Bacterial filamentation as a mechanism for cell-to-cell spread within an animal host.

Intracellular pathogens are challenged with limited space and resources while replicating in a single host cell. Mechanisms for direct invasion of neighboring host cells have been discovered in cell culture, but we lack an understanding of how bacteria directly spread between host cells in vivo. Here, we describe the discovery of intracellular bacteria that use filamentation for spreading between the intestinal epithelial cells of a natural host, the rhabditid nematode Oscheius tipulae. The bacteria, which belong to the new species Bordetella atropi, can infect the nematodes following a fecal-oral route, and reduce host life span and fecundity. Filamentation requires UDP-glucose biosynthesis and sensing, a highly conserved pathway that is used by other bacteria to detect rich conditions and inhibit cell division. Our results indicate that B. atropi uses a pathway that normally regulates bacterial cell size to trigger filamentation inside host cells, thus facilitating cell-to-cell dissemination.

Bordetella holmesii Contamination of Platelet Concentrates: Revisiting the Definition of a Positive Culture.

Bacterial contamination remains the most important infectious risk of platelet transfusion. After an initially positive result, a second test is performed on the blood products and the initial culture bottle to confirm the contamination. Based on the blood center's decision algorithm used, results can be either confirmed negative, positive, or indeterminate, or be unconfirmed or discordant. Here, we report the first cases of platelet concentrates contaminated with Bordetella holmesii The in vitro growth characteristics of this unusual contaminant in platelet concentrate were investigated. Two B. holmesii strains isolated from platelet concentrates, as well as a control strain (Serratia marcescens), were spiked into platelet concentrates (PCs) at 1 and 10 CFU/ml. PCs were stored at 20 to 24°C under agitation. Samples were collected on days 2, 3, 4, and 7 for colony count and for bacterial screening using the BacT/Alert 3D system. Two PCs were detected as being positive for B. holmesii However, recultures were negative. In vitro, B. holmesii did not grow but remained detectable in PCs. Its viability diminished rapidly in contact with human plasma. Upon screening using the BacT/Alert 3D system, the majority of products spiked with B. holmesii were negative. This is the first description of PCs contaminated with B. holmesii This bacterium survives in blood products and remains dormant at low concentrations in blood products stored at room temperature, thus making difficult its detection with the BacT/Alert 3D system. The present definition of a true-positive culture of PCs may be overly restrictive for certain bacterial strains.

PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-ß(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.

Poly-ß(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

Bacterial Metabolism in the Host Environment: Pathogen Growth and Nutrient Assimilation in the Mammalian Upper Respiratory Tract.

Pathogens evolve in specific host niches and microenvironments that provide the physical and nutritional requirements conducive to their growth. In addition to using the host as a source of food, bacterial pathogens must avoid the immune response to their presence. The mammalian upper respiratory tract is a site that is exposed to the external environment, and is readily colonized by bacteria that live as resident flora or as pathogens. These bacteria can remain localized, descend to the lower respiratory tract, or traverse the epithelium to disseminate throughout the body. By virtue of their successful colonization of the respiratory epithelium, these bacteria obtain the nutrients needed for growth, either directly from host resources or from other microbes. This chapter describes the upper respiratory tract environment, including its tissue and mucosal structure, prokaryotic biota, and biochemical composition that would support microbial life. Neisseria meningitidis and the Bordetella species are discussed as examples of bacteria that have no known external reservoirs but have evolved to obligately colonize the mammalian upper respiratory tract.

[Evaluation of mixed biofilm formation between Candida albicans and a variety of bacterial species isolated from peripheral catheters at Tlemcen CHU. First study in Algeria]. / Évaluation du potentiel de formation de biofilms mixtes entre Candida albicans et quelques espèces bactériennes isolées de cathéters vasculaires périphériques au CHU de Tlemcen. Première étude en Algérie.

INTRODUCTION: Mixed-species biofilms constitute a reservoir of infection for a group of bacteria and yeasts that coexist on the same support. Peripheral venous catheters make up a good surface for the attachment of microorganisms that promote biofilm formation and this requires complex strategies for antimicrobial treatments. OBJECTIVE: No such studies on formation mixed biofilms have ever been conducted in Algeria. Therefore, we evaluated the potential for the formation of mixed-species biofilms by Candida albicans and some bacterial species isolated from peripheral vascular catheters at the University Hospital of Tlemcen, in Algeria. RESULTS: The results obtained showed that C. albicans have the potential to form mixed biofilms with three bacteria (Enterobacter cloacae, Bordetella spp. and Serratia liquefaciens) isolated from the same catheter as the yeasts. The amount of biofilms produced varies depending on the species and the composition of the growth medium. Observations by scanning electron microscopy showed that the structure of the mixed biofilm depends on the surface support the biofilm was formed on, and varies with the species. CONCLUSION: A competition was noted between bacteria and yeasts; it depends on the composition of the medium and its pH, which both play an important role in promoting the dominance of one over the other.

Iron and pH-responsive FtrABCD ferrous iron utilization system of Bordetella species.

A putative operon encoding an uncharacterized ferrous iron transport (FtrABCD) system was previously identified in cDNA microarray studies. In growth studies using buffered medium at pH values ranging from pH 6.0 to 7.6, Bordetella pertussis and Bordetella bronchiseptica FtrABCD system mutants showed dramatic reductions in growth yields under iron-restricted conditions at pH 6.0, but had no growth defects at pH 7.6. Supplementation of culture medium with 2 mM ascorbate reductant was inhibitory to alcaligin siderophore-dependent growth at pH 7.6, but had a neglible effect on FtrABCD system-dependent iron assimilation at pH 6.0 consistent with its predicted specificity for ferrous iron. Unlike Bordetella siderophore-dependent and haem iron transport systems, and in agreement with its hypothesized role in transport of inorganic iron from periplasm to cytoplasm, FtrABCD system function did not require the TonB energy transduction complex. Gene fusion analysis revealed that ftrABCD promoter activity was maximal under iron-restricted growth conditions at acidic pH. The pH of human airway surface fluids ranges from pH 5.5 to 7.9, and the FtrABCD system may supply ferrous iron necessary for Bordetella growth in acidic host microenvironments in which siderophores are ineffective for iron retrieval.

[Analysis of microbial diversity of nitrifying bacteria by terminal restriction fragment length polymorphism].

We analyzed the microbial diversity and quantity of nitrifying bacteria in the enrichment reactor by Terminal Restriction Fragment Length Polymorphism (T_RFLP), a cultured-independent molecular technique. The result indicated that nitrobacteria enriched the best, and the diversity index decreased 62.80% compared with the initial data. Nitrobacteria were predominant in the reactor. Meanwhile, we studied the microbial diversity before and after adding Nitrobacteria into shrimp ponds, and analyzed several major bacterial species that existed stably in the pond. According to the analysis by T_RFLP program, species including Brevibacillus brevis, Microbacterium lactium, Azoarcus indigens and Bordetella holmesii were the dominant bacteria in the ponds.

Expression of the primary carbohydrate component of the Bordetella bronchiseptica biofilm matrix is dependent on growth phase but independent of Bvg regulation.

We previously showed that the Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica (Y. Irie, S. Mattoo, and M. H. Yuk, J. Bacteriol. 186:5692-5698, 2004). Analyses of the extracellular components of B. bronchiseptica biofilm matrix revealed that the major sugar component in the matrix was xylose, and linkage analysis indicated a majority of it to be in a 4-linked polymeric form. The production of xylose was independent of Bvg regulation but instead was dependent on bacterial growth phase. In addition, N-acetyl-glucosamine in the matrix was found to be important for the initial development of the biofilm. These results suggest that B. bronchiseptica biofilm formation is growth phase dependent in addition to being regulated by the Bvg virulence system.

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.

Stable coexistence of five bacterial strains as a cellulose-degrading community.

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, "knockout communities" in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.

Bioremediation of toluene and naphthalene: development and validation of a GC-FID method for their monitoring.

Bioremediation of toluene and naphthalene in liquid cultures of bacteria grown in the presence of these aromatic compounds as unique sources of carbon was investigated by gas chromatography (GC). For this purpose, a method based on the use of GC with flame ionization detection was developed and validated. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. In the case of naphthalene, LOD and LOQ values of 0.43 and 0.72 mg kg(-1) were achieved. Linearity was established over one order of magnitude in the range of interest, i.e. 10-100 mg kg(-1). Excellent precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 0.5%). A recovery of 97.9 +/- 0.2% (n=3) was calculated by addition of 640 mg kg(-1) of naphthalene to the Bushnell & Haas mineral salts basal solution containing the micro-organisms. Findings clearly showed a reduction of the naphthalene content equal to 50% and 75% after two and four weeks of contact with the micro-organisms, whereas a lower degradation was shown in the case of toluene. Finally bioremediation activity was ascribed to two different microbial populations, Bordetella Petrii and Bacillus Sphericus, which survived in the polluted medium.

Efficacies of whole cell and acellular pertussis vaccines against Bordetella parapertussis in a mouse model.

Pertussis vaccine development has mainly focused on Bordetella pertussis, and consequently these vaccines contain B. pertussis antigens only. However, the related species Bordetella parapertussis can also cause pertussis, although symptoms associated with the disease are generally considered to be milder. Recent field studies have shown that in some outbreaks B. parapertussis can prevail. Using a mouse model we compared the efficacy against B. parapertussis of two commercially available acellular vaccines and two whole cell vaccines, used in The Netherlands and Finland, respectively. The efficacies of the two whole cell vaccines against B. parapertussis were similar, but much lower compared to the efficacy against B. pertussis. Although, the acellular vaccines conferred some protection against B. parapertussis early in infection, the values were not significant. Later in infection, a highly significant enhancement of colonisation by B. parapertussis was observed in mice vaccinated with acellular vaccines. The whole cell vaccines protected significantly better than the acellular vaccines against B. parapertussis. The possible consequences of a switch from whole cell to acellular vaccines was discussed in the light of our findings.

RhuR, an extracytoplasmic function sigma factor activator, is essential for heme-dependent expression of the outer membrane heme and hemoprotein receptor of Bordetella avium.

Genes involved in iron (Fe) acquisition often are regulated in response to the local availability of Fe. In many bacteria, Fe-dependent responsiveness is mediated by Fur, a global Fe-dependent transcriptional repressor. Tighter regulatory control of Fur-responsive genes is afforded by incorporating additional regulators into Fur-dependent regulatory cascades. RhuI, a Fur-dependent extracytoplasmic function sigma factor of Bordetella avium, in response to the dual stimulation of Fe starvation and the presence of heme (or hemoproteins), regulates P(bhuR), a heme-responsive promoter which directs expression of the bhuRSTUV heme utilization operon. While BhuR, the outer membrane heme receptor, and RhuI have been shown to be indispensable for heme-dependent activation of P(bhuR), collateral components of the regulatory cascade have not been described. In this investigation, RhuR, an integral cytoplasmic membrane protein with homology to anti-sigma factors, is shown to be an essential activator of P(bhuR) expression. The functional domain of RhuR required for heme-dependent activation of P(bhuR) expression was mapped to the N-terminal 97 amino acids of the protein by use of a chimeric RhuR-BlaM fusion. Expression of the chimera in a rhuR mutant rendered P(bhuR) constitutive, thereby decoupling the promoter from heme dependency. Growth studies confirmed that B. avium requires RhuR for optimal utilization of hemoglobin, but not hemin, as a sole source of nutrient Fe. These data imply that B. avium expresses, in addition to the BhuR heme/hemoprotein utilization system, an alternative RhuR-independent heme utilization mechanism. A model is proposed in which RhuR is the functional bridge between BhuR and RhuI in a heme-dependent regulatory cascade.

Comparative phenotypic analysis of the Bordetella parapertussis isolate chosen for genomic sequencing.

The genomes of three closely related bordetellae are currently being sequenced, thus providing an opportunity for comparative genomic approaches driven by an understanding of the comparative biology of these three bacteria. Although the other strains being sequenced are well studied, the strain of Bordetella parapertussis chosen for sequencing is a recent human clinical isolate (strain 12822) that has yet to be characterized in detail. This investigation reports the first phenotypic characterization of this strain, which will likely become the prototype for this species in comparison with the prototype strains of B. pertussis (Tohama I), B. bronchiseptica (RB50), and other isolates of B. parapertussis. Multiple in vitro and in vivo assays distinguished each species. B. parapertussis was more similar to B. bronchiseptica than to B. pertussis in many assays, including in BvgS signaling characteristics, presence of urease activity, regulation of urease expression by BvgAS, virulence in the respiratory tracts of immunocompromised mice, induction of anti-Bordetella antibodies, and serum antimicrobial resistance. In other assays, B. parapertussis was distinct from all other species (in pigment production) or more similar to B. pertussis (by lack of motility and cytotoxicity to a macrophage-like cell line). These results begin to provide phenotypes that can be related to genetic differences identified in the genomic sequences of bordetellae.

A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro.

We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B. avium. The mutants also acquired resistance to a newly identified B. avium-specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O-antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild-type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.

Iron starvation of Bordetella avium stimulates expression of five outer membrane proteins and regulates a gene involved in acquiring iron from serum.

Iron starvation of Bordetella avium induced expression of five outer membrane proteins with apparent molecular masses of 95, 92, 91.5, 84, and 51 kDa. Iron-responsive outer membrane proteins (FeRPs) of similar sizes were detected in six of six strains of B. avium, suggesting that the five FeRPs are common constituents of the outer membrane of most, if not all, strains of B. avium. Iron-regulated genes of B. avium were targeted for mutagenesis with the transposon TnphoA. Two mutants with iron-responsive alkaline phosphatase activities were isolated from the transposon library. The transposon insertion did not alter the iron-regulated expression of the five FeRPs in mutant Pho-6. The mutant Pho-20 exhibited a loss in expression of the 95-kDa FeRP and the 84-kDa FeRP. Both Pho-6 and Pho-20 were able to use free iron as a nutrient source. However, Pho-20 was severely compromised in its ability to use iron present in turkey serum. The data indicated that the mutation in Pho-20 affected expression of one or more components of an uptake machinery that is involved in acquisition of iron from organic ferricomplexes.

Lipopolysaccharide expression within the genus Bordetella: influence of temperature and phase variation.

LPSs play an important role in bacterial pathogenesis. In this study, the LPS expression of the seven known Bordetella species and its dependency on growth temperature was analysed by oxidative silver staining of proteinase-K-treated whole bacteria separated by Tricine-SDS-PAGE. The bordetellae were found to have extensively variable LPS in a species-specific way. In addition, the human and ovine Bordetella parapertussis strains exhibited host-specific LPS expression. LPSs from human B. parapertussis strains grown at 37 and 25 degrees C were distinct. Growth temperature also affected LPS production by several Bordetella bronchiseptica strains. In some of these cases, BvgAS, the global regulator of virulence factors, was involved in this regulation of LPS biosynthesis. In contrast, no evidence was found for the involvement of the Bordetella pertussis BvgAS system in regulation of LPS synthesis. The obligate human pathogens B. pertussis and Bordetella holmesii are closely related but were shown to produce immunologically distinct LPSs. These species are isolated from the upper respiratory tract and blood, respectively. This raises several interesting questions concerning the potential role of LPS as a virulence factor in the infection processes.

Species selectivity of new siderophore-drug conjugates that use specific iron uptake for entry into bacteria.

Siderophores selectively bind ferric iron and are involved in receptor-specific iron transport into bacteria. Several types of siderophores were synthesized, and growth-promoting or inhibitory activities when they were conjugated to carbacephalosporin, erythromycylamine, or nalidixic acid were investigated. Overall, 11 types of siderophores and 21 drug conjugates were tested against seven different bacterial species: Escherichia coli, Bordetella bronchiseptica, Pasteurella multocida, Pasteurella haemolytica, Streptococcus suis, Staphylococcus aureus, and Staphylococcus epidermidis. In some species, the inhibitory activities of the drug conjugates were associated with the ability of the bacteria to use the siderophore portion of the molecules for growth promotion in disc diffusion tests (0.04 mumol of conjugate or siderophore per disc). E. coli used catechol-based siderophore portions as well as hydroxamate-based tri-delta-OH-N-OH-delta-N-acetyl-L-ornithine ferric iron ligands for growth under iron-restricted conditions achieved by supplemental ethylenediamine di (O-hydroxyphenylacetic acid) (100 micrograms/ml) and was sensitive to carbacephalosporin conjugated to these siderophore types (up to a 34-mm-diameter inhibition zone). B. bronchiseptica used desferrioxamine B and an isocyanurate-based or trihydroxamate in addition to catechol-based siderophore portions for promotion but was not inhibited by beta-lactam conjugates partly because of the presence of beta-lactamase. P. multocida and P. haemolytica did not use any of the synthetic siderophores for growth promotion, and the inhibitory activities of some conjugates seemed partly linked to their ability to withhold iron from these bacteria, since individual siderophore portions showed some antibacterial effects. Individual siderophores did not promote S. suis growth in restrictive conditions, but the type of ferric iron ligands attached to beta-lactams affected inhibitory activities. The antibacterial activities of the intracellular-acting agents erythromycylamine and nalidixic acid were reduced or lost, even against S. aureus and S. epidermidis, when the agents were conjugated to siderophores. Conjugate-resistant E. coli mutants showed the absence of some iron-regulated outer membrane proteins in gel electrophoresis profiles and in specific phage or colicin sensitivity tests, implying that the drugs used outer membrane receptors of ferric complexes to get into cells.

Genetic regulation of airway colonization by Bordetella species.

Bordetella species are respiratory pathogens that infect humans and other animals. A majority of the virulence factors expressed by these bacteria are regulated by a master control locus, BvgAS, a member of the two-component family of signal transduction systems. BvgAS senses environmental signals and mediates signal transduction by a phosphorylation cascade that leads to a biphasic transition between the Bvg+ and Bvg- phases. From natural host studies using Bordetella bronchiseptica, we have found that expression of Bvg+ phase factors, which include adhesins and toxins, is required for successful colonization of the mammalian respiratory tract. Suppression of the Bvg- phase motility phenotype is necessary for a successful interaction with the host. Although the Bvg- phase does not appear to be required in vivo, it does confer the ability to survive under conditions of severe nutrient deprivation. We hypothesize that the Bvg+ phase is necessary and sufficient for respiratory tract colonization and the Bvg- phase is adapted for survival in environments encountered during transmission between hosts.

Extended incubation of culture plates improves recovery of Bordetella spp.

Extended incubation of culture plates was studied to see if the recovery of Bordetella spp. from nasopharyngeal swabs could be improved. Forty-eight Bordetella isolates were recovered from 103 children (overall positive-culture rate, 46.6%) who met the clinical case definition of pertussis. Seven of 44 (16%) B. pertussis isolates and 2 of 4 (50%) B. parapertussis isolates were recovered only after extended incubation of nasopharyngeal cultures up to 12 days.