phenotypes observed in E. faecium and E. faecalis. Some genetic studies have shown NFF enterococci possess many virulence factors, even when isolated as a commensal (Table 4).

TABLE 4.

Virulence factors in enterococci

Function	Virulence factor	Gene(s)	References
Adhesion	Aggregation substance	asa1	(253–257)
	Collagen-binding adhesin	ace, acm	(258–263)
	Enterococcal surface protein	esp	(264–270)
	Pili	pilA, pilB, ebpA, ebpB, ebpC, srtC	(271–275)
Lysins	β-hemolysis	cylLL, cylLS, cylM, cylA, cylB	(4, 276–280)
	Gelatinase	gelE	(270, 281)
	Serine protease	sprE	(282–284)
	Hyaluronidase	hyl	(257, 285, 286)
Sex pheromones	Conjugative plasmid transfer, neutrophil chemotaxis, superoxide formation, inflammation	cpd, cob, ccf, cad	(263, 287–289)

Adhesion

One of the major virulence determinants in enterococci is their ability to adhere to host tissue. When the first enterococcal genome was published, a series of 41 putative cell wall proteins were identified. The proteins belong to a family known as microbial surface component recognizing adhesive matrix molecule (MSCRAMM) (290). The process of adhesion is complex and involves at least four major proteins, including the two collagen-binding adhesins (Ace and Acm), enterococcal surface protein (Esp), and aggregation substance (AS). Additionally, pili have been shown to play a role in biofilm formation in some enterococcal isolates.

Ace is a MSCRAMM and is one of the most well-studied adhesins in E. faecalis. It has a high sequence similarity to the A domain of the collagen adhesin (Cna) in Staphylococcus aureus. The cell surface protein mediates adherence of E. faecalis cells to bovine and rat collagen type I (CI), human collagen type IV (CIV), and mouse laminin (258, 291). In animal infection models, Ace has been shown to play a role in early endocarditis establishment (259). The ace gene is found in most clinical isolates of E. faecalis and tends to be found more frequently as a pseudogene in non-clinical isolates (258). Studies researching animal isolates of Enterococcus have identified ace in many NFF enterococci, including Enterococcus columbae, E. hirae, E. gallinarum, E. casseliflavus, E. cecorum, and E. durans (260).

Acm, a collagen adhesin, has 62% similarity to the S. aureus collagen adhesin Cna. The Acm protein consists of two domains (domains A and B) and is 60% and 75% similar to domains A and B of Cna, respectively (261). This adhesin was first identified in E. faecium, and while almost 100% of clinical isolates have the acm gene, not all exhibit binding to the Acm receptor, collagen type I (261). Introduction of the acm gene into acm deletion mutants showed an 11-fold increase in E. faecium adherence to collagen but not to fibronectin or fibrinogen (261). Acm has also been shown to play a role in endocarditis as acm deletion mutants were highly attenuated in mouse models of infection and had reduced valve colonization ability (262). Until recently, acm had not been identified in Enterococcus species other than E. faecium. However, a 2022 study found acm in isolates of E. gallinarum and E. casseliflavus in food products of animal origin (263).

Esp was initially identified in E. faecalis and was found to be significantly enriched in clinical isolates compared to isolates obtained from fecal samples of asymptomatic patients (264). Molecular studies have shown that, while Esp does not directly contribute to the histopathology of E. faecalis, it is associated with biofilm formation (265) and colonization and persistence in urinary tract infections (266). Esp is orthologous to Bap, a biofilm-associated protein in S. aureus. The Esp protein forms aggregates with an amyloid-like conformation, which is directly related to its ability to form biofilms (267). The presence of the esp gene in E. faecium appears to be restricted to hospital-associated isolates belonging to CC17 (268), although it has recently been detected in several NFF enterococci including E. casseliflavus, E. mundtii, E. durans, Enterococcus solitarius, and Enterococcus malodoratus (269, 270).

AS (encoded by asa1) is a plasmid-encoded enterococcal surface protein and was first discovered in relation to binding between bacterial cells, facilitating the transfer of the pAD1 plasmid between bacterial cells (253). However, in vitro studies have shown that the AS protein increases the adherence of bacteria to renal tubular cells (254) and protects E. faecalis from killing by polymorphonucleocytes (255). In E. faecalis, asa1 is highly associated with isolates from urine, blood, and wound sites (256). Reports of asa1 in NFF enterococci are rare, although the gene has been identified in E. gallinarum, E. raffinosus, and E. mundtii (257).

Pili have been well described in enterococci, being known primarily for their role in biofilm formation and endocarditis. Clinical isolates of E. faecium are significantly enriched for pilA and pilB genes compared to isolates from fecal swabs (271). Additionally, the endocarditis and biofilm-associated pilus, Ebp, has been well characterized. As its name suggests, Ebp plays a major role in endocarditis and biofilm formation and consists of ebpA, ebpB, ebpC, and an associated srtC (encoding sortase C) gene that also has an independent promoter (272). The protein complex has also been shown to play a role in animal models of urinary tract infection (273), adherence to human platelets (274), cell-cell aggregation, and intraspecies gene transfer (275). The epb locus is highly conserved between species of Enterococcus, including E. casseliflavus and E. gallinarum (274); the only notable difference is the absence of epbB in E. casseliflavus.

Lysins

β-hemolysis is identified in approximately one-third of E. faecalis isolates and has been associated with genes from the cyl operon that encodes cytolysin genes. The operon includes two cytolysin structural subunits (encoded by cylL_L and cylL_S), which are post-translationally modified intracellularly by the cylM gene product and transported out of the cell by an ATP-binding cassette transporter encoded by cylB; after externalization, cytolysin precursor components are activated by CylA, an extracellular activator serine protease (276, 277). The final gene in the operon cylI encodes the immunity protein for self-protection from cytolysin. Cytolysin has been studied in both animal and human models of infection and contributes to the severity of enterococcal disease (278, 279). While rare, β-hemolytic activity has been observed in E. avium, E. casseliflavus, E. cecorum, E. durans, Enterococcus flavescens, E. gallinarum, E. malodoratus, and E. raffinosus, and this has been associated with the presence of cylL_L, cylA, and cylL_S (4, 280).

E. faecalis is known to produce extracellular proteases, specifically gelatinase (encoded by gelE), serine protease (encoded by sprE), and hyaluronidase (encoded by hyl). Extracellular gelatinase is a matrix metalloproteinase that hydrolyzes gelatin, collagen, and other proteins. In E. faecalis, gelE plays a role in immune evasion by preventing both opsonization and the formation of membrane attack complex through the hydrolysis of C3a (281). The protease has also been shown to cleave the anaphylatoxin complement C5a, leading to decreased neutrophil migration in vitro (292). The sprE gene lies immediately downstream of gelE and encodes a secreted 26-kDa serine protease, which is homologous to the S. aureus V8 protease (282). The presence of both proteases is associated with biofilm formation (293). In vitro studies have shown that sprE deletion mutants exhibit increased autolysis, while gelE deletion prevents extracellular DNA release from the biofilm (294). The loss of one or both protease genes results in attenuated virulence in animal models of infection (295). The two genes are transcriptionally regulated by the fsr locus, and deletion of this locus prevents biofilm formation (294). Similar to other virulence genes in NFF enterococci, the presence of gelE and sprE is infrequent; however, gelE has been identified in E. casseliflavus, E. gallinarum, E. avium, E. durans, and E. raffinosus (270), and sprE has been identified in E. hirae, E. mundtii, and E. durans (283, 284).

Hyaluronidase is an enzyme produced by a variety of bacteria that breaks down hyaluronic acid. Hyaluronidase depolymerizes the mucopolysaccharide moiety of connective tissues, allowing bacteria to invade between cells (285). The gene encoding hyaluronidase has been identified in E. faecalis, particularly among oral strains associated with periodontal disease, and is more prevalent in clinical isolates of E. faecium (257, 285). The hyl gene is typically absent in most studies of NFF enterococci although it has been detected in E. casseliflavus, E. gallinarum, E. durans, E. malodoratus, and E. mundtii (257, 286).

Sex pheromones

E. faecalis possesses several chromosomally encoded genes that produce “sex pheromones.” These sex pheromones are small linear peptides that are produced by the recipient cells that stimulate (or inhibit) the transfer of conjugative plasmids between enterococci. When the peptide binds to the donor cells that carry a conjugative plasmid, the donor produces the AS adhesin, which results in the clumping of donor and recipient cells (287). This close contact facilitates the transfer of the conjugative plasmid. While these peptides have a role in plasmid transfer, they can play a role in neutrophil chemotaxis. The small peptides bind to formyl peptide receptors 1 and 2 (FPR1 and FPR2) and G protein-coupled receptor 41 or 4 (GPR41 or GPR4), which are found on the surface of neutrophils and other leukocytes (288). In addition to neutrophil chemotaxis, the binding of these peptides to the cell receptors can result in superoxide formation and the activation of inflammatory mediators such as IL-8 (289). There are several sex pheromones, and a single bacterium may produce multiple types. They are typically encoded by cpd, cob, ccf, and cad. Historically, the genes have been exclusively studied in E. faecalis, but they have recently been identified in E. gallinarum and E. casseliflavus (263). While these sex pheromones have been studied in mouse models and in vitro, their role in human infection and colonization remains unclear.

ANTIBIOTIC RESISTANCE

In the 1970s and 1980s, the continued use of antibiotics led to the evolution of antimicrobial-resistant E. faecium, which has become a frequent cause of hospital-acquired bacteremia (296). Antimicrobial resistance in E. faecium was particularly driven by the use of cephalosporins, which eradicated the intestinal bacterial microbiota and selected for vancomycin-resistant enterococci (VRE) (297, 298).

Antimicrobial-resistant NFF enterococci have been reported in several studies. The antimicrobial susceptibility of NFF enterococci against certain common antibiotics is summarized in Table 5 (57, 93, 100, 144, 178, 191, 299–319).

TABLE 5.

The antibiotic susceptibility of non-faecium non-faecalis enterococci in human clinical samples (57, 93, 100, 144, 178, 191, 299–319)^a

Antibiotics	Minimum inhibitory concentration (mg/L)
	E. avium	E. casseliflavus	E. cecorum	E. dispar	E. durans	E. gallinarum	E. hirae	E. mundtii	E. raffinosus	E. solitarius	E. sulfurous	E. thailandicus
Aminoglycoside
Amikacin	≥512	−b	−	−	−	−	−	−	−	−	−	−
Gentamycin	≥2,048	8–1,024	−	>500	>500	≤50–2,000	≥500	−	>2,048	≥500	−	−
Netilmicin	−	−	−	−	−	−	−	−	−	−	−	−
Streptomycin	≥2,048	64	−	>2,000	>2,000	≤125–250	≥2,048	−	>2,048	≥2,000	−	−
Beta-lactams
Cephalosporins
Cefotaxime	−	−	0.25	−	−	−	−	−	−	−	−	−
Carbapenems
Imipenem	0.5–128	≤0.06–64	−	−	−	≤1−≥16	≥1	−	>16	−	−	−
Penicillins
Amoxicillin	−	−	−	−	−	−	0.25	−	−	−	−	−
Amoxicillin-clavulanic acid	−	>20/10	−	>20/10	>20/10	−	−	−	−	−	−	−
Ampicillin	≤2− ≥32	≤2−≥32	0.38	>10	>10	≤2−≥ 32	≥16	−	>32	≥16	−	1
Penicillin	16–64	0.5–4	0.094	>10	≥16	16–64	≥16	8	16–64	−	−	32
Fluoroquinolones
Ciprofloxacin	≥1	8–12	−	>5	>5	1–8	0.5	−	4	≥4	−	0.5
Levofloxacin	−	4	−	−	−	1	−	−	−	−	−	−
Moxifloxacin	−	1	−	−	−	1	−	−	−	−	−	−
Nemonoxacin	−	−	−	−	−	−	−	−	−	−	−	−
Norfloxacin	−	8–16	−	−	−	2–8	−	−	−	−	−	−
Ofloxacin	0.5–64	≤0.06–128	−	−	−	0.25–128	−	−	−	−	−	−
Glycopeptide
Teicoplanin	≤0.5	≤0.5–1	−	≥32	96	≤0.5–32	≤2	−	32–256	1–16	1–16	−
Vancomycin	≤0.5–1	1–4	1.5	≥32	≥256	≤0.5–32	1	≥128	128–1,024	128−≥256	≥256	0.5
Glycylcyclines
Tigecycline	<0.12	<0.12	−	−	0.047	<0.12	−	−	<0.12	−	−
Lipopeptides
Daptomycin	−	0.5–2	−	−	32	0.5–8	−	−	1	−	−	1
Lipoglycopeptides
Dalbavancin	−	≤0.015–0.25	−	−	−	≤0.015–0.25	−	−	−	−	−	−
Macrolides, lincosamides, and streptogramins
Clindamycin	−	4	−	−	−	≥8	−	−	>256	−	−	−
Erythromycin	≤0.06−> 128	0.125–32	−	>15	>15	≤0.06–128	≥8	−	>256	−	−	−
Roxithromycin	−	−	−	−	0.25	−	−	−	−	−	−	−
Quinupristin-dalfopristin	1–8	1–4	−	−	−	1–>16	−	−	>16	−	−
Oxazolidinones
Linezolid	16	2	0.75	−	1	1–4	2	−	1–2	−	−	2
Tedizolid	4	−	−	−	−	0.5	−	−	−	−	−	0.25
Tetracyclines
Minocycline	≤0.06–16	≤0.06–16	−	−	−	≤0.06–32	−	−	−	−	−
Tetracycline	≥16	2–16	−	−	−	2–16	−	−	64	−	−
Miscellaneous agents
Chloramphenicol	−	8–16	−	−	−	64	−	−	−	−	−	32
Fosfomycin	−	128	−	−	−	16–512	−	−	−	−	−
Fusidic acid	−	−	−	−	−	2	−	−	−	−	−
Metronidazole	>128	>50	−	−	−	−	−	−	−	−	−
Nitrofurantoin	−	8–64	−	−	−	64–128	−	−	−	−	−
Rifampicin	−	4–8	−	−	−	64	−	−	2	−	−
Trimethoprim-sulfamethoxazole	−	<0.008–0.5	−	−	>32	−	−	−	−	−	−

^a Only NFF enterococci for which antibiotic susceptibility data were available were included in this table.

^b −, no susceptibiltiy data available for a given species.

Beta-lactams

Beta-lactam antibiotics inhibit bacterial growth by binding to D,D-transpeptidases (penicillin-binding proteins), which catalyze cross-linking of peptidoglycan during cell wall synthesis. The inhibition of cell wall synthesis leads to defects in the bacterial cell wall structure and eventually cell lysis. Enterococci are typically considered to have intrinsic low-level non-susceptibility to beta-lactams. In enterococci, there are seven genes involved in beta-lactam resistance: pbp5, ponA, pbpF, murAA, croRS, asrR, ireK/ireP, and stk/stpA (Table 6).

TABLE 6.

Antibiotic resistance-associated genes in non-faecium non-faecalis enterococci

Antibiotic	Resistance mechanism	Gene(s)	Genes detected in NFF enterococci	References
β-lactams	Low-affinity penicillin-binding proteins	pbp5, ponA, pbpF	−a	(320–323)
	Peptidoglycan synthesis	murAA, murAB	−	(324)
	Two-component signal transduction systems	croRS, ireK	−	(325–329)
	Transcriptional regulators	asrR	asrR homologs have been detected in E. casseliflavus, E. durans, E. gallinarum, and E. hirae	(330, 331)
	β-lactamase production	blaZ	blaZ detected in E. casseliflavus, E. gallinarum, and E. mundtii	(128, 332–334)
Aminoglycosides	Modification of antibiotic (intrinsic resistance)	aac(6′)-Ii, efmM	Genes not detected in NFF enterococci but high-level aminoglycoside resistance reported in E. avium, E. dispar, and E. pseudoavium	(243, 335, 336)
	Modification of antibiotic (acquired resistance)	aph(3′)-IIIa	aph(3′)-IIIa detected in E. avium, E. dispar, E. durans, E. gallinarum, and E. hirae	(337–340)
	Modification of antibiotic (acquired resistance)	ant(6)-Ia	ant(6)-Ia detected in E. casseliflavus and E. hirae	(341–343)
	Modification of antibiotic (acquired resistance)	ant(9)-Ia	ant(9)-Ia detected in E. avium, E. durans, and E. gallinarum	(344–348)
	Modification of antibiotic (acquired resistance)	aac(6')-aph(2')	aac(6')-aph(2') detected in E. avium, E. casselflavus, E. durans, E. hirae, and E. raffinosus	(340, 349–351)
Glycopeptides	Production of low vancomycin affinity peptidoglycan	vanC	vanC is intrinsically found in E. casseliflavus and E. gallinarum	(142, 316, 352)
		vanA	vanA detected in E. cecorum, E. dispar, E. durans, E. gallinarum, and E. raffinosus	(61, 308, 353–355)
		vanD	vanD detected in E. avium, E. gallinarum, and E. raffinosus	(310, 356–358)
		vanN	vanN detected in E. lactis	(359–361)
Tetracyclines	Efflux pumps	tetK, tetL	tetK and tetL detected in E. avium, E. casseliflavus, E. cecorum, E. durans, E. gallinarum, and E. hirae	(105, 140, 362)
	Ribosomal protection proteins	tetM, tetO, tetS	tetM, tetO, and tetS detected in E. avium, E. casseliflavus, E. cecorum, E. durans, E. gallinarum, and E. hirae	(105, 362, 363)
Glycylcyclines	Target modification	rpsJ mutations	−	(364–366)
	Ribosomal protection protein	tetM	tetM and tetL detected in E. avium, E. casseliflavus, E. cecorum, E. durans, E. gallinarum, and E. hirae but association with glycylcycline resistance has not been investigated	(105, 141, 362)
	Efflux pumps	tetL
Aminomethylcyclines	Target modification	16s rRNA gene	−	(367)
Macrolide-lincosamide-streptogramin B class	ATP-binding cassette protein— mechanism unknown	lsaA, etaA	lsaA detected in E. casseliflavus E. gallinarum, and E. hirae	(368, 369)
	Efflux pumps	mefA, mefE, msrA, msrC, mreA	mefA detected in E. durans	(370–374)
	Target modification	ermA, ermB, ermC	ermA detected in E. avium, ermB detected in E. avium, E. durans, E. gallinarum, and E. hirae	(375–379)
	Inactivation of antibiotic	mphC, ereA, lnuA, lnuB, linB	ereA detected in NFF enterococci (species not identified), linB detected in E. gallinarum	(380, 381)
	Inactivation of antibiotic	vatD, vatE	vatD and vatE detected in E. hirae	(382, 383)
Fluoroquinolones	Protection proteins	qnr	qnr detected in E. casseliflavus	(384–387)
	Target modification	Mutations in gyrA and parC	−	(388)
	Efflux pumps	emeA, erfAB	−	(389–391)
Lipopeptides	Genetic mechanisms have yet to be identified			(392–394)
Oxazolidinones	Target modification	Mutations in 23S rRNA gene	G2576T point mutation detected in E. hirae	(395)
	Target modification	cfrA, cfrB, cfrC, cfrD	cfr detected in E. avium, E. casseliflavus, and E. gallinarum	(396–398)
	Protection proteins	optrA, poxtA	optrA detected in E. hirae, poxtA detected in E. hirae and E. lactis	(343, 396–400)

^a −, the gene/s has not been detected in any NFF species.

In bacteria, there is a large diversity of penicillin-binding proteins (PBPs), with each bacterium often possessing several PBPs (401–403). Enterococci possess multiple PBPs, three of which are associated with β-lactam resistance: PBP5, PonA, and PbpF. While all three proteins are associated with β-lactam resistance, the predominant mechanism through which E. faecium is intrinsically not susceptible to β-lactams is via the production of a low-affinity class B penicillin-binding protein 5 (PBP5). Acquired resistance to high levels of ampicillin in this species has been observed in isolates with increased production of PBP5 (320). pbp5 deletion mutants show significant reductions in cephalosporin resistance (321). Additionally, point mutations in PBP5 can result in reduced affinity for penicillins and increased resistance to these antibiotics (322). While all enterococci possess PBPs, pbp5 has not been identified in NFF Enterococcus species, possibly in part due to a lack of AMR studies.

In E. faecalis, resistance to extended-spectrum cephalosporins, such as ceftriaxone, can occur through the presence of two PBPs: PonA and PbpF. Although single deletion mutants of ponA and pbpF do not increase susceptibility to ceftriaxone, double deletion mutants result in sensitization to this antibiotic (323). Site-directed mutagenesis of ponA and pbpF has shown that they are both essential partners of PBP5 (323). Neither ponA nor pbpF have been identified in NFF enterococcal species.

Intrinsic resistance to cephalosporins in E. faecalis can also occur through a UDP-N-acetylglucosamine 1-carboxyvinyl transferase known as MurAA (324). The enzyme is responsible for the phosphoenolpyruvate-dependent conversion of UDP-N-acetylglucosamine to UDP-N-acetylglucosamine-enolpyruvate, which is the first step in peptidoglycan synthesis. Deletion of murAA leads to increased susceptibility to cephalosporins in E. faecalis. MurAA appears to only play a role in E. faecalis, as deletion of its homolog, murAB in E. faecium has no effect on its susceptibility to cephalosporins (324). Neither murAA nor murAB have been detected in NFF enterococci.

In addition to PBPs, two-component signal transduction systems (TCSs) are known to contribute to β-lactam resistance, particularly to cephalosporins. The TCSs cause changes in the expression of several genes, resulting in physiological changes in the bacterial cell, which lead to increased resistance to β-lactams. Enterococci possess several TCSs; for example, E. faecalis strain V583 has at least 17 TCSs (404). In enterococci, two TCSs have been associated with β-lactam resistance, CroRS and IreKP.

CroRS is a TCS that is found in E. faecalis and E. faecium. CroS, a sensor with histidine kinase activity, phosphorylates the cognate response regulator CroR, which alters transcription in the bacterial cell leading to increased expression of pbp4/pbp5 (405). Deletion of croRS results in a 4,000-fold reduction in MIC for ceftriaxone (325, 326). In E. faecalis, disruption of CroRS leads not only to increased susceptibility to cephalosporins but also to ampicillin, bacitracin, and vancomycin (325, 404).

IreK (intrinsic resistance of enterococci kinase) is another TCS that is reported exclusively in E. faecalis. IreK is a eukaryotic-type serine/threonine kinase that belongs to a family of transmembrane kinases defined by the presence of multiple extracellular penicillin-binding protein and serine/threonine kinase-associated domains (327). IreK has been shown to directly phosphorylate its substrate, IreB. While the function of IreK/B remains unknown, it is hypothesized that they regulate peptidoglycan biosynthesis, metabolism, and resistance to cell wall stress (328). While its function is yet to be elucidated, it is clear that IreK plays a role in cephalosporin resistance, as ireK deletion mutants are susceptible to cephalosporins (328). While IreK is involved in cephalosporin resistance, it is also reciprocally regulated by IreP, a PP2C-type protein phosphatase encoded immediately upstream of ireK (328). Mutants lacking IreP are hyper-resistant to cephalosporins, and this resistance is mediated by IreK. While E. faecium does not possess IreK/B, it possesses its own serine/threonine kinase, Stk, and its cognate phosphatase, StpA. Impaired activity of StpA results in increased activity of L,D-transpeptidation pathway in PBP synthesis, leading to a non-mutational increase in ampicillin activity (329). Neither of the serine/threonine kinases has been identified in NFF enterococci.

In addition to TCSs, there are transcriptional regulators that are associated with penicillin resistance in E. faecium such as asrR (antibiotic and stress response regulator). AsrR is part of the MarR family that regulates 181 genes, including pbp5 (330). Deletion of asrR resulted in increased expression of pbp5 and increased resistance to ampicillin and vancomycin (330). AsrR homologs have not been found in E. faecalis; however, they have been identified in several NFF species, including E. durans, E. hirae, E. gallinarum, and E. casseliflavus (331). The role of these homologs in NFF enterococci has yet to be determined.

Beta-lactamases are enzymes that confer resistance to bacteria by hydrolyzing the β-lactam ring of penicillins. In enterococci, the enzyme is encoded by blaZ and can be transferred horizontally. However, the expression of blaZ is constitutively low, and a high bacterial inoculum is required to result in phenotypic penicillin resistance (332). β-lactamase production was first reported in E. faecalis in 1981 (332). β-lactamase production is extremely rare and is infrequently reported, although this may be underreported due to its low expression. In NFF enterococci, blaZ-associated ampicillin resistance has been reported in E. casseliflavus, E. gallinarum, and E. mundtii (333, 334).

While clinical isolates of E. faecalis and E. faecium are typically intrinsically resistant to penicillin, bacteremia isolates of E. gallinarum and E. casseliflavus are typically penicillin susceptible (60). β-lactam resistance has also been reported phenotypically in E. avium (375) and E. raffinosus (406, 407), but the mechanism of resistance is unknown. In rare circumstances, penicillin resistance has been observed in E. durans, E. casseliflavus, E. gallinarum, and E. mundtii; however, these resistant isolates were obtained from animal or environmental sources and have not been reported in human infection (408, 409).

Aminoglycosides

The aminoglycosides are a group of broad-spectrum antibiotics that irreversibly bind to the bacterial 30S ribosome, thereby interfering with protein translation, and resulting in the inhibition of protein synthesis. All enterococci are intrinsically low-level resistant to aminoglycosides with MICs ranging from 4 to 256 mg/L, depending on the antibiotic used. Two genes are responsible for intrinsic aminoglycoside resistance in E. faecium only: aac(6′)-Ii and efmM (Table 6). The aac(6′)-Ii gene encodes a 6′-N-aminoglycoside acetyltransferase, which modifies aminoglycoside antibiotics, including tobramycin, sisomicin, kanamycin, and netilmicin (335). The efmM (E. faecium methyltransferase) gene encodes an S-adenosyl methionine-dependent m⁵C methyltransferase (MTase) that specifically targets nucleotide 1,404 in 16S rRNA. While 16s rRNA methylation due to this enzyme imparts a smaller contribution to aminoglycoside resistance compared to aac(6′)-Ii, knockout mutants of the gene still contribute a twofold increase in susceptibility to kanamycin and tobramycin (336). High-level resistance to aminoglycosides has been reported in several NFF enterococcal species, including E. avium (376), E. dispar, and E. pseudoavium (243).

Acquired resistance to aminoglycosides in enterococci can occur via aph(3′)-IIIa, which encodes a 3′-aminoglycoside O-phosphotransferase type IIIa (Table 6). The gene is primarily associated with high-level spectinomycin resistance, and the enzyme can inactivate multiple aminoglycoside antibiotics, such as butirosin, amikacin, and isepamicin via the addition of a phosphate group (337). The gene is frequently detected in both E. faecalis and E. faecium, with prevalence being as low as 37% and as high as 50% (338, 339). In the few studies in which aph(3′)-IIIa has been investigated in NFF enterococci, the prevalence ranged from 28% to 100%, although the prevalence is likely skewed due to exceedingly small study sample sizes. Nevertheless, aph(3′)-IIIa has been detected in E. durans, E. avium, E. hirae, E. dispar, and E. gallinarum (338, 340).

The second major mechanism of acquired aminoglycoside resistance in enterococci is via ant(6)-Ia. The gene encodes an aminoglycoside nucleotidyltransferase, which modifies aminoglycoside antibiotics resulting in high-level resistance to gentamicin and streptomycin. The ant(6)-Ia gene has been identified in E. faecium and E. faecalis isolated from human and animal origin (341, 342). It has also been detected in NFF enterococci, such as E. casseliflavus and E. hirae, isolated from meat products (343, 408).

The third mechanism of acquired aminoglycoside resistance is via Ant(9)-Ia. This protein is another aminoglycoside nucleotidyltransferase, which acts specifically on spectinomycin by binding to the 9-hydroxyl group (344). The introduction of plasmids carrying ant(9) from E. faecalis into E. coli have shown a 10,000-fold increase in the MIC of spectinomycin (345). In NFF enterococci, this gene has been detected in E. avium, E. gallinarum, and E. durans (346–348).

The fourth and final mechanism of acquired aminoglycoside resistance in enterococci is via the bifunctional enzyme Aac(6')-Aph(2') (349). This enzyme has both adenyltransferase and phosphotransferase activities and results in high-level gentamicin resistance (HLGR) (MIC > 1,000 mg/L) (349). This is the major mechanism through which HLGR occurs in enterococci. The aac(6')-aph(2') gene has been detected in several NFF enterococcal species, including E. avium, E. casseliflavus, E. durans, E. hirae, and E. raffinosus (340, 350, 351).

Glycopeptides

Glycopeptides are a class of antibiotics that inhibit peptidoglycan synthesis by binding with strong affinity to acyl-D-alanyl-D-alanine (a peptidoglycan precursor) on lipid II, preventing the addition of new peptidoglycan units (410). The most frequently used glycopeptides are vancomycin and teicoplanin.

In enterococci, vancomycin resistance can be either intrinsic or acquired. Acquired vancomycin resistance can be mediated by vanA, vanB, vanD, vanE, vanG, vanL, vanM, or vanN genes, while intrinsic resistance is mediated by vanC (411) (Table 6). A new van gene cluster, vanP, has recently been identified, but its role in vancomycin resistance has not been clearly established (412). The van gene clusters mediate resistance by synthesizing peptidoglycan precursors that do not end in the high vancomycin affinity D-alanine. Several NFF enterococci have developed resistance to vancomycin, such as E. casseliflavus (MIC 1,024 mg/L) (413), E. gallinarum (MIC 1,024 mg/L) (413), E. mundtii (MIC ≥ 128 mg/L) (414), and E. raffinosus (MIC 128–1,024 mg/L) (415). While vancomycin resistance can be mediated by any of the van genes in E. faecium and E. faecalis, resistance in NFF enterococci has only been associated with vanA, vanB, vanC, vanD, and/or vanN.

The vanA and vanB operons are historically the most prevalent in infections due to vancomycin-resistant enterococci and are typically associated with E. faecium. However, vancomycin resistance in NFF enterococci is most commonly caused by the chromosomally mediated vanC operon, which is intrinsic to E. gallinarum and E. casseliflavus only. Intrinsic resistance due to vanC was first reported in 1988 in the first case report of E. gallinarum, which was isolated from multiple blood cultures from a patient receiving prophylactic vancomycin (316). Many studies have reported almost all E. gallinarum and E. casseliflavus isolates as vancomycin resistant, but this is less frequently reported in other NFF enterococci (352). While initially believed to be non-transferrable, vanC has been identified in E. faecium and E. faecalis, indicating the potential for HGT (321, 416). The low-level vancomycin resistance caused by vanC is clinically important as isolates may be identified as vancomycin “susceptible” but could result in treatment failure (417).

Strains with the VanA phenotype possess inducible, high-level resistance to vancomycin (MIC ≥ 64 mg/L) and teicoplanin (MIC ≥ 16 mg/L). While not intrinsic to NFF enterococci, vanA has been identified in several NFF enterococcal species. A 2019 study in Japan identified vanA on a conjugative plasmid (pELF2), which was identified in E. faecium, E. gallinarum, and E. raffinosus and should be reported as resistant (418). Recently, there have been several reports of vanA-positive NFF enterococci causing outbreaks in hospitals, particularly E. raffinosus (61, 308, 353). Additionally, vanA-positive E. durans and E. dispar have been found in Belgium (354).

In the poultry industry, vanA-positive E. cecorum has been detected with a vancomycin MIC of > 256 mg/L and a teicoplanin MIC of 4 mg/L. The isolate also possessed Tn1546 on its chromosomal DNA (355). This case has raised concern about the possibility of poultry farms acting as a reservoir of antibiotic-resistant genes relevant to human health (419).

The vanD gene is chromosomally mediated and strongly associated with E. faecium. Isolates harboring vanD have moderate levels of vancomycin resistance (MIC range, 16–256 mg/L) and teicoplanin resistance (MIC range, 4–64 mg/L) (356). While rare, vanD has been found in E. avium, E. raffinosus, and E. gallinarum (310, 357, 358).

The vanN gene cluster has only recently been identified in E. faecium from both human and animal origin (359–361). However, these strains were identified as E. faecium clade B, which has now been reclassified as E. lactis (361), indicating that vanN is predominantly associated with E. lactis. VanN causes low-level vancomycin resistance (16 mg/L).

Tetracyclines

Tetracyclines have a broad spectrum of activity and low toxicity to the host and are therefore widely used to treat a range of infections. Tetracyclines are protein inhibitors and bind to the 30S ribosomal subunit, preventing the amino-acyl tRNA from binding to the ribosome. In enterococci, resistance to tetracycline can occur via two mechanisms: efflux pumps and ribosomal protection. Efflux pumps are encoded by plasmid-borne tetK and tetL and mediate the removal of tetracyclines (except for minocycline) from the bacterial cell (420). Ribosomal protection proteins are chromosomally encoded by tetM, tetO, and tetS and mediate the enzymatic alteration of ribosomal conformation and displace the tetracycline binding site (411) (Table 6). The three genes can be transferred via the Tn916 transposon and confer resistance to tetracycline, doxycycline, and minocycline (421, 422).

All five tet genes have been found in multiple NFF enterococci, including E. avium, E. casseliflavus, E. cecorum, E. durans, E. gallinarum, and E. hirae (105, 141, 362). Tetracycline resistance is frequently associated with NFF enterococci, with tetM being the most frequently detected AMR gene (105, 363) Tetracycline resistance has also been reported in E. dispar and E. pseudoavium, but the genetic resistance determinants were not investigated in the study (243).

Glycylcyclines

Glycylcyclines are derived from tetracycline and are designed to overcome resistance due to efflux pumps. Tigecycline is the most frequently used antibiotic in this class and is recommended for the treatment of VRE. Decreased susceptibility to tigecycline in enterococci is primarily related to target modification (rpsJ) and ribosomal protection proteins encoded by tetM and tetL (364–366). Tigecycline-resistant E. casseliflavus was initially isolated in Egypt with an MIC of 1.0 mg/L. The study investigated the effect of proton pump inhibitors (PPIs) on tigecycline resistance and showed that an increased dose of PPIs will substantially increase the tigecycline MICs by 4- to 128-fold (304). Another study has reported tigecycline non-susceptible isolates of E. gallinarum from poultry and of E. hirae from swine with MICs between 0.5 and 1.0 mg/L; however, the mechanism of resistance was not investigated (423). Similarly, the tetM and tetL genes have been detected in E. avium, E. casseliflavus, E. cecorum, E. durans, E. gallinarum, and E. hirae, but their association with glycylcycline resistance has not been investigated (105, 141, 362).

Aminomethylcyclines

Aminomethylcyclines are another tetracycline derivative and have a broad spectrum of activity against Gram-positive and Gram-negative bacteria, anaerobic bacteria, and atypical bacteria (424). Like tetracyclines, omadacycline inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit; however, it still remains active against common tetracycline resistance mechanisms such as efflux pumps and ribosomal protection proteins. A recent study of 276 E. faecalis isolates showed that ribosomal protection protein gene, tetM, and the efflux pump genes, tetK and tetL, did not affect omadacycline (367). However, strains that had multiple mutations in the 16s rRNA genes demonstrated omadacycline resistance.

Among NFF enterococci, omadacycline susceptibility is rarely measured; however, antimicrobial susceptibility testing (AST) on NFF enterococcal isolates collected as part of the SENTRY antimicrobial surveillance program has not detected any omadacycline resistance (425, 426).

Macrolide-lincosamide-streptogramin B class antibiotics

The macrolide-lincosamide-streptogramin class (MLS) antibiotics are a class of structurally different but functionally similar class of antibiotics. Macrolides are characterized by a lactone ring with one or more attached deoxy sugars; lincosamides are alkyl derivatives of proline and consist of a pyrrolidine ring linked to a pyranose moiety via an amide bond, and streptogramins consist of naturally occurring cyclic peptide compounds that are grouped into two classes, group A and group B (427–429). MLS antibiotics bind to the 50S ribosomal subunit, preventing peptidyltransferase action and thereby blocking protein translation (430, 431). In enterococci, there are three mechanisms for the acquired resistance to MLS antibiotics: (i) modification of the drug target, (ii) inactivation/deactivation of the drug, and (iii) active efflux of the antibiotic (Table 6).

Target modification is the major mechanism of MLS resistance in enterococci. This primarily occurs via erythromycin methylases encoded by ermB or less commonly ermA and ermC, which act on specific residues in the 23S rRNA subunit. The modification of the target results in cross-resistance to all MLS antibiotics. In enterococci, ermB is responsible for approximately 80% of erythromycin resistance, followed by ermA and then ermC (432, 433). However, ermB is responsible for almost all high-level erythromycin resistance (MICs > 128 mg/L) and has been found in E. faecium, E. faecalis, E. durans, E. avium, E. hirae, and E. gallinarum (370). Quinupristin-dalfopristin (QD) (a streptogramin) resistance is also frequently associated with ermB carriage, and approximately 55% of all QD-resistant NFF enterococci are ermB positive (382). Macrolide resistance has been identified in E. avium and is associated with ermA and/or ermB (375–379). The erm genes are often found on conjugative plasmids, and efficient conjugative transfer of ermB from E. durans to E. faecium has been demonstrated in vitro (434).

Deactivation/inactivation of MLS antibiotics in enterococci occurs through phosphotransferases and nucleotidyltransferases. This mechanism of resistance is extremely rare in enterococci with only mphC (macrolide phosphotransferase), ereA (esterase), and lnuA/lnuB/linB (lincosamide nucleotidyltransferases) being infrequently detected in E. faecium. In recent studies, the genes have been prevalent among MLS-resistant strains of enterococci, with only ereA being identified in NFF species (380). Additionally, linB has been detected in E. gallinarum isolated from broiler chickens (381).

While target modification and drug inactivation usually result in resistance to only one antibiotic, the efflux pumps often confer resistance to multiple MLS antibiotics. Five genes are involved in the active efflux of MLS antibiotics: mefA, mefE, msrA, msrC, and mreA (370–374). The mefA, mefE, and mreA genes have been associated with macrolide resistance in enterococci, while msrA and mrsC have been associated with macrolide and streptogramin B resistance.

Additionally, MLS resistance can occur via vatD (formerly called satA) and vatE, which encode acetyltransferases that confer resistance to streptogramin A via an unknown mechanism (383). Although vatD and vatE are rarely found in NFF enterococci, the genes have been detected in three isolates of E. hirae, one isolate with vatD, and two isolates with vatE (382).

Finally, in enterococci, there exists a phenotype that exhibits cross-resistance to lincosamides, streptogramin A, and pleuromutilins (a class of protein synthesis inhibitors) known as the LS_AP phenotype. This phenotype is shared with NFF enterococci, such as E. avium, E. gallinarum, and E. casseliflavus (435). In E. faecalis, intrinsic LS_AP resistance is mediated by the ATP-binding cassette (ABC) protein LsaA. Disruption of the lsaA gene in E. faecalis is associated with a ≥40-fold decrease in MIC for QD, clindamycin, and dalfopristin. While not intrinsically resistant, E. faecium can acquire the LS_AP phenotype via a point mutation in a lsaA homolog known as eatA (368, 369). Although it is infrequent, lsaA has been detected in some NFF enterococci, including E. gallinarum, E. hirae, and E. casseliflavus (436), while eatA appears to be unique to E. faecium (368).

Fluoroquinolones

Fluoroquinolones are broad-spectrum antibiotics that prevent DNA replication by inhibiting the ligase activity of the bacterial DNA gyrase and topoisomerase IV (437).

E. faecium and E. faecalis have low levels of intrinsic resistance to fluoroquinolones via the production of proteins in the Qnr family (encoded by qnr) (384, 385). The proteins protect the DNA gyrase and topoisomerase IV, thereby decreasing DNA binding of the quinolone and subsequently decreasing the formation of the quinolone–gyrase complex (386). In NFF enterococci, the qnr gene has only been reported in one study, in three isolates of E. casseliflavus (387) (Table 6).

High-level fluoroquinolone resistance can also occur through target modification and through the production of efflux pumps. Target modification can occur via mutations in the GyrA subunit of DNA gyrase (gyrA) and the ParC subunit of topoisomerase IV (parC), which reduce the antibiotic affinity for the target (388). Mutations in these two genes have not been reported in NFF enterococci.

The final mechanism of fluoroquinolone resistance is the production of efflux pumps. There are two efflux pumps that are associated with fluoroquinolone resistance in E. faecium and E. faecalis: the enterococcal multidrug resistance efflux pump (encoded by emeA) and an ABC family of efflux pumps encoded by erfAB (389–391). The two efflux pumps have not been identified in NFF enterococci.

While the genes associated with fluoroquinolone resistance have not been reported in NFF enterococci, ciprofloxacin resistance has been reported in NFF enterococcal species such as E. avium (375), E. dispar (243), and E. pseudoavium (243, 438, 439).

Lipopeptides

Lipopeptides are produced by other microorganisms and consist of a lipid connected to a peptide (440). While several lipopeptides are used as antibiotics, daptomycin is the only lipopeptide available for use in enterococci and is reserved as a last-line therapy for VRE. The mechanism of action of daptomycin remains unclear; however, it is known to disrupt the gram-positive bacterial cell membrane, leading to cell death (392). Genes associated with daptomycin resistance have not yet been defined, but potential candidates have been proposed in forced evolution studies (393, 394).

Although daptomycin resistance is rare in NFF enterococci (441), a study in 2019 found that 58.3% of E. hirae isolates (from a total of 84 isolates) obtained from water samples were resistant to daptomycin (MIC > 4 mg/L) (442). Daptomycin resistance in E. gallinarum and E. mundtii was also identified in the study.

Oxazolidinones

Oxazolidinones are synthetic molecules that bind to the bacterial 50S subunit and prevent protein translation. Linezolid was the first oxazolidinone to be approved in human health and is currently recommended for the treatment of Gram-positive infections, including VRE. Resistance to linezolid can occur through mutations in domain V of the 23S rRNA genes or through the acquisition of Cfr and Cfr-like methylases encoded by cfrA/B/C/D (443–445), and finally via ATP-binding cassette proteins encoded by optrA and poxtA (Table 6). The Cfr methylases confer resistance by post-translational methylation of the 23S rRNA, while OptrA and PoxtA confer resistance by a ribosomal protection mechanism (446, 447). In enterococci, these resistance genes have been found on mobile genetic elements and can be transferred between species via HGT (446, 448–450). Additionally, linezolid resistance can occur through mutations in the 23S rRNA, which reduces the affinity to the antibiotic; however, this has not been reported in NFF enterococci (451).

In recent years, linezolid resistance and linezolid-resistant genes have become increasingly prevalent among NFF enterococci. Resistance to linezolid has been reported in E. avium (314), E. casseliflavus (413), E. gallinarum, and E. hirae (413, 452, 453). Linezolid-resistant E. hirae isolated from animals have been reported with the G2576T point mutation in the V domain of the 23S rRNA (395). E. hirae harboring optrA and poxtA have also been detected in healthy humans (343). Similarly, poxtA2 (a poxtA ancestor) and optrA have been found in E. gallinarum isolated from swine (396, 399). The cfr gene has also been detected in E. casseliflavus, E. gallinarum, and E. avium (396–398). Additionally, a recently identified E. lactis isolated from pigs in China was found to be positive for a mobilizable poxtA (400).

CONCLUSION

The NFF enterococci include a diverse list of species that are ubiquitous in humans, animals, and the environment. While a less frequent cause of human infection compared to E. faecium and E. faecalis, NFF enterococci may cause a range of clinically significant diseases, which may not be routinely reported by the diagnostic microbiology laboratory. NFF enterococci may carry a range of virulence genes that are known to contribute to pathogenesis in E. faecium and E. faecalis; however, their role in NFF enterococci largely remains unstudied. Additionally, AMR genes for almost all known classes of antibiotics have been detected in NFF enterococci, and while it is not always clear if they result in phenotypic expression in these species, NFF enterococci may act as a reservoir for these genes. Historically, NFF enterococci have been overlooked, in part, due to the inability to characterize them in diagnostic microbiology laboratories. However, newer methods for identification, such as matrix-assisted laser desorption ionization–time of flight mass spectrometry, allow for classification at a species level. Consequently, the clinical significance and prevalence of previously unreported NFF enterococci species may alter in the future.

Biographies

Christopher A. Mullally is an early-career researcher at the School of Medical, Molecular, and Forensic Sciences at Murdoch University. He completed his PhD in Microbiology at the University of Western Australia in 2020. He currently holds two positions at Murdoch University, in the Neonatal Infection and Immunity group, and the Australian Group for Antimicrobial Resistance. His research focuses on sepsis pathogens with a special interest in diagnosis, prevention, and antimicrobial resistance.

Marhami Fahriani is a PhD candidate at the School of Medical, Molecular and Forensic Science, at Murdoch University. She received her medical degree at Universitas Syiah Kuala, Aceh, Indonesia and her Masters of Infectious Diseases at the School of Biomedical Science at The University of Western Australia, Perth, Australia. She is currently working on her doctoral thesis focusing on non-faecium non-faecalis enterococcal bloodstream infections.

Shakeel Mowlaboccus, Ph.D. is an early-career research scientist in the field of infectious diseases and antimicrobial resistance with over ten years of experience in microbiology, large-scale whole genome sequencing, bioinformatics analyses, molecular cloning, and protein expression. He currently works at Murdoch University in Western Australia where he specialises in understanding the molecular epidemiology and evolution of bacterial pathogens through state-of-the-art genomics. His research focuses on understanding the molecular mechanisms and the evolution of antimicrobial resistance in bacterial pathogens of public health concern including methicillin-resistant S. aureus, Enterococcus faecium, and Neisseria gonorrhoeae. Dr. Mowlaboccus is the curator of the Enterococcus faecium and Enterococcus faecalis sequence definition databases on the PubMLST website. He is also the current ASM International Young Ambassador representing Australia.

Geoffrey W. Coombs is the Chair of Public Health at the School of Veterinary and Life Sciences, Murdoch University and the Senior Clinical Scientist for PathWest Laboratory Medicine – WA, Fiona Stanley Hospital. At Murdoch University, he and his colleagues have established a three-million-dollar one health antimicrobial and infectious diseases research laboratory, which enables his group to perform basic and clinical antimicrobial resistance research on human and animal isolates. The laboratory has become the reference laboratory for the Australian Group on Antimicrobial Resistance (AGAR) Australian S. aureus (ASSOP) and Enterococcus (AESOP) Sepsis Onset Programs. During his professional career, he has received several awards including a Distinguished Service Award from the Australian Society for Microbiology (2016), a Royal College of Pathology of Australasia Faculty of Science Fellowship (2010), and a Chancellor’s Letter of Commendation for his PhD (2013).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/cmr.00121-23.

Table S1. cmr.00121-23-s0001.docx.

List of species in the Enterococcus genus.

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