Non-classical Bordetella sp. (closely related to Bordetella hinzii and Bordetella pseudohinzii) lower respiratory tract infection in a patient with extensive bronchiectasis: a case report

Abstract

An increasing number of reports have described the pathogenic nature of several non-classical Bordetella spp. Among them, Bordetella hinzii and Bordetella pseudohinzii have been implicated in a myriad of respiratory-associated infections in humans and animals. We report the isolation of a genetically close relative of B. hinzii and B. pseudohinzii from the sputum of a woman in her early 60s with extensive bronchiectasis who presented with fever and brown colored sputum. The isolate had initially been identified as Bordetella avium by API 20NE, the identification system for non-enteric Gram-negative rod bacteria. Sequencing of the 16S rDNA, ompA, nrdA, and genes used in the Bordetella multilocus sequence typing scheme could not resolve the identity of this Bordetella isolate. Whole-genome single nucleotide polymorphism analysis positioned the isolate between B. hinzii and B. pseudohinzii in the phylogenetic tree, forming a distinct cluster. Whole-genome sequencing enabled the further identification of this rare organism, and should be considered for wider applications, especially the confirmation of organism identity in the clinical diagnostic microbiology laboratory.

Introduction

The classical bordetellae, namely Bordetella pertussis, B. parapertussis, and B. bronchiseptica are well researched respiratory pathogens affecting animals and humans. ¹ Non-classical bordetellae have recently begun to receive attention because of their ability to cause a number of diseases including arthritis, endocarditis, mandibular osteomyelitis, mastoiditis, and meningitis.^1,2 Indeed, the recently characterized non-classical bordetellae B. pseudohinzii, predominantly found in laboratory rodents, was found to cause respiratory disease ranging from asymptomatic rhinitis to clinical bronchopneumonia. ³ Incidentally, a genetically close relative, B. hinzii, which has been implicated in cases of human pulmonary disease, was suggested to have originated from birds. ⁴ Herein, we report a patient with extensive bronchiectasis infected with a non-classical bordetellae. Based on whole-genome single nucleotide polymorphism (SNP) analysis, the isolated Bordetella sp. was found to be closely related to B. hinzii and B. pseudohinzii.

Case report

A Gram-negative bacillus was repeatedly isolated on blood agar from two sputum samples taken 20 days apart from a woman in her early 60s with underlying extensive bronchiectasis who presented with fever and brown colored sputum. She had undergone middle lobectomy 11 years earlier for recurrent hemoptysis resulting from bronchiectasis. She did not have diabetes mellitus and was not immunocompromised. About 3.5 months earlier, and again a month earlier, she had taken ciprofloxacin for a week for recurrent fever and blood-stained purulent sputum.

In the current presentation, the patient was initially empirically treated with moxifloxacin for 10 days. Although there was a reduction in the purulence of her sputum, she was admitted 20 days later for a 20-day course of intravenous imipenem as she had been having intermittent hemoptysis over the past 3.5 months occasionally with fever and purulent sputum and had not fully recovered with previous courses of ciprofloxacin and the recent course of moxifloxacin. Furthermore, a high-resolution computed tomography scan of her thorax this time showed an area of consolidation in the right upper lobe in addition to extensive bronchiectasis involving the right upper lobe, the lingular segment of the left upper lobe, and both lower lobes. During the course of imipenem treatment, her fever subsided, and her sputum volume and purulence decreased. On discharge, she was told to complete another seven days of trimethoprim–sulfamethoxazole. However, she was readmitted a week later to another hospital for fever and hyponatremia attributed to syndrome of inappropriate antidiuretic hormone (SIADH) secretion caused by the incomplete resolution of her lung infection. She was treated with a further 7-day course of imipenem, which resolved her fever and hyponatremia.

The first sputum sample obtained for culture prior to the commencement of moxifloxacin was sent to the diagnostic medical microbiology laboratory of the hospital, and revealed >25 polymorphonuclear leukocytes and five epithelial cells per low power field (lpf) on microscopy. Bacterial culture grew moderate growth of multidrug-resistant Acinetobacter baumanii, which was sensitive only to amikacin, netilmicin, and colistin and had an uncertain clinical significance, and a Gram-negative bacillus (isolate BH371) that was initially identified by the API 20NE system (bioMérieux, Marcy-l'Étoile, France) as Bordetella avium (API profile: 0000067, which the API software identified with 96.6% identity [good identification]). This Gram-negative, oxidase-positive bacillus grew within 24 hours of incubation at 35°C on blood and chocolate agars incubated in 5% CO₂, and on MacConkey agar incubated aerobically. Increased carbon dioxide incubation did not improve the growth on blood or chocolate agar. On blood agar incubated in O₂, the colonies were whitish in color with a flat rough appearance; after 48 hours of incubation, they were larger and rough with irregular edges, with flat spreading edges in some areas. On blood agar incubated in 5% CO₂, the colonies had no spreading edges. On MacConkey agar without crystal violet (in O₂ incubation), they appeared slightly pinkish after 24 hours but were slightly pale and rough with irregular edges at 48 hours, with no obvious flat spreading edges. When later subcultured onto MacConkey agar with crystal violet, they appeared as non-lactose fermenting colonies with some areas of flat spreading edges after 48 hours of incubation. The bacterium was also able to grow on nutrient agar left at room temperature, although not as well as on nutrient agar incubated aerobically at 35°C. Using Etest (bioMérieux) for minimum inhibitory concentration (MIC) determination and following the Clinical and Laboratory Standards Institute interpretive standards for ‘Other non-Enterobacteriaceae’, ⁵ it was found to be sensitive to ceftazidime (MIC: 3 µg/mL), ciprofloxacin (MIC: 0.75 µg/mL), trimethoprim–sulfamethoxazole (MIC: 0.19 µg/mL), piperacillin–tazobactam (MIC: 1 µg/mL), and imipenem (MIC: 0.75 µg/mL). The isolate was sent for 16S rDNA PCR and sequencing.

The second sputum sample taken 20 days after the first, and just prior to the commencement of imipenem, revealed 15 polymorphonuclear leukocytes and no squamous epithelial cells per lpf on microscopy. Bacterial culture grew moderate growth of a Gram-negative bacillus with identical phenotypic, antimicrobial susceptibility, and API 20NE identification profiles as that of the organism isolated from the first sputum sample (B. avium). From 16S rDNA sequencing of the first isolate (BH371), it was identified as either B. avium or B. hinzii (accession no. LN868267). To confirm the identification, ompA (accession no. LN868268) sequencing was performed in the research laboratory. However, 16S rDNA and ompA sequencing were insufficient to resolve the species identities of the non-classical Bordetella isolates, ⁶ and although analysis of the trimmed nrdA sequences ⁷ resulted in a novel locus 217, its species identity remained unresolved (https://bigsdb.pasteur.fr/bordetella/). The multilocus sequence typing scheme for Bordetella ⁸ could not amplify gene fragments for analysis. Moreover, amplification of the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas) locus, a unique characteristic of B. pseudohinzii ⁹ , was unsuccessful.

Transmission electron microscopy revealed that BH371 possessed rod-like structures (Figure 1a) and fimbriae (Figure 1b) similar to B. pseudohinzii. ¹⁰ Whole-genome sequencing performed on Bordetella sp. BH371 resulted in a draft genome 4,880,027 bp in length and composed of 619 contigs with an N₅₀ of 13,907 bp. The G+C content of the genome sequence was approximately 67.2%, and a total of 5,695 protein-coding genes and 60 RNAs were predicted. The presence of genetic components that make up the type I secretion system were identified in the draft genome sequences. However, the locus encoding the adenylate cyclase toxin was not found. The BH371 draft genome sequence was then compared against 13 bordetellae complete genomes for SNP discovery using the reference sequence alignment-based phylogeny builder, with B. pseudohinzii HI4681 (accession number NZ_CP016440.1) as the reference genome.

Figure 1.

Transmission electron microscopy of Bordetella sp. BH371. (a) Longitudinal section of Bordetella sp. BH371 and (b) Cross section of Bordetella sp. BH371. Arrows indicates fimbriae.

The entire genome-based SNP phylogenetic tree was reconstructed using PhyML 3.0 ¹¹ using the general time-reversible model coupled with the addition of invariant sites and a gamma distribution of rates across sites (GTR +G+I). The significance of the phylogenetic tree was accessed using the bootstrap method (1000 replicates). Bordetella sp. BH371 was positioned in between B. hinzii and B. pseudohinzii, suggesting that all three bordetellae are closely related (Figure 2). The draft genome sequence of Bordetella sp. BH371 is accessible from the European Nucleotide Archive (https://www.ebi.ac.uk/ena/browser/view/PRJEB17918) under genome assembly accession number PRJEB17918. A comparison of median genome lengths based on GenBank information revealed that B. pseudohinzii has a smaller genome (∼4.5 Mb) relative to B. hinzii and Bordetella sp. BH371 (∼4.9 Mb) (data not shown).

Figure 2.

Whole-genome SNP phylogenetic tree of selected bordetellae. The significance of the phylogenetic tree was accessed using the bootstrap test (1000 replicates). Bordetella sp. BH371 is shown in bold. A complex arrangement profile is observed, reflecting the vast fragmentations of the draft genome of Bordetella sp. BH371 with 619 contigs.

A timeline of events summarizing the clinical symptoms, pathogen isolation, and antibiotic treatments for the patient is presented in Figure 3. The patient’s verbal consent was obtained for their treatment, laboratory tests, and for the publication of this study during a clinic visit and documented in her case notes. This study received approval from the Medical Research Ethics Committee, University Malaya Medical Centre (MREC ID No. 2021108-10675). All details of the patient have been de-identified. The reporting of this study conforms to the CARE guidelines. ¹²

Figure 3.

Timeline of events summarizing the clinical symptoms, pathogen isolation, and antibiotic treatments for our patient.

Discussion

Bronchiectasis is characterized by dilatation of the bronchi and lung inflammation associated with persistent bacterial colonization of the diseased airways. ¹³ The repeated isolation of Bordetella species in the sputum of this patient together with her clinical presentation and sputum microscopy suggest that it has a pathogenic role in causing infection in this case. Although a multidrug-resistant Acinetobacter spp. was co-isolated with this organism in the first sputum sample, its clinical importance was uncertain and targeted antimicrobial therapy against it was not given. The subsequent sputum specimen did not isolate this organism again, which suggests it might have been a transient colonizer possibly from the upper respiratory tract or oral cavity.

Most non-classical bordetellae are not included in the databases of commercial phenotypic identification systems such as the API 20NE;^14,15 hence they cannot be relied on for the identification of Bordetella sp. BH371. Multilocus sequence typing would have been successful in this instance if isolate BH371 was B. avium, but comparison of the trimmed nrdA sequences with the PubMLST database was unable to resolve the species identity. The failure to detect the CRISPR–Cas locus, a distinguishing feature of B. pseudohinzii,^9,16 further confounded the identification of isolate BH371. Accurate identification therefore required the more discriminative whole-genome SNP phylogenetic analysis. ¹⁷ This method, which was previously shown to be effective in discriminating between different B. melitensis strains, ¹⁷ placed Bordetella sp. BH371 in between B. hinzii and B. pseudohinzii in the resulting phylogenetic tree. This suggests that Bordetella sp. BH371 could be a separate species; however, confirmation of this requires further biochemical and genetic analyses.

Despite initial empirical treatment of our patient with moxifloxacin, the organism was isolated again in the second sputum specimen, and she required a further 20-day course of imipenem followed by trimethoprim–sulfamethoxazole for 7 days, then another course of imipenem a week later. The chronic nature of bordetellae infections could be explained by the ability of many invasive Bordetella spp. to produce biofilm and infiltrating inflammatory cells. ³ Additionally, the fimbriae of Bordetella sp. BH371 (Figure 1b) could facilitate attachment to ciliated cells in the respiratory tract and subsequent colonization of the respiratory system. ³ Future study could investigate the specific role of fimbriae, and determine if the isolate had potential for biofilm production to better understand its pathogenicity mechanism.

In a previous report, exposure to poultry was the suspected cause of a B. hinzii infection. ⁴ Although other animals such as rabbits and rodents could also be potential reservoirs, recent studies have shown that the genus Bordetella evolved from environmental microbe ancestors. ¹⁸ Its adaptation to specific hosts has invoked a reduction in genome size with the loss of many genes involved in survival outside of the host. ¹⁸ Despite its smaller genome size, B. pseudohinzii was previously shown to be significantly more efficient at mouse lung colonization than B. hinzii, implying that both bordetellae have different hosts and occupy different ecological niches. ¹⁰ This case report highlights the involvement of another non-classical Bordetella sp. in human respiratory illness, and shows the importance of whole-genome sequence analysis in facilitating its accurate identification.