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.

Bordetella petrii infection with long-lasting persistence in human.

We report the repeated isolation of Bordetella petrii in the sputum of a 79-year-old female patient with diffuse bronchiectasis and persistence of the bacterium for >1 year. The patient was first hospitalized due to dyspnea, which developed into severe cough with purulent sputum that yielded B. petrii on culture. After this first episode, the patient was hospitalized an additional 4 times with bronchorrhea symptoms. The isolates collected were analyzed by using biochemical, genotypic, and proteomic tools. Expression of specific proteins was analyzed by using serum samples from the patient. The B. petrii isolates were compared with other B. petrii isolates collected from humans or the environment and with isolates of B. pertussis, B. parapertussis, B. bronchiseptica, and B. holmesii, obtained from human respiratory tract infections. Our observations indicate that B. petrii can persist in persons with chronic pulmonary obstructive disease as has been previously demonstrated for B. bronchiseptica.

In vivo evolution of an emerging zoonotic bacterial pathogen in an immunocompromised human host.

Zoonotic transfer of animal pathogens to human hosts can generate novel agents, but the genetic events following such host jumps are not well studied. Here we characterize the mechanisms driving adaptive evolution of the emerging zoonotic pathogen Bordetella hinzii in a patient with interleukin-12 receptor ß1 deficiency. Genomic sequencing of 24 B. hinzii isolates cultured from blood and stool over 45 months revealed a clonal lineage that had undergone extensive within-host genetic and phenotypic diversification. Twenty of 24 isolates shared an E9G substitution in the DNA polymerase III ε-subunit active site, resulting in a proofreading deficiency. Within this proofreading-deficient clade, multiple lineages with mutations in DNA repair genes and altered mutational spectra emerged and dominated clinical cultures for more than 12 months. Multiple enzymes of the tricarboxylic acid cycle and gluconeogenesis pathways were repeatedly mutated, suggesting rapid metabolic adaptation to the human environment. Furthermore, an excess of G:C > T:A transversions suggested that oxidative stress shaped genetic diversification during adaptation. We propose that inactivation of DNA proofreading activity in combination with prolonged, but sub-lethal, oxidative attack resulting from the underlying host immunodeficiency facilitated rapid genomic adaptation. These findings suggest a fundamental role for host immune phenotype in shaping pathogen evolution following zoonotic infection.

Airway Microbiota-Host Interactions Regulate Secretory Leukocyte Protease Inhibitor Levels and Influence Allergic Airway Inflammation.

Homeostatic mucosal immune responses are fine-tuned by naturally evolved interactions with native microbes, and integrating these relationships into experimental models can provide new insights into human diseases. Here, we leverage a murine-adapted airway microbe, Bordetella pseudohinzii (Bph), to investigate how chronic colonization impacts mucosal immunity and the development of allergic airway inflammation (AAI). Colonization with Bph induces the differentiation of interleukin-17A (IL-17A)-secreting T-helper cells that aid in controlling bacterial abundance. Bph colonization protects from AAI and is associated with increased production of secretory leukocyte protease inhibitor (SLPI), an antimicrobial peptide with anti-inflammatory properties. These findings are additionally supported by clinical data showing that higher levels of upper respiratory SLPI correlate both with greater asthma control and the presence of Haemophilus, a bacterial genus associated with AAI. We propose that SLPI could be used as a biomarker of beneficial host-commensal relationships in the airway.

A case of cervical subcutaneous abscess due to Bordetella hinzii.

We present a case of subcutaneous infection caused by Bordetella hinzii in a healthy male. The isolate was successfully identified by gyrB gene sequencing. B. hinzii cannot be distinctively identified using 16S rRNA gene sequencing or by biochemical methods. The number of cases infected with B. hinzii might be underestimated owing to the difficulty in accurate identification, which can be achieved by gyrB gene sequencing to gain knowledge about the species.

The BvgS/BvgA phosphorelay system of pathogenic Bordetellae: structure, function and evolution.

In the genus Bordetella several important human and animal pathogens are classified with B. pertussis, the etiological agent of whooping cough, being medically the most relevant. In these bacteria expression of the most important virulence factors including several toxins, adhesins and colonization factors is controlled by a single master regulatory two-component system, the BvgS/BvgA system. This system represents a paradigm of a complex phosphorelay system that mediates a fine-tuned transcriptional response resulting in different expression levels of virulence factors during different stages of the infection process. In this chapter the current knowledge about signal perception and the molecular basis of differential gene expression controlled by a single two-component system is discussed.

Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice.

Several species of the Gram-negative genus Bordetella are the cause of respiratory infections in mammals and birds, including whooping cough (pertussis) in humans. Very recently, a novel atypical species, Bordetella pseudohinzii, was isolated from laboratory mice. These mice presented no obvious clinical symptoms but elevated numbers of neutrophils in bronchoalveolar lavage fluid and inflammatory signs in histopathology. We noted that this species can occur at high prevalence in a mouse facility despite regular pathogen testing according to the FELASA-recommendations. Affected C57BL/6 J mice had, in addition to the reported pulmonary alterations, tracheal inflammation with reduced numbers of ciliated cells, slower ciliary beat frequency, and largely (>50%) compromised cilia-driven particle transport speed on the mucosal surface, a primary innate defence mechanism. In an in vitro-model, Bordetella pseudohinzii attached to respiratory kinocilia, impaired ciliary function within 4 h and caused epithelial damage within 24 h. Regular testing for this ciliotropic Bordetella species and excluding it from colonies that provide mice for lung research shall be recommended. On the other hand, controlled colonization and infection with Bordetella pseudohinzii may serve as an experimental model to investigate mechanisms of mucociliary clearance and microbial strategies to escape from this primary innate defence response.

Bacteriophage-mediated competition in Bordetella bacteria.

Apparent competition between species is believed to be one of the principal driving forces that structure ecological communities, although the precise mechanisms have yet to be characterized. Here we develop a model system that isolates phage-mediated interactions by neutralizing resource competition with a large excess of nutrients, and consists of two genetically identical Bordetella strains that differ only in that one is the carrier of phage and the other is susceptible to the phage. We observe and quantify the competitive advantage of the bacterial strain bearing the prophage in both invading and in resisting invasion by the bacterial strain sensitive to the phage, and use our experimental measurements to develop a mathematical model of phage-mediated competition. The model predicts, and experimental evidence confirms, that the competitive advantage conferred by the lysogenic phage depends only on the phage pathology on the sensitive bacterial strain and is independent of other phage and host parameters, such as the infection-causing contact rate, the spontaneous and infection-induced lysis rates and the phage burst size. This work combines experimental and mathematical approaches to the study of phage-driven competition, and provides an experimentally tested framework for evaluation of the effects of pathogens/parasites on interspecific competition.

Bordetella holmesii: Still Emerging and Elusive 20 Years On.

Since the first description of Bordetella holmesii in 1995, almost 100 publications have contributed to the increasing knowledge of this emerging bacterium. Although first reported to induce bacteremia mainly in immunocompromised patients, it has also been isolated in healthy persons and has shown the capacity to induce pertussis-like symptoms and other clinical entities, such as meningitis, arthritis, or endocarditis. Respiratory diseases are generally less severe than those induced by Bordetella pertussis. However, B. holmesii was found to have a higher capacity of invasiveness given the various infection sites in which it was isolated. The diagnosis is difficult, particularly as it is a slow-growing organism but also because respiratory infections are systematically misdiagnosed as B. pertussis. Treatment is delicate, as its susceptibility to macrolides (prescribed in respiratory infections) and ceftriaxone (used in invasive disease) is challenged. Regarding prevention, there is no consensus on prophylactic treatment following index cases and no vaccine is available. Epidemiological data are also sparse, with few prevalence studies available. In this chapter, we provide an overview of the current state of knowledge on B. holmesii.

BipA Is Associated with Preventing Autoagglutination and Promoting Biofilm Formation in Bordetella holmesii.

Bordetella holmesii causes both invasive and respiratory diseases in humans. Although the number of cases of pertussis-like respiratory illnesses due to B. holmesii infection has increased in the last decade worldwide, little is known about the virulence factors of the organism. Here, we analyzed a B. holmesii isolate that forms large aggregates and precipitates in suspension, and subsequently demonstrated that the autoagglutinating isolate is deficient in Bordetella intermediate protein A (BipA) and that this deletion is caused by a frame-shift mutation in the bipA gene. A BipA-deficient mutant generated by homologous recombination also exhibited the autoagglutination phenotype. Moreover, the BipA mutant adhered poorly to an abiotic surface and failed to form biofilms, as did two other B. holmesii autoagglutinating strains, ATCC 51541 and ATCC 700053, which exhibit transcriptional down-regulation of bipA gene expression, indicating that autoagglutination indirectly inhibits biofilm formation. In a mouse intranasal infection model, the BipA mutant showed significantly lower levels of initial lung colonization than did the parental strain (P < 0.01), suggesting that BipA might be a critical virulence factor in B. holmesii respiratory infection. Together, our findings suggest that BipA production plays an essential role in preventing autoagglutination and indirectly promoting biofilm formation by B. holmesii.

Bordetella holmesii infection: current knowledge and a vision for future research.

Bordetella holmesii is a recently recognized Gram-negative bacterium causing both pertussis-like respiratory symptoms and invasive infections, such as bacteremia, pneumonia, meningitis, arthritis, pericarditis and endocarditis. Few data are available on its epidemiological characteristics, mostly related to respiratory infections. However, these are frequently misdiagnosed as a Bordetella pertussis infection as most diagnostic tests routinely used are not species-specific, thus biasing the epidemiological studies of both strains, as well as the efficacy studies on pertussis vaccination. There is no accepted agreement on treatment and it remains unknown if antimicrobial prophylaxis is indicated in certain clinical settings. We review here the current knowledge on B. holmesii and the need for further research.

Adherence of Bordetella bronchiseptica and Pasteurella multocida to swine nasal ciliated epithelial cells in vitro.

A bacterial adherence assay using swine nasal turbinate fragments was established. Turbinate fragments were incubated with Bordetella bronchiseptica or Pasteurella multocida type D at different concentrations or for different incubation times at 37 degrees C on a shaker at 120 rev/min. B. bronchiseptica phase I strains exhibited strong adherence to swine nasal ciliated epithelial cells. The number of adherent bacteria per cell increased when the bacterial concentration or incubation time increased (0, 15, 30, and 60 min); however, the number of adherent bacteria decreased after 3 or 6 hours' incubation due to the loss of cilia from cells. The optimal bacterial concentration and incubation time were 1 x 10(9) organisms/ml and one hour respectively, which resulted in 7.48 +/- 0.66 (Mean +/- SEM; B. bronchiseptica strain 03) and 9.31 +/- 0.54 (B. bronchiseptica strain 013) adherent bacteria per cell. In contrast to B. bronchiseptica phase I strains, rough phase strains of B. bronchiseptica and all P. multocida strains tested showed no adherence to swine nasal ciliated epithelial cells. All B. bronchiseptica phase I strains could agglutinate calf RBC but rough phase strains could not. Furthermore, pretreatment of B. bronchiseptica phase I organisms with 1 mg/ml or 2 mg/ml of trypsin significantly inhibited the adherence of B. bronchiseptica to ciliated epithelial cells; however, trypsin (2 mg/ml) treatment of bacteria did not decrease their ability to agglutinate calf RBC. From these results we conclude that, in addition to hemagglutinin, other proteinaceous components exist on the surface of virulent B. bronchiseptica that are sensitive to 2 mg/ml trypsin; these are suggested to be the adhesins for the adherence of B. bronchiseptica to swine nasal ciliated epithelial cells.

Adherence of ovine and human Bordetella parapertussis to continuous cell lines and ovine tracheal organ culture.

The adherence of ovine and human isolates of Bordetella parapertussis to ovine and human continuous culture cell lines and to ovine tracheal organ culture was compared. Adherence to non-ciliated respiratory continuous culture cells did not reveal any host-specificity of the isolates. In contrast, adherence of ovine B. parapertussis strains to ciliated ovine tracheal organ culture was significantly greater than that of human strains. These results indicate that tracheal organ culture is a useful tool for studying host-specific adherence of B. parapertussis and suggest that adherence of B. parapertussis to ciliated epithelia is species-specific making it unlikely that the transfer of B. parapertussis between humans and sheep will result in an infection.

Effect of passively administered immunoglobulin G on the colonization and clearance of Bordetella avium in turkeys.

This study was conducted to detect the effect of parenterally administered immunoglobulin isotype G (IgG) on the colonization and clearance of Bordetella avium at the tracheal surface in young turkeys. In two separate experiments, 3-week-old turkeys were infected with B. avium either after or before IgG administration. Comparisons were made between a control group which received an irrelevant IgG (specific for keyhole limpet hemocyanin [KLH]) and the experimental group which received a B. avium-specific IgG. When given before the bacteria, IgG reduced the numbers of colony-forming units (CFUs) in the trachea. As a supplement to non-specific respiratory defense mechanisms, B. avium-specific IgG also appears to inhibit colonization of the tracheal mucosa. In a second experiment designed to study the role of IgG in bacterial clearance, administration of B. avium-specific IgG after bacterial inoculation significantly reduced the number of CFUs on the tracheal surface. These studies support the role of B. avium-specific IgG in resistance to and recovery from B. avium infection.

Turkey coryza: toxin production by Bordetella avium.

Twelve strains of Bordetella avium representing isolates from turkeys in the United States, the Federal Republic of Germany, and the Republic of South Africa were tested for toxin production. Sterile filtered sonicates from 9 of 12 strains contained a toxin that was lethal for 7-to-10-day-old poults. Mice were also susceptible to the lethal effects of the toxin. No differences in susceptibility to the toxin were found between Beltsville small white and broad-breasted white poults. The toxin was solubilized by sonication and inactivated by heating at 56 C for 30 min. Treatment with formalin or proteolytic enzymes inactivated the toxin, indicating that it is probably a protein. The evidence suggests that the toxin is involved in the pathogenesis of turkey coryza.

Pili of Bordetella avium: expression, characterization, and role in in vitro adherence.

Electron microscopy revealed pili on all isolates of Bordetella avium and B. avium-like bacteria examined. Trypticase soy broth (TSB) and 2% peptone agar were the best media for promoting pilus expression. Cultures grown at 37 or 42 C had similar pilus production, whereas cultures grown at 18 C produced few or no pili. Pilus expression of the Art Vax strain was best when that strain was grown in TSB, but the strain yielded fewer pili than B. avium and B. avium-like isolates grown under the same cultural conditions. B. avium pili had a diameter of 2.0 nm, ranged in length from 370 nm to 1500 nm, and had a protein subunit molecular mass of about 13,100 daltons. Purified pili from B. avium did not hemagglutinate guinea pig erythrocytes, and a 1:20 dilution of hyperimmune antisera against B. avium pili did not block the hemagglutinating activity of whole-cell preparations of B. avium. In the indirect immunofluorescence test, B. avium isolates and the Art Vax strain adhered to the tracheal explants of turkeys, but B. avium-like isolates did not. Purified pili from B. avium adhered to the surface of the mucosal lining of the tracheal explants, and hyperimmune antisera against B. avium pili blocked the in vitro adherence of whole-cell preparations of B. avium. It was concluded that pili of B. avium are involved in the in vitro attachment of that bacterium to the mucosal surface of turkey tracheal explants.

Bordetella avium hemagglutination and motility mutants: isolation, characterization, and pathogenicity.

Transposon mutagenesis was used to produce Bordetella avium mutants, which were screened for the lack of potential virulence factors, including a hemagglutinin, flagella, pili, and toxins. A mini-Tn10 transposon containing a kanamycin-resistance gene was introduced into the chromosomal DNA of the virulent 002/S isolate by electroporation. A hemagglutination-negative (HA-) mutant and a motility-negative mutant were obtained. Southern blot analysis showed that only the motility-negative mutant contained the transposon, whereas the HA- mutant was a spontaneous kanamycin-resistant mutant. Both mutants were stable in vitro and in vivo. Following inoculation of 2-week-old poults, the HA- mutant was determined to be less virulent than the 002/S parent, whereas the motility-negative mutant was similar in virulence to the 002/S parent. These results indicate that the hemagglutinin of B. avium is a virulence factor, but motility does not appear to contribute to virulence.

Passive immunization versus adhesion of Bordetella avium to the tracheal mucosa of turkeys.

Three-week-old turkeys were passively immunized with convalescent serum or treated with tracheal washings from turkeys infected with Bordetella avium. Western blot analysis of the convalescent serum and tracheal washings revealed at least two bands of interaction with outer membrane protein preparations of B. avium. Adherence of B. avium in vivo to tracheal mucosa was determined and compared in treated and untreated turkeys. Passive immunization with convalescent serum reduced adherence of B. avium to the tracheal mucosa in a dose- and time-dependent manner. Adherence was significantly inhibited (P less than 0.01) when turkeys were treated intravenously with 1 ml of undiluted serum either 1 or 6 hours previously. Incubation of the bacterial inoculum with convalescent tracheal washings or application of the washings to tracheal segments before adherence determination in vivo resulted in a significant (P less than 0.01) decrease in adherence. These results indicate that adherence of B. avium to tracheal mucosa is inhibited by substances (antibody) present in both serum and tracheal secretions of convalescent turkeys.

Pathogenicity, transmissibility, and tissue distribution of avian pneumovirus in turkey poults.

The pathogenicity, transmissibility, tissue distribution, and persistence of avian pneumovirus (APV) in turkey poults were investigated in three experiments. In the first experiment, we inoculated 2-wk-old commercial turkey poults oculonasally with APV alone or in combination with Bordetella avium. In the dually infected group, clinical signs were more severe, the virus persisted longer, the bacteria invaded more respiratory tissues, and the birds had higher antibody titer than the group exposed to APV or B. avium alone. In the second experiment, we studied the distribution of APV in different tissues in experimentally inoculated 2-wk-old commercial turkey poults. Only samples from sinuses, tracheas, and lungs were positive for APV by both reverse transcriptase-polymerase chain reaction and virus isolation. In the third experiment, we studied the ability of APV to spread among birds in 1-wk-old commercial turkey poults inoculated oculonasally. The virus was isolated and the viral RNA was detected in the inoculated and direct contact birds. The virus was not isolated, viral RNA was not detected, and no antibodies were detected in the indirect contact birds. These birds were placed in different cages in the same room where the airflow was directed from the infected toward the uninfected indirect contact group.

The production of colibacillosis in turkeys following sequential exposure to Newcastle disease virus or Bordetella avium, avirulent hemorrhagic enteritis virus, and Escherichia coli.

Female large white turkeys were intranasally inoculated with either Newcastle disease virus (ND) or Bordetella avium (BA) at 4 weeks of age. This was followed by oral inoculation with an avirulent (vaccine) strain of hemorrhagic enteritis virus (HE) at 5 weeks and intravenous inoculation with Escherichia coli (EC) at 6 weeks. Control birds received ND, BA, or HE followed by EC; EC alone; or nothing at all. Turkeys receiving one agent prior to EC challenge did not experience a significant increase in mortality or pericarditis. Those exposed to ND or BA followed by HE and EC experienced a significant elevation in mortality and pericarditis. A highly significant positive correlation between the number of infectious agents encountered during primary exposure and the incidence of colibacillosis after EC challenge was demonstrated.