Molecular characterization of two Bordetella bronchiseptica strains isolated from children with coughs

During a surveillance program associated with the Italian clinical trial for the evaluation of new acellular pertussis vaccines, two bacterial isolates were obtained in cultures of samples from immunocompetent infants who had episodes of cough. Both clinical isolates were identified as Bordetella bronchiseptica by biochemical criteria, although both strains agglutinated with antisera specific for Bordetella parapertussis, suggesting that the strains exhibited some characteristics of both B. bronchiseptica and B. parapertussis. Both children from whom these strains were isolated exhibited an increase in serum antibody titer to pertussis toxin (PT), a protein that is produced by Bordetella pertussis but that is not thought to be produced by B. bronchiseptica. We therefore examined whether the clinical isolates were capable of producing PT. Neither strain produced PT under laboratory conditions, although both strains appeared to contain a portion of the ptx region that encodes the structural subunits of PT. In order to determine whether the ptx genes may encode functional proteins, we inserted an active promoter directly upstream of the ptx region of one of these strains. Biologically active PT was produced, suggesting that this strain contains the genetic information necessary to encode an active PT molecule. Sequence analysis of the ptx promoter region of both strains indicated that, while they shared homology with the B. bronchiseptica ATCC 4617 sequence, they contained certain sequence motifs that are characteristic of B. parapertussis and certain motifs that are characteristic of B. pertussis. Taken together, these findings suggest that variant strains of B. bronchiseptica exist and might be capable of causing significant illness in humans.     

Inoculation experiments with Bordetella bronchiseptica strains in SPF pigs

The progeny of 9 SPF gilts fed a balanced ration (Table I) was used in an inoculation experiment with field strains of Bordetella bronchiseptica isolated in herds suffering atrophic rhinitis. Acute rhinitis was produced within a week after intranasal inoculation of B. bronchiseptica into 1 to 11-day-old piglets. Coughing occurred in some of the exposed pigs, but signs of pneumonia did not develop. A few pigs were killed at intervals of 1 to 3 weeks after exposure. These pigs all showed histological lesions in the turbinate and B. bronchiseptica was recovered from various parts of the respiratory tract. Pigs killed 3 weeks after inoculation showed advanced turbinate atrophy (Tables II and III). Among inoculated litter mates reared to slaughter weight, only one developed clinical signs (slight) of atrophic rhinitis, and a tendency towards an elimination of the B. bronchiseptica infection from the accessible part of the nasal cavity was noticed during the growth period. By examination of nasal swabs collected when the pigs were 10 to 13 weeks old, Mycoplasma flocculare was isolated as well from pigs inoculated with B. bronchiseptica as from the control pigs. The growth rate of the experimental pigs was high and no difference in feed consumption or feed conversion occurred between the exposed pigs and the control pigs. By post mortem examination of the snouts from the pigs slaughtered at approx. 85 kg live weight, severe atrophic rhinitis was not found. Approximately one third (32%) of the exposed pigs showed slight atrophic rhinitis lesions (Table IV). The results are discussed and it is concluded that the isolated B. bronchiseptica strains are pathogenic in young pigs and able to induce turbinate atrophy 2 to 3 weeks after inoculation. Turbinate atrophy induced in pigs a few weeks old, may apparently restore completely or almost completely during the growth period. Under the provided experimental conditions, infection with B. bronchiseptica did not result in the development of a lasting, growth-retarding atrophic rhinitis.     

Isolation and characterization of Bordetella bronchiseptica from cats in southern Louisiana

The role of Bordetella bronchiseptica in respiratory disease of domestic cats is currently being explored. Clinical and experimental studies in the United Kingdom have shown Bordetella bronchiseptica to be a primary respiratory pathogen in cats; similar studies in the United States are limited. The purpose of this study is to report on the isolation, seroprevalence, and partial characterization of Bordetella bronchiseptica from shelter cats in southern Louisiana. A total of 614 cats from four local shelters were studied. All cats appeared to be asymptomatic for signs of respiratory disease. Bordetella bronchiseptica was isolated in 19/614 (3.1%) cats by oropharyngeal swab and in 6/614 cats by bronchial lavage. Using an antibody capture ELISA method, 148/614 (24.1%) cats were seropositive for Bordetella bronchiseptica. The 25 isolates of Bordetella bronchiseptica were further characterized by ribotype analysis, and a total of 17 different ribotypes were identified. Specific pathogen-free kittens were experimentally infected with five of the isolates, and four of the five isolates induced clinical signs typical of feline bordetellosis. It is concluded that Bordetella bronchiseptica is present in the cat population in southern Louisiana, the organism can be isolated from asymptomatic cats, some of these isolates can produce disease in specific pathogen-free kittens, and that Bordetella bronchiseptica isolates from cats in a relatively small geographic area are genetically diverse. This and other studies indicate that Bordetella bronchiseptica should be considered in cases of feline respiratory disease.     

Use of ribotyping to distinguish Bordetella bronchiseptica isolates

A total of 113 Bordetella bronchiseptica strains, isolated from 11 different host species worldwide, were characterized by ribotyping with restriction enzyme PvuII. Sixteen distinct ribotypes were identified, and each ribotype contained five to seven restriction fragments ranging in size from 1.8 to 5.6 kb. Approximately 88% of the swine isoaltes were identified as ribotype 3 strains. Isolates from dogs also displayed little variation; 74.1% were found to be ribotype 4 strains. Strains obtained from the remaining nine host species belonged to 15 different ribotypes. There was no association between geographic location and ribotype. The technique which we used may be useful for epidemiologic studies in which the transmission of B. bronchiseptica, both within and between species, is investigated.     

Comparative analysis of the virulence control systems of Bordetella pertussis and Bordetella bronchiseptica

Bordetella pertussis and Bordetella bronchiseptica contain nearly identical BvgAS signal-transduction systems that mediate a biphasic transition between virulent (Bvg+) and avirulent (Bvg-) phases. In the Bvg+ phase, the two species express a similar set of adhesins and toxins, and in both organisms the transition to the Bvg- phase occurs in response to the same environmental signals (low temperature or the presence of nicotinic acid or sulphate anion). These two species differ, however, with regard to Bvg(-)-phase phenotypes, host specificity, the severity and course of the diseases they cause, and also potentially in their routes of transmission. To investigate the contribution of the virulence-control system to these phenotypic differences, we constructed a chimeric B. bronchiseptica strain containing bvgAS from B. pertussis and compared it with wild-type B. bronchiseptica in vitro and in vivo. The chimeric strain was indistinguishable from the wild type in its ability to express Bvg(+)- and Bvg(-)- phase-specific factors. However, although the chimeric strain responded to the same signals as the wild type, it differed dramatically in sensitivity to these signals; significantly more nicotinic acid or MgSO4 was required to modulate the chimeric strain compared with the wild-type strain. Despite this difference in signal sensitivity, the chimeric strain was indistinguishable from the wild type in its ability to cause respiratory-tract infections in rats, indicating that the bvgAS loci of B. pertussis and B. bronchiseptica are functionally interchangeable in vivo. By exchanging discrete fragments of bvgAS, we found that the periplasmic region of BvgS determines signal sensitivity.     

Two-color hybridization assay for simultaneous detection of Bordetella bronchiseptica and toxigenic Pasteurella multocida from swine

Bordetella bronchiseptica and toxigenic Pasteurella multocida are the etiologic agents of swine atrophic rhinitis. Methods currently used for their identification are time-consuming and suffer from a lack of sensitivity. We describe a colony lift-hybridization assay for detection of B. bronchiseptica and toxigenic P. multocida that can be performed with a single colony lift derived from a primary isolation plate without the need for pure subcultures of suspect bacteria. Membranes are hybridized simultaneously to probes derived from the B. bronchiseptica alcA gene and the P. multocida toxA gene. A multicolor development procedure permits sequential detection of bound probes. The assay was tested with 84 primary isolation plates generated from nasal swabs from swine with clinical signs of atrophic rhinitis. Comparison of the results from the colony lift-hybridization assay with those from conventional testing, based on a combination of colony morphology, biochemical reactions, mouse lethality, and enzyme-linked immunosorbent assay, indicated that the colony lift assay has superior sensitivity and comparable specificity. This technique has wide application for diagnostic and experimental studies.     

Filamentous hemagglutinin of Bordetella bronchiseptica is required for efficient establishment of tracheal colonization

Adherence to ciliated respiratory epithelial cells is considered a critical early step in Bordetella pathogenesis. For Bordetella pertussis, the etiologic agent of whooping cough, several factors have been shown to mediate adherence to cells and cell lines in vitro. These putative adhesins include filamentous hemagglutinin (FHA), fimbriae, pertactin, and pertussis toxin. Determining the precise roles of each of these factors in vivo, however, has been difficult, due in part to the lack of natural-host animal models for use with B. pertussis. Using the closely related species Bordetella bronchiseptica, and by constructing both deletion mutation and ectopic expression mutants, we have shown that FHA is both necessary and sufficient for mediating adherence to a rat lung epithelial (L2) cell line. Using a rat model of respiratory infection, we have shown that FHA is absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals. FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting that its role in establishment may be dedicated to overcoming the clearance action of the mucociliary escalator.     

The lipopolysaccharide of Bordetella bronchiseptica acts as a protective shield against antimicrobial peptides

Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains.     

Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli

The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry. Chromosome replication patterns in these strains were followed by marker frequency analyses. In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild-type parent. The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication. The site for conversion of uni- to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC. Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi- or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern. These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects. Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality. However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.     

Ampicillin resistance mediated by the R plasmid in strains of Shigella flexneri]

Forty Shigella flexneri strains isolated from children attended to at the Children's Hospital of Camagüey during an outbreak of acute diarrheal disease were studied; the minimal inhibitory concentration of ampicillin was determined. 33 strains (82.5%) were resistant to higher concentrations: 8 to 16 micrograms/mL, and 7 were susceptible to 4 micrograms/mL concentrations. Resistance plasmid (R) extraction was carried out in all the isolated strains and a common plasmid was found this plasmid was purified and transferred to Escherichia coli HE 101. Resistance transmission was tested.     

A highly adherent phenotype associated with virulent Bvg+-phase swine isolates of Bordetella bronchiseptica grown under modulating conditions

The ability of Bvg(-)-phase and Bvg(+)-phase Bordetella bronchiseptica swine isolates, grown under modulating or nonmodulating conditions, to adhere to swine ciliated nasal epithelial cells was determined. When virulent strains were cultivated at 37 degrees C in the Bvg+ phase, numerous adherent bacteria (approximately eight per cell, depending on the strain used) were observed. However, when such strains were grown under modulating conditions (23 degrees C), a significant increase in the level of attachment was seen, suggesting that B. bronchiseptica produces a Bvg-repressed adhesin under these conditions. bvg mutant strains, including an isogenic bvgS mutant, adhered minimally. Western blots indicated that two putative B. bronchiseptica adhesins, filamentous hemagglutinin and pertactin, were not detectable in cultures displaying the highly adherent phenotype. Several proteins apparent in Western blots obtained by using bacterial extracts enriched in outer membrane proteins derived from B. bronchiseptica grown at 23 degrees C were not present in similar extracts prepared from an isogenic bvgS mutant grown at 23 degrees C or from the parent strain grown at 37 degrees C. Adherence of bacteria cultivated at 23 degrees C was almost completely abolished by pretreatment of organisms at 60 degrees C; adherence was reduced by 57% when bacteria were pretreated with pronase E. Temperature shift experiments revealed that the heightened level of adhesion that occurs following growth at 23 degrees C was maintained for up to 18 h when bacteria were subsequently incubated at 37 degrees C. We propose that a Bvg-repressed adhesin, expressed only by modulated bvg+ strains of B. bronchiseptica, may play a key role in the initial colonization of naturally infected swine.     

Flagellin, a major protein present in SDS-PAGE profiles of Sarkosyl-OMP-enriched fractions from Bordetella bronchiseptica Bvg- or modulated Bvg+ strains

The bvg or vir locus positively regulates the expression of many Bordetella virulence-associated determinants (encoded by vag genes), including cell envelope proteins, in response to environmental stimuli. On the other hand, several genes named vrg genes are negatively controlled by the bvg regulon (Knapp and Mekalanos, 1988). Flagellin is encoded by a vrg gene, which is expressed when the principal virulence factors are eliminated during antigenic modulation or in phase variants (Akerley et al., 1992). We have previously analyzed SDS-PAGE profiles of Sarkosyl-outer membrane protein (OMP)-enriched fractions from B. bronchiseptica Bvg- and modulated Bvg+ strains and reported a major band associated with the avirulent phenotype (Passerini de Rossi et al., 1995). In order to characterize this band we have purified flagellar filaments from Bvg- and modulated Bvg+ strains, and analyzed them by SDS-PAGE. These profiles revealed a single major band of 40 or 45 kDa depending on the strain. The N-terminal amino acid sequence of the putative flagellin expressed by BB7200a was identical over the first 21 residues analyzed to that of the flagellin from the modulated strain BB7865 reported by Akerley et al. (1992). Comparison of the SDS-PAGE profile of flagellar filaments with that of the OMP-enriched fraction of the corresponding strain showed that the flagellum-associated polypeptide had the same electrophoretic mobility as that of the characteristic band of the avirulent phenotype. Furthermore, this band was absent in the OMP-enriched fraction profile from a Bvg- strain subjected to a treatment that removes flagella. Our results indicate that the major protein observed in SDS-PAGE profiles of Sarkosyl-OMP-enriched fractions from B. bronchiseptica Bvg- and modulated Bvg+ strains corresponds to flagellin.     

A second two-component regulatory system of Bordetella bronchiseptica required for bacterial resistance to oxidative stress, production of acid phosphatase, and in vivo persistence

Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetella spp., the bvg locus. A risA'-'lacZ translational fusion was constructed and integrated into the chromosome of B. bronchiseptica. Determination of beta-galactosidase activity under different environmental conditions suggested that ris is regulated independently of bvg and is optimally expressed at 37 degrees C, in the absence of Mg2+, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all Bordetella spp., is required for the expression of acid phosphatase by B. bronchiseptica. Although catalase and superoxide dismutase production were unaffected, the ris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of bvg-positive and bvg-negative ris mutants with the intact ris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the ris mutant is cleared by the host much earlier than the wild-type strain, suggesting that ris-regulated products play a significant role in natural infections. The identification of a second two-component system in B. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.     

Regulatory implications of protein assemblies at the gamma origin of plasmid R6K - a review

Recognition of the replication origin (ori) by initiator protein is a recurring theme for the regulated initiation of DNA replication in diverse biological systems. The objective of the work reviewed here is to understand the initiation process focusing specifically on the gamma-ori of the antibiotic-resistance plasmid R6K. The control of gamma-ori copy number is determined by both plasmid-encoded and host-encoded factors. The two central regulatory elements of the plasmid are a multifunctional initiator protein pi, and sequence-related DNA target sites, the inverted half-repeats (IRs) and the direct repeats (DRs). The replication activator and inhibitor activities of pi seem to be at least partially distributed between two naturally occurring pi polypeptides (designated by their molecular weights pi35.0 and pi30.5). Regulatory variants of pi with altered states of oligomerization in nucleoprotein complexes with DRs and IRs have been isolated. The properties of these mutants laid the foundation for our model of pi protein activity which proposes that different protein surfaces are required for the formation of functionally distinct complexes of pi with DRs and IRs. These mutants also suggest that pi polypeptides have a modular structure; the C-terminus contains the DNA-binding domain while the N-terminus controls protein oligomerization. Additionally, pi35.0 binds to a novel DNA sequence in the A+T-rich segment of gamma-ori. This binding site is at or near the site from which synthesis of the leading strand begins.     

The aadA gene of plasmid R100 confers resistance to spectinomycin and streptomycin in Myxococcus xanthus

Plasmids with the aadA gene from plasmid R100, which confers resistance to the aminoglycosides spectinomycin and streptomycin in Escherchia coli, can be introduced into wild-type Myxococcus xanthus, strain DK1622, by electroporation. Recombinant M. xanthus strains with integrated plasmids carrying the aadA gene acquire resistance to high levels of these antibiotics. Selection for aadA in M. xanthus can be carried out independently of, or simultaneously with, selection for resistance to kanamycin. The kinds and frequencies of recombination events observed between integrative plasmids with aadA and the M. xanthus chromosome are similar to those observed after the transformation of yeast. Cleavage of integrative plasmid DNA at a site adjacent to a region of homology between the plasmid and the M. xanthus genome favors the targeted disruption of M. xanthus genes by allele replacement.     

Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing

Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.     

Physicochemical properties correlated with drug resistance and the reversal of drug resistance in Plasmodium falciparum

At high molar excess, verapamil can selectively increase the accumulation and cytotoxicity of structurally dissimilar natural product drugs in many multidrug-resistant tumor cell lines. Such concentrations of verapamil are also capable of increasing the accumulation and activity of chloroquine in chloroquine-resistant strains of the human malaria parasite Plasmodium falciparum. Despite such similarities, it is not clear why chloroquine-resistant P. falciparum is often susceptible to closely related compounds such as amodiaquine, whereas cancer cells are cross-resistant to many structurally unrelated drugs. For 13 aminoquinoline and aminoacridine compounds, relative drug resistance was negatively correlated with lipid solubility at physiological pH (r2 = 0.90, p < 0.0001). The ability of verapamil (5 microM) to reverse drug resistance was also negatively correlated with lipid solubility (r2 = 0.88, p < 0.0001). Furthermore, molar refractivity was weakly correlated with relative drug resistance (r2 = 0.46, p < 0.05) and reversal of drug resistance (r2 = 0.52, p < 0.005). Verapamil increases chloroquine accumulation by resistant parasites, a mechanism suggested to account for its selective chemosensitization effect. We show that the initial rate of chloroquine accumulation by resistant parasites is increased by verapamil. This effect of verapamil is abolished when deoxy-glucose is substituted for glucose. Therefore, verapamil produces an energy-dependent increase in the permeability of resistant parasites to chloroquine. For a panel of four chloroquine-resistant and two chloroquine-susceptible isolates, the effect of verapamil on the accumulation of chloroquine and monodesethyl amodiaquine was found to be correlated (r2 = 0.96, p < 0.001). Verapamil chemosensitization was also correlated for the two drugs (r2 = 0.92, p < 0.005), suggesting a common mechanism. In summary, the degree of drug resistance and the extent of verapamil chemosensitization for a particular drug seem to be dependent on general physical features such as lipid solubility and molar refractivity rather than on closely defined structural parameters. These studies provide insight into this important resistance mechanism of malaria parasites and may provide direction for the development of new drugs that are effective against resistant parasites.