Multiplex LightCycler PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal specimens.
(33/308)
A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens, 12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different (P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction, compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens. (+info)
In vivo modifications of small GTPase Rac and Cdc42 by Bordetella dermonecrotic toxin.
(34/308)
Bordetella dermonecrotic toxin (DNT) is known to activate the small GTPase Rho through deamidation or polyamination. In this study, we examined whether Rac and Cdc42, the two other members of the Rho family, serve as intracellular targets for the toxin. Immunoprecipitation and immunoblot assays revealed that DNT deamidated or polyaminated intracellular Rac and Cdc42. After the modifications, both Rac and Cdc42 lost their GTP-hydrolyzing, but not GTP-binding, activities. The interactions of the modified Rac and Cdc42 with their respective effectors were strictly dependent on GTP. MC3T3-E1 cells treated with DNT at high concentrations demonstrated extensive formations of lamellipodia and filopodia, which indicate the intracellular activation of Rac and Cdc42, respectively. (+info)
Use of bacteriophage Ba1 to identify properties associated with Bordetella avium virulence.
(35/308)
Bordetella avium causes bordetellosis, an upper respiratory disease of birds. Commercially raised turkeys are particularly susceptible. We report here on the use of a recently described B. avium bacteriophage, Ba1, as a tool for investigating the effects of lysogeny and phage resistance on virulence. We found that lysogeny had no effect on any of the in vivo or in vitro measurements of virulence we employed. However, two-thirds (six of nine) spontaneous phage-resistant mutants of our virulent laboratory strain, 197N, were attenuated. Phage resistance was associated, in all cases, with an inability of the mutants to bind phage. Further tests of the mutants revealed that all had increased sensitivities to surfactants, and increased amounts of incomplete (O-antigen-deficient) lipopolysaccharide (LPS) compared to 197N. Hot phenol-water-extracted 197N LPS inactivated phage in a specific and dose-dependent manner. Acid hydrolysis and removal of lipid A had little effect upon the ability of isolated LPS to inactivate Ba1, suggesting that the core region and possibly the O antigen were required for phage binding. All of the mutants, with one exception, were significantly more sensitive to naive turkey serum and, without exception, significantly less able to bind to tracheal rings in vitro than 197N. Interestingly, the three phage-resistant mutants that remained virulent appeared to be O antigen deficient and were among the mutants that were the most serum sensitive and least able to bind turkey tracheal rings in vitro. This observation allowed us to conclude that even severe defects in tracheal ring binding and serum resistance manifested in vitro were not necessarily indicative of attenuation and that complete LPS may not be required for virulence. (+info)
Bordetella interspecies allelic variation in AlcR inducer requirements: identification of a critical determinant of AlcR inducer responsiveness and construction of an alcR(Con) mutant allele.
(36/308)
Previous studies established the critical roles of AlcR and alcaligin inducer in positive regulation of alcaligin siderophore biosynthesis and transport genes in Bordetella pertussis and Bordetella bronchiseptica. Transcriptional analyses using plasmid-borne alcR genes of B. pertussis UT25 and B. bronchiseptica B013N to complement the alcR defect of B. bronchiseptica strain BRM13 (Delta alcR1 alcA::mini-Tn5 lacZ1) revealed interspecies differences in AlcR inducer requirements for activation of alcABCDER operon transcription. Whereas the B. pertussis UT25 AlcR protein retained strong inducer dependence when produced from multicopy plasmids, B. bronchiseptica B013N alcR partially suppressed the alcaligin requirement for transcriptional activation. Functional analysis of AlcR chimeras produced by interspecies domain swapping and interspecies reciprocal site-specific mutagenesis determined that the phenotypic difference in AlcR inducer dependence was due to a single amino acid difference within the proposed inducer-binding and multimerization domain of AlcR. Structural predictions guided the design of a mutant AlcR protein with a single amino acid substitution at this critical position, AlcR(S103T), that was fully constitutive not only when produced from multicopy plasmids but also at a single-copy gene dosage. These results indicate that AlcR residue 103 affects a critical determinant of alcaligin inducer dependence of AlcR-mediated transcriptional activation. The alcR(S103T) mutant allele is the first alcR(Con) mutant allele identified. (+info)
Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases.
(37/308)
Lipid A (endotoxin) is a major structural component of Gram-negative outer membranes. It also serves as the hydrophobic anchor of lipopolysaccharide and is a potent activator of the innate immune response. Lipid A molecules from the genus Bordetella are reported to exhibit unusual structural asymmetry with respect to the acyl chains at the 3- and 3'-positions. These acyl chains are attached by UDP-N-acetylglucosamine acyltransferase (LpxA). To determine the origin of the acyl variability, the single lpxA ortholog present in each of the genomes of Bordetella bronchiseptica (lpxA(Br)), Bordetella parapertussis (lpxA(Pa)), and Bordetella pertussis (lpxA(Pe)) was cloned and expressed in Escherichia coli. In contrast to all LpxA proteins studied to date, LpxA(Br) and LpxA(Pe) display relaxed acyl chain length specificity in vitro, utilizing C(10)OH-ACP, C(12)OH-ACP, and C(14)OH-ACP at similar rates. Furthermore, hybrid lipid A molecules synthesized at 42 degrees C by an E. coli lpxA mutant complemented with lpxA(Pe) contain C(10)OH, C(12)OH, and C(14)OH at both the 3- and 3'-positions, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In contrast, LpxA from B. parapertussis did not display relaxed specificity but was selective for C(10)OH-ACP. This study provides an enzymatic explanation for some of the unusual acyl chain variations found in Bordetella lipid A. (+info)
Real-time LightCycler PCR for detection and discrimination of Bordetella pertussis and Bordetella parapertussis.
(38/308)
Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of Bordetella pertussis and Bordetella parapertussis in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for B. pertussis and B. parapertussis, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for B. pertussis and/or B. parapertussis. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (P = 0.08) to 2.3-fold (simplex PCR) (P = 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format. (+info)
Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: a tool for rapid bacterial identification.
(39/308)
A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria. (+info)
Comparative phenotypic analysis of the Bordetella parapertussis isolate chosen for genomic sequencing.
(40/308)
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. (+info)