Clinical Evaluation of the iCubate iC-GPC Assay for Detection of Gram-Positive Bacteria and Resistance Markers from Positive Blood Cultures.

The iC-GPC Assay (iCubate, Huntsville, AL) is a qualitative multiplex test for the detection of five of the most common Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faecalis, and Enterococcus faecium) responsible for bacterial bloodstream infections, performed directly from positive blood cultures. The assay also detects the presence of the mecA, vanA, and vanB resistance determinants. This study comparatively evaluated the performance of the iC-GPC Assay against the Verigene Gram-positive blood culture (BC-GP) assay (Luminex Corp., Austin, TX) for 1,134 patient blood culture specimens positive for Gram-positive cocci. The iC-GPC Assay had an overall percent agreement with the BC-GP assay of 95.5%. Discordant specimens were further analyzed by PCR and a bidirectional sequencing method. The results indicate that the iC-GPC Assay together with the iCubate system is an accurate and reliable tool for the detection of the five most common Gram-positive bacteria and their resistance markers responsible for bloodstream infections.

Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection.

Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone-usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29ß-NAc, is a biphenyl N-acetyl-ß-galactosaminoside with a Ki of ∼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate-lectin interactions. 29ß-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29ß-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29ß-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29ß-NAc-inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.

The Transcriptional Regulator BpsR Controls the Growth of Bordetella bronchiseptica by Repressing Genes Involved in Nicotinic Acid Degradation.

Many of the pathogenic species of the genus Bordetella have an absolute requirement for nicotinic acid (NA) for laboratory growth. These Gram-negative bacteria also harbor a gene cluster homologous to the nic cluster of Pseudomonas putida which is involved in the aerobic degradation of NA and its transcriptional control. We report here that BpsR, a negative regulator of biofilm formation and Bps polysaccharide production, controls the growth of Bordetella bronchiseptica by repressing the expression of nic genes. The severe growth defect of the ΔbpsR strain in Stainer-Scholte medium was restored by supplementation with NA, which also functioned as an inducer of nic genes at low micromolar concentrations that are usually present in animals and humans. Purified BpsR protein bound to the nic promoter region, and its DNA binding activity was inhibited by 6-hydroxynicotinic acid (6-HNA), the first metabolite of the NA degradative pathway. Reporter assays with the isogenic mutant derivative of the wild-type (WT) strain harboring deletion in nicA, which encodes a putative nicotinic acid hydroxylase responsible for conversion of NA to 6-HNA, showed that 6-HNA is the actual inducer of the nic genes in the bacterial cell. Gene expression profiling further showed that BpsR dually activated and repressed the expression of genes associated with pathogenesis, transcriptional regulation, metabolism, and other cellular processes. We discuss the implications of these findings with respect to the selection of pyridines such as NA and quinolinic acid for optimum bacterial growth depending on the ecological niche.IMPORTANCE BpsR, the previously described regulator of biofilm formation and Bps polysaccharide production, controls Bordetella bronchiseptica growth by regulating the expression of genes involved in the degradation of nicotinic acid (NA). 6-Hydroxynicotinic acid (6-HNA), the first metabolite of the NA degradation pathway prevented BpsR from binding to DNA and was the actual in vivo inducer. We hypothesize that BpsR enables Bordetella bacteria to efficiently and selectively utilize NA for their survival depending on the environment in which they reside. The results reported herein lay the foundation for future investigations of how BpsR and the alteration of its activity by NA orchestrate the control of Bordetella growth, metabolism, biofilm formation, and pathogenesis.

Functional role of the type 1 pilus rod structure in mediating host-pathogen interactions.

Uropathogenic E. coli (UPEC), which cause urinary tract infections (UTI), utilize type 1 pili, a chaperone usher pathway (CUP) pilus, to cause UTI and colonize the gut. The pilus rod, comprised of repeating FimA subunits, provides a structural scaffold for displaying the tip adhesin, FimH. We solved the 4.2 Å resolution structure of the type 1 pilus rod using cryo-electron microscopy. Residues forming the interactive surfaces that determine the mechanical properties of the rod were maintained by selection based on a global alignment of fimA sequences. We identified mutations that did not alter pilus production in vitro but reduced the force required to unwind the rod. UPEC expressing these mutant pili were significantly attenuated in bladder infection and intestinal colonization in mice. This study elucidates an unappreciated functional role for the molecular spring-like property of type 1 pilus rods in host-pathogen interactions and carries important implications for other pilus-mediated diseases.

Bacterial virulence phenotypes of Escherichia coli and host susceptibility determine risk for urinary tract infections.

Urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC) strains. In contrast to many enteric E. coli pathogroups, no genetic signature has been identified for UPEC strains. We conducted a high-resolution comparative genomic study using E. coli isolates collected from the urine of women suffering from frequent recurrent UTIs. These isolates were genetically diverse and varied in their urovirulence, that is, their ability to infect the bladder in a mouse model of cystitis. We found no set of genes, including previously defined putative urovirulence factors (PUFs), that were predictive of urovirulence. In addition, in some patients, the E. coli strain causing a recurrent UTI had fewer PUFs than the supplanted strain. In competitive experimental infections in mice, the supplanting strain was more efficient at colonizing the mouse bladder than the supplanted strain. Despite the lack of a clear genomic signature for urovirulence, comparative transcriptomic and phenotypic analyses revealed that the expression of key conserved functions during culture, such as motility and metabolism, could be used to predict subsequent colonization of the mouse bladder. Together, our findings suggest that UTI risk and outcome may be determined by complex interactions between host susceptibility and the urovirulence potential of diverse bacterial strains.

Inflammation-Induced Adhesin-Receptor Interaction Provides a Fitness Advantage to Uropathogenic E. coli during Chronic Infection.

Uropathogenic E. coli (UPEC) is the dominant cause of urinary tract infections, clinically described as cystitis. UPEC express CUP pili, which are extracellular fibers tipped with adhesins that bind mucosal surfaces of the urinary tract. Here we identify the role of the F9/Yde/Fml pilus for UPEC persistence in the inflamed urothelium. The Fml adhesin FmlH binds galactose ß1-3 N-acetylgalactosamine found in core-1 and -2 O-glycans. Deletion of fmlH had no effect on UPEC virulence in an acute mouse model of cystitis. However, FmlH provided a fitness advantage during chronic cystitis, which is manifested as persistent bacteriuria, high bladder bacterial burdens, and chronic inflammation. In situ binding confirmed that FmlH bound avidly to the inflamed, but not the naive bladder. In accordance with its pathogenic profile, vaccination with FmlH significantly protected mice from chronic cystitis. Thus, UPEC employ separate CUP pili to adapt to the rapidly changing niche during bladder infection.

Metabolic Requirements of Escherichia coli in Intracellular Bacterial Communities during Urinary Tract Infection Pathogenesis.

UNLABELLED: Uropathogenic Escherichia coli (UPEC) is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs). Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs) of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly, lacZ was highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZ strain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, a galK mutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated gene yeaR revealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies. IMPORTANCE: Urinary tract infections (UTIs) are one of the most common bacterial infections, impacting mostly women. Every year, millions of UTIs occur in the U.S. with most being caused by uropathogenic E. coli(UPEC). During a UTI, UPEC invade bladder cells and form an intracellular bacterial community (IBC) that allows for the bacteria to replicate protected from the host immune response. In this study, we investigated genes that are expressed by UPEC within the IBC and determined how they contribute to the formation of this specialized community. Our findings suggest that galactose is important for UPEC growth in the IBC. Additionally, we found that a gene involved in oxidative stress is also important in the regulation of a key factor needed for UPEC invasion of bladder cells. These results may open the door for the development of treatments to diminish UTI frequency and/or severity.

The unexplored relationship between urinary tract infections and the autonomic nervous system.

Urinary tract infections (UTIs), the majority of which are caused by uropathogenic E. coli (UPEC), are extremely common infections that preferentially effect women. Additional complicating factors, such as catheterization, diabetes, and spinal cord injuries can increase the frequency and severity of UTIs. The rise of antimicrobial resistant uropathogens and the ability of this disease to chronically recur make the development of alternative preventative and therapeutic modalities a priority. The major symptoms of UTIs, urgency, frequency, and dysuria, are readouts of the autonomic nervous system (ANS) and the majority of the factors that lead to complicated UTIs have been shown to impact ANS function. This review summarizes the decades' long efforts to understand the molecular mechanisms of the interactions between UPEC and the host, with a particular focus on the recent findings revealing the molecular, bacteriological, immunological and epidemiological complexity of pathogenesis. Additionally, we describe the progress that has been made in: i) generating vaccines and anti-virulence compounds that prevent and/or treat UTI by blocking bacterial adherence to urinary tract tissue and; and ii) elucidating the mechanism by which anti-inflammatories are able to alleviate symptoms and improve disease prognosis. Finally, the potential relationships between the ANS and UTI are considered throughout. While these relationships have not been experimentally explored, the known interactions between numerous UTI characteristics (symptoms, complicating factors, and inflammation) and ANS function suggest that UTIs are directly impacting ANS stimulation and that ANS (dys)function may alter UTI prognosis.

Establishment and Characterization of UTI and CAUTI in a Mouse Model.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

Uropathogenic Escherichia coli superinfection enhances the severity of mouse bladder infection.

Urinary tract infections (UTIs) afflict over 9 million women in America every year, often necessitating long-term prophylactic antibiotics. One risk factor for UTI is frequent sexual intercourse, which dramatically increases the risk of UTI. The mechanism behind this increased risk is unknown; however, bacteriuria increases immediately after sexual intercourse episodes, suggesting that physical manipulation introduces periurethral flora into the urinary tract. In this paper, we investigated whether superinfection (repeat introduction of bacteria) resulted in increased risk of severe UTI, manifesting as persistent bacteriuria, high titer bladder bacterial burdens and chronic inflammation, an outcome referred to as chronic cystitis. Chronic cystitis represents unchecked luminal bacterial replication and is defined histologically by urothelial hyperplasia and submucosal lymphoid aggregates, a histological pattern similar to that seen in humans suffering chronic UTI. C57BL/6J mice are resistant to chronic cystitis after a single infection; however, they developed persistent bacteriuria and chronic cystitis when superinfected 24 hours apart. Elevated levels of interleukin-6 (IL-6), keratinocyte cytokine (KC/CXCL1), and granulocyte colony-stimulating factor (G-CSF) in the serum of C57BL/6J mice prior to the second infection predicted the development of chronic cystitis. These same cytokines have been found to precede chronic cystitis in singly infected C3H/HeN mice. Furthermore, inoculating C3H/HeN mice twice within a six-hour period doubled the proportion of mice that developed chronic cystitis. Intracellular bacterial replication, regulated hemolysin (HlyA) expression, and caspase 1/11 activation were essential for this increase. Microarrays conducted at four weeks post inoculation in both mouse strains revealed upregulation of IL-1 and antimicrobial peptides during chronic cystitis. These data suggest a mechanism by which caspase-1/11 activation and IL-1 secretion could predispose certain women to recurrent UTI after frequent intercourse, a predisposition predictable by several serum biomarkers in two murine models.

Pilicide ec240 disrupts virulence circuits in uropathogenic Escherichia coli.

UNLABELLED: Chaperone-usher pathway (CUP) pili are extracellular organelles produced by Gram-negative bacteria that mediate bacterial pathogenesis. Small-molecule inhibitors of CUP pili, termed pilicides, were rationally designed and shown to inhibit type 1 or P piliation. Here, we show that pilicide ec240 decreased the levels of type 1, P, and S piliation. Transcriptomic and proteomic analyses using the cystitis isolate UTI89 revealed that ec240 dysregulated CUP pili and decreased motility. Paradoxically, the transcript levels of P and S pilus genes were increased during growth in ec240, even though the level of P and S piliation decreased. In contrast, the most downregulated transcripts after growth in ec240 were from the type 1 pilus genes. Type 1 pilus expression is controlled by inversion of the fimS promoter element, which can oscillate between phase on and phase off orientations. ec240 induced the fimS phase off orientation, and this effect was necessary for the majority of ec240's inhibition of type 1 piliation. ec240 increased levels of the transcriptional regulators SfaB and PapB, which were shown to induce the fimS promoter phase off orientation. Furthermore, the effect of ec240 on motility was abolished in the absence of the SfaB, PapB, SfaX, and PapX regulators. In contrast to the effects of ec240, deletion of the type 1 pilus operon led to increased S and P piliation and motility. Thus, ec240 dysregulated several uropathogenic Escherichia coli (UPEC) virulence factors through different mechanisms and independent of its effects on type 1 pilus biogenesis and may have potential as an antivirulence compound. IMPORTANCE: CUP pili and flagella play active roles in the pathogenesis of a variety of Gram-negative bacterial infections, including urinary tract infections mediated by UPEC. These are extremely common infections that are often recurrent and increasingly caused by antibiotic-resistant organisms. Preventing piliation and motility through altered regulation and assembly of these important virulence factors could aid in the development of novel therapeutics. This study increases our understanding of the regulation of these virulence factors, providing new avenues by which to target their expression.

Comparative analyses of a cystic fibrosis isolate of Bordetella bronchiseptica reveal differences in important pathogenic phenotypes.

Bordetella bronchiseptica is a Gram-negative bacterium that infects and causes disease in a wide variety of animals. B. bronchiseptica also infects humans, thereby demonstrating zoonotic transmission. An extensive characterization of human B. bronchiseptica isolates is needed to better understand the distinct genetic and phenotypic traits associated with these zoonotic transmission events. Using whole-genome transcriptome and CGH analysis, we report that a B. bronchiseptica cystic fibrosis isolate, T44625, contains a distinct genomic content of virulence-associated genes and differentially expresses these genes compared to the sequenced model laboratory strain RB50, a rabbit isolate. The differential gene expression pattern correlated with unique phenotypes exhibited by T44625, which included lower motility, increased aggregation, hyperbiofilm formation, and an increased in vitro capacity to adhere to respiratory epithelial cells. Using a mouse intranasal infection model, we found that although defective in establishing high bacterial burdens early during the infection process, T44625 persisted efficiently in the mouse nose. By documenting the unique genomic and phenotypic attributes of T44625, this report provides a blueprint for understanding the successful zoonotic potential of B. bronchiseptica and other zoonotic bacteria.

Transcriptome profiling reveals stage-specific production and requirement of flagella during biofilm development in Bordetella bronchiseptica.

We have used microarray analysis to study the transcriptome of the bacterial pathogen Bordetella bronchiseptica over the course of five time points representing distinct stages of biofilm development. The results suggest that B. bronchiseptica undergoes a coordinately regulated gene expression program similar to a bacterial developmental process. Expression and subsequent production of the genes encoding flagella, a classical Bvg(-) phase phenotype, occurs and is under tight regulatory control during B. bronchiseptica biofilm development. Using mutational analysis, we demonstrate that flagella production at the appropriate stage of biofilm development, i.e. production early subsequently followed by repression, is required for robust biofilm formation and maturation. We also demonstrate that flagella are necessary and enhance the initial cell-surface interactions, thereby providing mechanistic information on the initial stages of biofilm development for B. bronchiseptica. Biofilm formation by B. bronchiseptica involves the production of both Bvg-activated and Bvg-repressed factors followed by the repression of factors that inhibit formation of mature biofilms.

BpsR modulates Bordetella biofilm formation by negatively regulating the expression of the Bps polysaccharide.

Bordetella bacteria are Gram-negative respiratory pathogens of animals, birds, and humans. A hallmark feature of some Bordetella species is their ability to efficiently survive in the respiratory tract even after vaccination. Bordetella bronchiseptica and Bordetella pertussis form biofilms on abiotic surfaces and in the mouse respiratory tract. The Bps exopolysaccharide is one of the critical determinants for biofilm formation and the survival of Bordetella in the murine respiratory tract. In order to gain a better understanding of regulation of biofilm formation, we sought to study the mechanism by which Bps expression is controlled in Bordetella. Expression of bpsABCD (bpsA-D) is elevated in biofilms compared with levels in planktonically grown cells. We found that bpsA-D is expressed independently of BvgAS. Subsequently, we identified an open reading frame (ORF), BB1771 (designated here bpsR), that is located upstream of and in the opposite orientation to the bpsA-D locus. BpsR is homologous to the MarR family of transcriptional regulators. Measurement of bpsA and bpsD transcripts and the Bps polysaccharide levels from the wild-type and the ΔbpsR strains suggested that BpsR functions as a repressor. Consistent with enhanced production of Bps, the bpsR mutant displayed considerably more structured biofilms. We mapped the bpsA-D promoter region and showed that purified BpsR protein specifically bound to the bpsA-D promoter. Our results provide mechanistic insights into the regulatory strategy employed by Bordetella for control of the production of the Bps polysaccharide and biofilm formation.

Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice.

Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA). In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.

FHA-mediated cell-substrate and cell-cell adhesions are critical for Bordetella pertussis biofilm formation on abiotic surfaces and in the mouse nose and the trachea.

Bordetella spp. form biofilms in the mouse nasopharynx, thereby providing a potential mechanism for establishing chronic infections in humans and animals. Filamentous hemagglutinin (FHA) is a major virulence factor of B. pertussis, the causative agent of the highly transmissible and infectious disease, pertussis. In this study, we dissected the role of FHA in the distinct biofilm developmental stages of B. pertussis on abiotic substrates and in the respiratory tract by employing a murine model of respiratory biofilms. Our results show that the lack of FHA reduced attachment and decreased accumulation of biofilm biomass on artificial surfaces. FHA contributes to biofilm development by promoting the formation of microcolonies. Absence of FHA from B. pertussis or antibody-mediated blockade of surface-associated FHA impaired the attachment of bacteria to the biofilm community. Exogenous addition of FHA resulted in a dose-dependent inhibitory effect on bacterial association with the biofilms. Furthermore, we show that FHA is important for the structural integrity of biofilms formed on the mouse nose and trachea. Together, these results strongly support the hypothesis that FHA promotes the formation and maintenance of biofilms by mediating cell-substrate and inter-bacterial adhesions. These discoveries highlight FHA as a key factor in establishing structured biofilm communities in the respiratory tract.

The Bps polysaccharide of Bordetella pertussis promotes colonization and biofilm formation in the nose by functioning as an adhesin.

Many respiratory pathogens establish persistent infection or a carrier state in the human nasopharynx without overt disease symptoms but the presence of these in the lungs usually results in disease. Although the anatomy and microenvironments between nasopharynx and lungs are different, a virulence factor with an organ-specific function in the colonization of the nasopharynx is unknown. In contrast to the severity of pertussis and mortality in non-vaccinated young children, Bordetella pertussis results in milder and prolonged cough in vaccinated adolescents and adults. Individuals harbouring bacteria in the nasopharynx serve as reservoirs for intrafamilial and nosocomial transmission. We show that the Bps polysaccharide of B. pertussis is critical for initial colonization of the mouse nose and the trachea but not of the lungs. Our data reveal a biofilm lifestyle for B. pertussis in the nose and the requirement of Bps in this developmental process. Bps functions as an adhesin by promoting adherence of B. pertussis and Escherichia coli to human nasal but not to human lung epithelia. Patient serum specifically recognized Bps suggesting its expression during natural human infections. We describe the first bacterial factor that exhibits a differential role in colonization and adherence between the nasopharynx and the lungs.

Cross-species protection mediated by a Bordetella bronchiseptica strain lacking antigenic homologs present in acellular pertussis vaccines.

The Bordetella species are Gram-negative bacterial pathogens that are characterized by long-term colonization of the mammalian respiratory tract and are causative agents of respiratory diseases in humans and animals. Despite widespread and efficient vaccination, there has been a world-wide resurgence of pertussis, which remains the leading cause of vaccine-preventable death in developed countries. It has been proposed that current acellular vaccines (Pa) composed of only a few bacterial proteins may be less efficacious because of vaccine-induced antigenic shifts and adaptations. To gain insight into the development of a newer generation of vaccines, we constructed a Bordetella bronchiseptica strain (LPaV) that does not express the antigenic homologs included in any of the Pa vaccines currently in use. This strain also lacks adenylate cyclase toxin, an essential virulence factor, and BipA, a surface protein. While LPaV colonized the mouse nose as efficiently as the wild-type strain, it was highly deficient in colonization of the lower respiratory tract and was attenuated in induction of inflammation and injury to the lungs. Strikingly, to our surprise, we found that in an intranasal murine challenge model, LPaV elicited cross-species protection against both B. bronchiseptica and Bordetella pertussis. Our data suggest the presence of immunogenic protective components other than those included in the pertussis vaccine. Combined with the whole-genome sequences of many Bordetella spp. that are available, the results of this study should serve as a platform for strategic development of the next generation of acellular pertussis vaccines.

Active and passive immunizations with Bordetella colonization factor A protect mice against respiratory challenge with Bordetella bronchiseptica.

Bordetella colonization factor A (BcfA) is an outer membrane immunogenic protein, which is critical for efficient colonization of the murine respiratory tract. These properties of BcfA prompted us to examine its utility in inducing a protective immune response against Bordetella bronchiseptica in a mouse model of intranasal infection. Mice vaccinated with BcfA demonstrated reduced pathology in the lungs and harbored lower bacterial burdens in the respiratory tract. Immunization with BcfA led to the generation of BcfA-specific antibodies in both the sera and lungs, and passive immunization led to the reduction of B. bronchiseptica in the tracheas and lungs. These results suggest that protection after immunization with BcfA is mediated in part by antibodies against BcfA. To further investigate the mechanism of BcfA-induced immune clearance, we examined the role of neutrophils and macrophages. Our results demonstrate that neutrophils are critical for anti-BcfA antibody-mediated clearance and that opsonization with anti-BcfA serum enhances phagocytosis of B. bronchiseptica by murine macrophages. We show that immunization with BcfA results in the production of gamma interferon and subclasses of immunoglobulin G antibodies that are consistent with the induction of a Th1-type immune response. In combination, our findings suggest that the mechanism of BcfA-mediated immunity involves humoral and cellular responses. Expression of BcfA is conserved among multiple clinical isolates of B. bronchiseptica. Our results demonstrate the striking protective efficacy of BcfA-mediated immunization, thereby highlighting its utility as a potential vaccine candidate. These results also provide a model for the development of cell-free vaccines against B. bronchiseptica.