Cytochrome oxidase requirements in Bordetella reveal insights into evolution towards life in the mammalian respiratory tract.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis, the causal agent of human whooping cough, and B. bronchiseptica, which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica, which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis, which evolved from a B. bronchiseptica-like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1, and cyoABCD1. To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo. No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis-conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens.

Transient reemergence of Bordetella parapertussis in France in 2022.

Between March and October 2022, a peak of detection of Bordetella parapertussis by qPCR, real-time PCR was observed in France.Hypothesis/Gap Statement. Whether this peak was due to resurgence from previous circulating lineages or reintroduction into the country was unknown.Objective. The objective of this study is to understand B. parapertussis-transient increase observed in France in 2022 whereas it had virtually stopped being reported since the start of the COVID-19 pandemic in 2020.Methods. We analysed real-time PCR (qPCR) data from the two largest French outpatient laboratories performing whooping cough diagnosis and characterized all B. parapertussis isolates collected in the 2016-2022 period by the French National Reference Centre for Whooping Cough.Results. Microbiological analyses reveal that 13 of 18 bacterial isolates collected in 2022 produce the vaccine antigen pertactin, whereas none of the 22 isolates collected in the 2016-2021 period did.Conclusion. We hypothesize a re-introduction of B. parapertussis from regions of the world where whole-cell vaccines are still in use.

Uncovering the Unseen: Bordetella hinzii Emerges in a Lung Transplant Recipient.

Bordetella hinzii (B. hinzii), a Gram-negative bacillus commonly associated with respiratory infections in animals, has garnered attention for its sporadic cases in humans, particularly in immunocompromised individuals. Despite its opportunistic nature, there remains limited understanding regarding its pathogenicity, diagnostic challenges, and optimal treatment strategies, especially in the context of immunosuppression. Herein, we present the first documented case of acute bronchitis caused by B. hinzii in an immunocompromised patient following double-lung transplantation. The patient, a former smoker with sarcoidosis stage IV, underwent transplant surgery and subsequently developed a febrile episode, leading to the identification of B. hinzii in broncho-alveolar lavage samples. Antimicrobial susceptibility testing revealed resistance to multiple antibiotics, necessitating tailored treatment adjustments. Our case underscores the importance of heightened awareness among clinicians regarding B. hinzii infections and the imperative for further research to elucidate its epidemiology and optimal management strategies, particularly in immunocompromised populations.

Evaluation of the ELITe InGenius and Bordetella ELITe MGB Kit for the detection and identification of B. pertussis, B. parapertussis and B. holmesii.

Bordetella pertussis is the causative pathogen of whooping cough or pertussis, a contagious respiratory disease. Aside from serodiagnosis, laboratory confirmation of pertussis is done through PCR, as B. pertussis is difficult to culture. The ELITe InGenius instrument (ELITechGroup, France) with accompanying Bordetella ELITe MGB Kit was evaluated against a laboratory-developed assay. Both assays combine two screening (IS481, IS1001) and two confirmation targets (recA, ptxA-Pr or IS1002) for optimal sensitivity and specificity. The company's stated claims on sensitivity and reproducibility were confirmed. Accuracy testing showed full concordance between both assays for the screening targets. Minor discrepancies were seen for the B. pertussis confirmation target. Some cross-reactivity with other Bordetella species was observed for the IS481-target, however, none of these were confirmed in the ptxA-Pr target. These results show the suitability of the Bordetella ELITe MGB Kit for the detection and differentiation of B. pertussis, B. parapertussis and B. holmesii.

Comparative structural and immunological analysis of outer membrane proteins and dermonecrotic toxin in Bordetella bronchiseptica canine isolate.

Bordetella bronchiseptica is a pathogen causing respiratory infections in mammals. With the improving understanding of companion animals' welfare, addressing the side effects of bordetella vaccine gains importance in dogs. Studies on diverse subunit vaccines are actively pursued in humans to safely and effectively control bordetellosis. Therefore, our objective was to develop a canine bordetella vaccine inspired by human vaccine development. We evaluated the immunogenicity of the two bacterial components: the outer membrane proteins (OMPs) and the dermonecrotic toxin (DNT) from a canine isolate of B. bronchiseptica. In-silico analysis identified eight domains of DNT, and Domain 3 was selected as the most promising antigen candidate. Additionally, the OMPs were extracted and examined using SDS-PAGE and Western blot analysis. The distinct immunological characteristic of OMPs and DNT-3 were examined individually and in combination. Gene expression and cytokine production were also evaluated in DH82 cells after stimulation with those antigens. Treatment with OMPs resulted in higher level of Th1 related cytokines, while DNT-3 induced a predominant response associated with Th17 and Th2 in the cytokine production. Synergistic effects were observed exclusively on IL-23, indicating increase of a potential risk of side effects when OMPs and DNT act together. These findings provide valuable insights into the reactogenicity of conventional Bordetella vaccines. Further, the presented preclinical data in this study offer an alternative method of the development for an optimal next-generation Bordetella vaccine for companion animals and humans, replacing the acellular vaccines containing both toxin and protein components.

Reemergence of Bordetella parapertussis, United States, 2019-2023.

To determine changes in Bordetella pertussis and B. parapertussis detection rates, we analyzed 1.43 million respiratory multiplex PCR test results from US facilities from 2019 through mid-2023. From mid-2022 through mid-2023, Bordetella spp. detection increased 8.5-fold; 95% of detections were B. parapertussis. While B. parapertussis rates increased, B. pertussis rates decreased.

Effect of carbohydrates on the adhesion of Bordetella bronchiseptica to the respiratory epithelium in rabbits.

This study proposes an ecological approach for preventing respiratory tract infections caused by Bordetella bronchiseptica in mammals using a mixture of carbohydrates. In an in vivo study, 51-day-old New Zealand rabbits were treated with a solution containing 1 × 107 CFUs of B. bronchiseptica and 250 µg of one of the following carbohydrates: N acetylglucosamine (GlcNAc), N acetylgalactosamine (GalNAc), alpha methyl mannose (AmeMan), alpha methyl glucose (AmeGlc) and sialic acid (Neu5AC). Positive (B. bronchiseptica) and negative (Physiological Saline Solution (PSS)) controls were included. Animals treated with GlcNAc or AmeGlc showed no clinical signs of infection and exhibited a significant reduction (p < 0.05) in the severity of microscopic lesions evaluated in the nasal cavity and lung compared with the positive controls. Additionally, the presence of bacteria was not detected through microbiological isolation or PCR in the lungs of animals treated with these sugars. Use of a mixture of GlcNAc and AmeGlc resulted in greater inhibition of microscopic lesions, with a significant reduction (p < 0.05) in the severity of these lesions compared to the results obtained using individual sugars. Furthermore, the bacterium was not detected through microbiological isolation, Polymerase Chain Reaction (PCR) or indirect immunoperoxidase (IIP) in this group.

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.

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.

RpoN (sigma factor 54) contributes to bacterial fitness during tracheal colonization of Bordetella bronchiseptica.

The Gram-negative pathogenic bacterium Bordetella bronchiseptica is a respiratory pathogen closely related to Bordetella pertussis, the causative agent of whooping cough. Despite sharing homologous virulence factors, B. bronchiseptica infects a broad range of mammalian hosts, including some experimental animals, whereas B. pertussis is strictly adapted to humans. Therefore, B. bronchiseptica is often used as a representative model to explore the pathogenicity of Bordetella in infection experiments with laboratory animals. Although Bordetella virulence factors, including toxins and adhesins have been studied well, our recent study implied that unknown virulence factors are involved in tracheal colonization and infection. Here, we investigated bacterial genes contributing to tracheal colonization by high-throughput transposon sequencing (Tn-seq). After the screening, we picked up 151 candidate genes of various functions and found that a rpoN-deficient mutant strain was defective in tracheal colonization when co-inoculated with the wild-type strain. rpoN encodes σ54 , a sigma factor that regulates the transcription of various genes, implying its contribution to various bacterial activities. In fact, we found RpoN of B. bronchiseptica is involved in bacterial motility and initial biofilm formation. From these results, we propose that RpoN supports bacterial colonization by regulating various bacteriological functions.

Anti-Bordetella bronchiseptica effects of targeted bacteriophages via microbiome and metabolic mediated mechanisms.

Bordetella bronchiseptica poses a significant challenge in the context of respiratory infections, particularly in weanling pigs. In this study, we investigated the impact of a novel targeted bacteriophage in controlling B. bronchiseptica challenge (BBC) in an experimental design involving five distinct treatment groups: NC (no challenge), PC (BBC challenge), BF (108 pfu bacteriophage/kg diet + BBC), BN (2 × 107 pfu/day bacteriophage by nasal spray + BBC), and AT (antibiotic + BBC). The experiment was conducted for 2 weeks. The highest turbinate score was observed in the PC. The BF treatment showed higher plasma IL (interleukine)-1ß and IL-6 compared with the BN and AT treatments. Plasma concentrations of IL-1ß were increased in the BF pigs compared with the BN, AT, and NC. Among the BBC groups, the PC treatment exhibited a higher abundance of Staphylococcus. aureus and B. bronchiseptica in the lung. A lower S. aureus, Streptococcus. suis, and B. bronchiseptica colonization was detected in the AT compared with the BF and BN treatments. The BF showed lower plasma zonulin compared with the BN and AT. A higher plasma concentration of superoxide dismutase was observed in the BF and AT compared with PC and BN. The BN influenced the glycine, serine-threonine metabolism; glycerolipid metabolism; glyoxylate-dicarboxylate metabolism; and arachidonic acid metabolism compared with the NC. In conclusion, nasal-sprayed bacteriophage effectively controlled B. bronchiseptica infection, however, their efficiency was lower than the antibiotic.

Adaptive immune protection of the middle ears differs from that of the respiratory tract.

The efficacy of the adaptive immune system in the middle ear (ME) is well established, but the mechanisms are not as well defined as those of gastrointestinal or respiratory tracts. While cellular elements of the adaptive response have been detected in the MEs following infections (or intranasal immunizations), their specific contributions to protecting the organ against reinfections are unknown. How immune protection mechanisms of the MEs compares with those in the adjacent and attached upper and lower respiratory airways remains unclear. To address these knowledge gaps, we used an established mouse respiratory infection model that we recently showed also involves ME infections. Bordetella bronchiseptica delivered to the external nares of mice in tiny numbers very efficiently infects the respiratory tract and ascends the Eustachian tube to colonize and infect the MEs, where it causes severe but acute inflammation resembling human acute otitis media (AOM). Since this AOM naturally resolves, we here examine the immunological mechanisms that clear infection and protect against subsequent infection, to guide efforts to induce protective immunity in the ME. Our results show that once the MEs are cleared of a primary B. bronchiseptica infection, the convalescent organ is strongly protected from reinfection by the pathogen despite its persistence in the upper respiratory tract, suggesting important immunological differences in these adjacent and connected organs. CD4+ and CD8+ T cells trafficked to the MEs following infection and were necessary to robustly protect against secondary challenge. Intranasal vaccination with heat killed B. bronchiseptica conferred robust protection against infection to the MEs, even though the nasopharynx itself was only partially protected. These data establish the MEs as discrete effector sites of adaptive immunity and shows that effective protection in the MEs and the respiratory tract is significantly different. This model system allows the dissection of immunological mechanisms that can prevent bacteria in the nasopharynx from ascending the ET to colonize the ME.

Bordetella spp. block eosinophil recruitment to suppress the generation of early mucosal protection.

Bordetella spp. are respiratory pathogens equipped with immune evasion mechanisms. We previously characterized a Bordetella bronchiseptica mutant (RB50ΔbtrS) that fails to suppress host responses, leading to rapid clearance and long-lasting immunity against reinfection. This work revealed eosinophils as an exclusive requirement for RB50ΔbtrS clearance. We also show that RB50ΔbtrS promotes eosinophil-mediated B/T cell recruitment and inducible bronchus-associated lymphoid tissue (iBALT) formation, with eosinophils being present throughout iBALT for Th17 and immunoglobulin A (IgA) responses. Finally, we provide evidence that XCL1 is critical for iBALT formation but not maintenance, proposing a novel role for eosinophils as facilitators of adaptive immunity against B. bronchiseptica. RB50ΔbtrS being incapable of suppressing eosinophil effector functions illuminates active, bacterial targeting of eosinophils to achieve successful persistence and reinfection. Overall, our discoveries contribute to understanding cellular mechanisms for use in future vaccines and therapies against Bordetella spp. and extension to other mucosal pathogens.

Prior infection with Bordetella bronchiseptica enhanced colonization but not disease with Streptococcus suis.

Bordetella bronchiseptica and Streptococcus suis are widely distributed swine pathogens. B. bronchiseptica is a primary pathogen and causes atrophic rhinitis and bronchopneumonia. S. suis is a contributing agent to porcine respiratory disease complex and causes systemic diseases including arthritis, meningitis, polyserositis, and septicemia. Colonization with B. bronchiseptica has been associated with increased colonization by other pathogenic bacteria and increased disease severity with viral and bacterial pathogens. It has also been reported to predispose cesarean derived, colostrum deprived (CDCD) piglets to S. suis systemic disease. Here, we evaluated the role of B. bronchiseptica colonization on S. suis colonization, dissemination, and disease in one study using conventional pigs and another using CDCD pigs. Pigs were challenged with S. suis, B. bronchiseptica, or B. bronchiseptica followed by S. suis. Incidence of S. suis disease was not increased in either study for animals pre-inoculated with B. bronchiseptica. Nasal colonization with S. suis was increased in coinfected animals, while B. bronchiseptica was similar between mono- and co-infected animals. Although increased S. suis disease was not seen in coinfected pigs, there is evidence that B. bronchiseptica can increase colonization with S. suis, which may contribute to enhanced disease when animals are stressed or immunocompromised.

Case report: Plastic bronchitis associated with Bordetella parapertussis.

RATIONALE: Bordetella parapertussis caused by a severe infection is rare in clinical practice. Here, we report a case of plastic bronchitis (PB). PATIENT CONCERNS: A 4-year-old girl with a 2-day history of fever, paroxysmal cough, and subconjunctival hemorrhage. DIAGNOSES: The diagnoses were (1) B parapertussis , (2) pulmonary atelectasis, and (3) PB. INTERVENTIONS: The patient received azithromycin and underwent bronchoscopy. OUTCOMES: Symptoms disappeared after treatment. The patient had an outpatient follow-up of 2 months without respiratory symptoms. LESSONS: PB can lead to respiratory failure if not intervened in the early stages.

Bordetella bronchiseptica and Bordetella pertussis: Similarities and Differences in Infection, Immuno-Modulation, and Vaccine Considerations.

Bordetella pertussis and Bordetella bronchiseptica belong to the genus Bordetella, which comprises 14 other species. B. pertussis is responsible for whooping cough in humans, a severe infection in children and less severe or chronic in adults. These infections are restricted to humans and currently increasing worldwide. B. bronchiseptica is involved in diverse respiratory infections in a wide range of mammals. For instance, the canine infectious respiratory disease complex (CIRDC), characterized by a chronic cough in dogs. At the same time, it is increasingly implicated in human infections, while remaining an important pathogen in the veterinary field. Both Bordetella can evade and modulate host immune responses to support their persistence, although it is more pronounced in B. bronchiseptica infection. The protective immune responses elicited by both pathogens are comparable, while there are important characteristics in the mechanisms that differ. However, B. pertussis pathogenesis is more difficult to decipher in animal models than those of B. bronchiseptica because of its restriction to humans. Nevertheless, the licensed vaccines for each Bordetella are different in terms of formulation, route of administration and immune responses induced, with no known cross-reaction between them. Moreover, the target of the mucosal tissues and the induction of long-lasting cellular and humoral responses are required to control and eliminate Bordetella. In addition, the interaction between both veterinary and human fields are essential for the control of this genus, by preventing the infections in animals and the subsequent zoonotic transmission to humans.

Adjuvant Efficacy of the ECMS-Oil on Immune Responses against Bordetella bronchiseptica in Mice through the TLR2/MyD88/NF-κB Pathway.

Bordetella infection can be efficiently prevented through vaccination. The current study investigated the effects of an extract of Cochinchina momordica seed (ECMS) combined with oil on the immune responses to the inactivated Bordetella vaccine in mice. Serum IgG and IgG1 level was significantly increased in ECMS-oil group compared to any other group (P < 0.05) 2 weeks after immunization, while groups ECMS200 µg/400 µg-oil had a markedly higher level of serum IgG2b and IgG3 than any other groups (P < 0.05). Moreover, lipopolysaccharide/ConA-stimulated proliferation of splenocytes was significantly enhanced in ECMS 400 µg-oil immunized mice in comparison with mice in any other group (P < 0.05). RT-PCR assay revealed that while ECMS800 µg-oil group had significantly higher levels of serum IL-4, IL-10, Toll-like receptor (TLR)2, and IL-1 beta than any other group (P < 0.05), the levels of serum IL-2, IL-4, and IL-10 were markedly increased in ECMS 400 µg-oil group as compared to any other groups (P < 0.05). Blood analysis showed that ECMS800 µg-oil and oil groups had a significantly higher number of immunocytes than any other groups (P < 0.05). There were significant differences in the number of IgG+, IgG2b+, and IgA+ cells in the lung between ECMS800 µg-oil group and any other groups (P < 0.05). Western blot analysis demonstrated that stimulation with ECMS 25 µg/mL or 50 ng/mL led to a significant increase in the expression of TLR2, MyD88, and NF-κB in Raw264.7 cells (P < 0.05). Compared with any other group, the expression of MyD88 was markedly increased in the cells stimulated with ECMS 50 ng/mL, as indicated by the RT-PCR analysis (P < 0.05). Overall, we observed that ECMS-oil efficiently enhanced the humoral or cellular immune responses against Bordetella and suggested that the mechanism of adjuvant activity of ECMS-oil might involve TLR2/MyD88/NF-κB signaling pathway.

Characterization of Temperate LPS-Binding Bordetella avium Phages That Lack Superinfection Immunity.

Bordetella avium causes a highly infectious upper respiratory tract disease in turkeys and other poultry with high economic losses. Considering the antimicrobial resistance crisis, bacteriophages (phages) may be an alternative approach for treating bacterial infections such as bordetellosis. Here, we describe seven B. avium phages, isolated from drinking water and feces from chicken and turkey farms. They showed strong bacteriolytic activity with a broad host range and used lipopolysaccharides (LPS) as a host receptor for their adsorption. All phages are myoviruses based on their structure observed by transmission electron microscopy. Genome sequence analyses revealed genome assembly sizes ranging from 39,087 to 43,144 bp. Their permutated genomes were organized colinearly, with a conserved module order, and were packed according to a predicted headful packing strategy. Notably, they contained genes encoding putative markers of lysogeny, indicative of temperate phages, despite their lytic phenotype. Further investigation revealed that the phages could indeed undergo a lysogenic life cycle with varying frequency. However, the lysogenic bacteria were still susceptible to superinfection with the same phages. This lack of stable superinfection immunity after lysogenization appears to be a characteristic feature of B. avium phages, which is favorable in terms of a potential therapeutic use of phages for the treatment of avian bordetellosis. IMPORTANCE To maintain the effectiveness of antibiotics over the long term, alternatives to treat infectious diseases are urgently needed. Therefore, phages have recently come back into focus as they can specifically infect and lyse bacteria and are naturally occurring. However, there is little information on phages that can infect pathogenic bacteria from animals, such as the causative agent of bordetellosis of poultry, B. avium. Therefore, in this study, B. avium phages were isolated and comprehensively characterized, including whole-genome analysis. Although phenotypically the phages were thought to undergo a lytic cycle, we demonstrated that they undergo a lysogenic phase, but that infection does not confer stable host superinfection immunity. These findings provide important information that could be relevant for potential biocontrol of avian bordetellosis by using phage therapy.