Genomic fluidity of Bordetella pertussis assessed by a new method for chromosomal mapping.

The genomic organization of Bordetella pertussis strains has been examined by using a new method. This method does not depend on the prior determination of a restriction map of the bacterial chromosome but is based on the ability to measure directly the distance between two genes. This is accomplished through the integration at each gene of a suicide vector containing a cleavage site for the intron-encoded endonuclease I-SceI, which is not otherwise found in the chromosome. Integration is mediated by homologous recombination between the chromosomal and cloned plasmid copies of a gene of interest. Digestion with I-SceI gives rise to a fragment the size of which represents the distance between the two genes. Multiple pairwise determinations within a set of genes provide sufficient information to derive a map of the relative gene positions. Mapping a set of 11 to 13 genes for five strains of B. pertussis and one strain of B. parapertussis revealed extensive divergence of gene order between B. pertussis Tohama I, B. pertussis 18-323, and B. parapertussis ATCC 15311. Less extensive divergence of gene order was observed between B. pertussis Tohama I and B. pertussis Tohama III, BP165, and Wellcome 28, with most of the observed differences explainable by large inversions.     

Molecular genetic characteristics of the B. pertussis strains isolated in different periods of the pertussis epidemic process

Despite the fact that the mass immunization of the children population with the DPTs vaccine has been carried out in the Russian Federation since 1959, the pertussis infection persists to be one of the pressing problems for the children population. Although the vaccination coverage of the children population with pertussis vaccines is high in Russia, at present time the pertussis incidence rates are increasing among schoolchildren and remain high among infants younger than 12 months old. Many researchers believe that the variability of the genetic structure of the pertussis causative agent may be one of the causes of increasing pertussis incidence rates. This investigation provides the molecular genetic characteristics of 97 B. pertussis strains isolated in pertussis patients in Moscow in different periods of pertussis epidemic process since the 1950s up to present time. It shows the changes in the structures of genes, which are encoding the main protective antigens of the pertussis microbe that are the pertussis toxin (ptxS1) and the pertactin (pm). The structurre of the ptxS1 and pm gene of the B. pertussis vaccine strains was compared with the structures of these genes in the B. pertussis strains isolated from the pertussis patients at present time and also in past years. All B. pertussis strains isolated in the prevaccination period (1948-1959) and most strains (95%) isolated during the first twenty years of the mass immunization in Russia are characterized by the presence of the so called "vaccine" alleles of the pertussis toxin and pertactin genes that are ptxS1 B or ptxS1 D and pm 1 alleles that corresponds to the genetic structure of the vaccine producing strains. In the early 1970s the B. pertussis strains of another toxin and pertactin genetic structures with so-called "non-vaccinal" alleles ptxS1 A and pm 3 (pm 2 since 1980s) began to appear. The B. pertussis strains with "non-vaccinal" alleles have completely displaced the "old" strains. At present time in Moscow the pertussis disease is caused by the B. pertussis strains bearing ptxS1 A and pm 2 or pm 3 alleles of pertussis toxin and pertactin genes. There was no correlation between the genotype and serotype. Thus, the structure of the B. pertussis toxin and pertactin genes in strains which have been isolated since the 1980s up to now differs from the structure of these genes in strains which are used for producing DPTs vaccine. The data obtained in this investigation suggest that the genetic structure specificity of circulating B. pertussis strains that are producing the disease at present time should be used as one of the criteria for selecting vaccine producing strains.     

Restriction analysis of Bordetella pertussis DNA isolated from patients with whooping cough in 1968 and 1995-98 and B. pertussis used for production of national vaccine strains

The genotypic and serotypic analysis of B. pertussis strains isolated from the nasopharynx of children with whooping cough in the years 1968 and 1995-98 and B. pertussis vaccine strains was the aim of this study. The genotyping of the examined strains was done by electrophoretic division of DNA in pulsed field. The 3 types (A, B, C) and 2 subtypes (A1 and A2) of DNA restriction patterns were determined for the B. pertussis strains isolated in 1968. The 2 types (D and E) and 10 subtypes (D1-D10) of DNA restriction patterns were identified for B. pertussis strains from the years 1995-98. The DNA restriction patterns of B. pertussis strains isolated in the years 1968 and 1995-98 were not identical what was the evidence of the fact that in the sixties and nineties whooping cough was caused by different B. pertussis clones. The different DNA profiles were also observed for vaccine strains as well as for vaccine strains and current isolates. Differences in DNA patterns of vaccine strains and B. pertussis strains isolated in the years 1995-98 indicated a relationship possibility in some cases while lack of relationship between these strains in other cases. Serotyping of the examined B. pertussis strains was performed by the agglutination method with the sera against B. pertussis agglutinogens 1, 2 and 3. Most strains--15 (75%) isolated in 1968 possessed only agglutinogens 1 and 3. Serotype 1, 2, 3 was most frequently observed among isolates from the years 1995-98. This study indicates the expediency of periodical change of B. pertussis vaccine strains in the aspect of whooping cough resurgence in the years 1994-95 and 1997-98.     

Spontaneous phase variation in Bordetella pertussis is a multistep non-random process.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.     

Susceptibility to macrolide antibiotics of Bordetella pertussis and Bordetella parapertussis strains isolated from whooping cough patients in 1968 and in 1997-99]

The activity of erythromycin, azithromycin, clarithromycin, dirithromycin, oleandomycin, roxithromycin, spiramycin and josamycin against 21 and 34 B. pertussis strains and against 6 and 8 B. parapertussis strains isolated respectively in the years 1968 and 1997-99 was examined. The antibiotic agar dilution method was used. The minimum concentration of macrolides which inhibited growth of B. pertussis and B. parapertussis was calculated for 50% (MIC50) and 90% (MIC90) of isolates. The susceptibility to macrolides of B. pertussis and B. parapertussis strains isolated in the years 1968 and 1997-99 did not differ significantly. The MIC90 values of erythromycin were the same for B. pertussis (MIC90 = 0.125 mg/l) and B. parapertussis strains (MIC90 = 0.25 mg/l) recovered in 1968 as for those recovered in the years 1997-99. The most active antibiotic against all strains was azithromycin (MIC90 = 0.06 mg/l). The least active antibiotics were oleandomycin (MIC90 = 2-4 mg/l) and spiramycin (MIC90 = 8 mg/l). The study showed that erythromycin remains the antibiotic of choice for treatment of whooping cough and in case of emergence of B. pertussis and/or B. parapertussis strains erythromycin resistant, can be replaced by azithromycin.     

Taxonomic distribution of the antigen eliciting bactericidal antibody for Bordetella pertussis.

Strains of Bordetella pertussis varied in their ability to elicit (in mice) an antibody bactericidal for an antiserum-sensitive strain of B. pertussis, although antibody was usually detectable after only one injection. High titres were produced by a course of seven injections with all strains of B. pertussis tested (six of phase I and three of phase IV) but not with three strains of other Bordetella species nor with two unrelated organisms, a finding of possible taxonomic value. Preliminary investigations have not revealed whether strain vaiations are due to quantitative or qualitative differences in either the bacterial lipopolysaccharide or the carrier protein necessary for antibody production, or whether they may be due to differences in heat lability of 'bactericidal antigen'.     

Bordetella pertussis, the causative agent of whooping cough, evolved from a distinct, human-associated lineage of B. bronchiseptica

Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu), and B. parapertussis(ov) are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu) are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu) are assumed to have evolved from a B. bronchiseptica-like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hu), and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than previously assumed.     

Genetic diversity analysis of isolates belonging to Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species

In this study, Amplified Fragment Length Polymorphism (AFLP) method was used to track differences among human and animal isolates of B. pertussis, B. parapertussis and B. bronchiseptica species. One hundred and sixty representative strains of these species orginated from international and Polish bacterial collections were genotyped according to AFLP involving EcoRI/Msel and SpeI/ApaI restriction/ligation/amplification procedures. This study has confirmed high potential AFLP SpeI/ApaI procedure for intra-species differentiation of B. pertussis and B. bronchiseptica strains. Both AFLP EcoRI/MseI and SpeI/ApaI procedures have been found to be useful for species-specific classification in case of B. pertussis strains. In case of B. bronchiseptica or B. parapertussis species-specific classification, SpeI/ApaI procedure has been found more precise than EcoRI/MseI one.     

Characterisation of major biological properties of Bordetella bacteriophages

The main biological properties (morphology of negative colonies, parameters of adsorption and single development cycle) of B. pertussis and B. bronchiseptica phages, isolated spontaneously and by induction with mitomycin C, were studied. To compare these characteristics, one B. parapertussis indicator strain was used, and the experiments were carried out under identical conditions. Highly active sera were obtained with the use of complete Freund's adjuvant. B. pertussis phages isolated from the strains of different serovars were serologically related, but not identical, and differed in their constant characterizing their rate of neutralization with homologous antisera. The adsorption of the phages on homologous strains was more intensive than on the cells of B. parapertussis indicator strain. However, the authors failed to observe the further development of the phages in the host cells.     

Multiple-locus variable-number tandem repeat analysis of Dutch Bordetella pertussis strains reveals rapid genetic changes with clonal expansion during the late 1990s

Bordetella pertussis, the causative agent of whooping cough, has remained endemic in The Netherlands despite extensive nationwide vaccination since 1953. In the 1990s, several epidemic periods have resulted in many cases of pertussis. We have proposed that strain variation has played a major role in the upsurges of this disease in The Netherlands. Therefore, molecular characterization of strains is important in identifying the causes of pertussis epidemiology. For this reason, we have developed a multiple-locus variable-number tandem repeat analysis (MLVA) typing system for B. pertussis. By combining the MLVA profile with the allelic profile based on multiple-antigen sequence typing, we were able to further differentiate strains. The relationships between the various genotypes were visualized by constructing a minimum spanning tree. MLVA of Dutch strains of B. pertussis revealed that the genotypes of the strains isolated in the prevaccination period were diverse and clearly distinct from the strains isolated in the 1990s. Furthermore, there was a decrease in diversity in the strains from the late 1990s, with a remarkable clonal expansion that coincided with the epidemic periods. Using this genotyping, we have been able to show that B. pertussis is much more dynamic than expected.     

Evaluation of whole-cell and acellular pertussis vaccines effectiveness in clearance of experimental B. pertussis infection in mice

The study is based on assumption that B. pertussis strains harbouring different allele variants of genes encoding subunit S1 of pertussis toxin and pertactin might be eliminated with different efficiency from lung tissue of mice which were immunized with whole-cell and acellular pertussis vaccines. It has been assumed that strains containing combinations of genes alleles which were not prevalent since 1990-ties are consisting of mutated strains in respect to pertussis toxin subunit S1 and pertactin, and are capable to decrease efficiency of pertussis vaccines. Experiments performed in vivo dealt with activity of tested vaccines against B. pertussis strains of different combinations of ptxS1/prn. The study indicated for lowered efficiency of whole-cell and acellular pertussis vaccines in elimination of mutated strains of B. pertussis from animal lung tissue in comparison with strains currently used for vaccine production.     

Characterization of Bordetella pertussis strains of recent isolation.

During the clinical trial conducted in Italy to evaluate the efficacy of new acellular pertussis vaccines, the most favorable conditions for the recovery and characterization of Bordetella pertussis strains, isolated from children with cough, were adopted. The nasopharyngeal aspirates were collected and sent to the laboratory in refrigerated conditions within 24 hours. Charcoal agar selective and non selective plates were used, and most of the isolates were recovered after 3-4 days of incubation. Confirmation of all suspected colonies included the use of biochemical tests and specific agglutination reaction with B. pertussis antiserum. Serotyping of fimbriae, susceptibility to erythromycin and DNA fingerprinting by Pulsed Field Gel Electrophoresis (PFGE), were performed to characterize B. pertussis isolates and to determine relatedness among different strains. Serotype 1,3 was the most represented in the bacterial population examined. A predominant pulsetype (PTA) characterized most of the isolates accounting for 71.4% of the strains examined. Eight subclones (23.5%) and three unrelated pulsetypes were also found. No resistant strains to erythromycin were detected.     

Pathogenic properties of freshly isolated strains of the pertussis microbe

The serovar composition, toxicity, virulence and lymphocytosis stimulating activity (LSA) of B. pertussis strains circulating in the 1980-ies were studied in comparison with the strains circulating in previous years. The study revealed changes in the toxic properties of B. pertussis: their decrease in the years of the intensive immunization of children against whooping cough and rise at the period when the number of immunized children was reduced. The toxic properties and LSA in most B. pertussis strains were less pronounced in the 1980-ies than in the 1960-ies. The serovar composition of the circulating strains remained stable for 15 years with the prevalence of serovar 1.0.3.     

Bordetella pertussis serotypes in Canada.

A study was done to determine the major antigenic factors of Bordetella pertussis strains isolated throughout Canada and whether these isolates have the same antigenic structure as the bacilli in the currently used vaccines. Testing for the major pertussis antigens, factors 1, 2 and 3, was conducted with 440 freshly isolated strains of B. pertussis received from seven canadian provinces between August 1976 and February 1978 and six batches of pertussis vaccine or immunizing agents containing pertussis vaccine. With the aid of specific antisera prepared in rabbits, the antigenic factors were detected by a slide agglutination technique. Almost all (98.9%) of the pertussis strains examined were serotype 1,3.All six batches of pertussis vaccine or immunizing agents containing pertussis vaccine proved to be rich in each of the three main pertussis agglutinogens.     

Actions of cyclic adenosine monophosphate on the cytodifferentiation of ovarian cells: studies in cultured swine granulosa cells using a novel exogenous adenylate cyclase from Bordetella pertussis

The regulation by cAMP of cholesterol side-chain cleavage activity and the synthesis of immunoisolated cytochrome P-450scc and adrenodoxin proteins was investigated in primary cultures of swine ovarian (granulosa) cells. Administration of a novel adenylate cyclase toxin isolated from Bordetella pertussis increased granulosa-cell cAMP accumulation up to 200-fold over basal. These effects were additive with those of FSH, forskolin, and cholera toxin. In contrast, bacterial extracts BP 347 and BP 348 from mutant strains of B. pertussis that lack either all virulent factors or the adenylate cyclase toxin and hemolysin were devoid of effect. Granulosa-cell cAMP accumulation supported by active bacterial adenylate cyclase was accompanied by 2- to 11-fold, time-dependent increases in [35S]methionine incorporation into immunospecific cytochrome P-450scc and adrenodoxin. These increases in the synthesis of cholesterol side-chain cleavage proteins were associated with enhanced pregnenolone production in response to exogenous sterol substrate, 25-hydroxycholesterol, and augmented progesterone secretion both in the absence and presence of exogenous lipoprotein. Moreover, the effects of Bordetella adenylate cyclase toxin on granulosa cell steroidogenesis were functionally integrated with other regulatory responses, since the non-cAMP dependent effector, estradiol 17-beta, interacted synergistically with bacterial adenylate cyclase in stimulating progesterone production. We conclude that exogenous adenylate cyclase isolated from B. pertussis can be functionally integrated into the cAMP-dependent effector pathway of granulosa cells with a resulting increase in intracellular cAMP concentrations, augmented biosynthesis of progesterone and pregnenolone, enhanced synthesis of immunospecific cytochrome P-450scc and adrenodoxin, and synergistic interactions with a non-cAMP-dependent ovarian effector hormone (estradiol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Toxigenicity conversion by pertussis phages in Bordetella parapertussis

For the first time toxigenicity conversion in B. parapertussis induced by B. pertussis phages was discovered. The clones of B. parapertussis recipient strain No. 17903 used in this study were subjected to lysogenization with 4 B. pertussis phages; as a result, 95% of these clones became immune to the repeated phage infection, developed spontaneous phage production and showed toxic properties (lethal toxicity due to the action of thermolabile and thermostable toxins) characteristic of the donor strains from which B. pertussis phages had been obtained. Differences in the degree of toxicity shown by the converted strains were determined by means of the spleen index. The convertants thus obtained did not possess protective potency.     

Limitation of phage multiplication by microbes of the genus Bordetella

Possible causes limiting the multiplication of Bordetella phages or inducing their restriction, such as the influence of lysogenic immunity and the restriction-modification (R-M) system or the incompatibility of the receptor apparatus, have been studied. The limitation of the multiplication of phages by some B. bronchiseptica and B. pertussis strains has been shown to be due to the presence of the R-M system and lysogenic immunity. In five B. bronchiseptica strains and two B. pertussis strains site-specific endonucleases (restrictases) with Hind III specificity have been detected. One B. bronchiseptica strain without the R-M system has been detected. B. bronchiseptica strains producing site-specific endonucleases are practically nonpathogenic for humans, grow in common culture media and selectively produce only one restrictase, type Hind III, which guarantees from the admixture of other specific endonucleases. The B. parapertussis strains under study (altogether 100 strains) have not been found to limit the multiplication of Bordetella test phages. The absence of site-specific endonucleases has also been confirmed biochemically. These strains are recommended as indicator strains for the multiplication of Bordetella phages.     

Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis.

Expression of virulence factors by Bordetella pertussis is altered by environmental signals (antigenic modulation) and is dependent on an activator encoded by a gene called vir. We have used TnphoA (Tn5 IS50L::phoA) gene fusions to define two sets of genes whose expression is either activated (vag loci) or repressed (vrg loci) by modulation signals. Both groups of genes appear to be regulated by the vir gene product in that, in the absence of modulators, null mutations in vir lead to the repression of vag gene fusions and derepression of vrg gene fusions. Mutants of B. pertussis were isolated that constitutively express virulence factors in the presence of the modulator MgSO4, nicotinic acid, or low incubation temperature. We designate the gene that carries such mutations mod (modulation) and have characterized one (mod-1) of these mod constitutive mutations. A method was developed for the insertional inactivation of the vir gene by using the integration of a suicide replicon. Inactivation of the vir gene in the mod-1 mutant, followed by transcomplementation with the cloned wild-type vir gene, gives the Mod-1 constitutive phenotype, showing that the mod-1 mutation defines a gene distinct from vir. The gene carrying the mod-1 mutation is linked to vir and was cloned on a recombinant cosmid (pLAF-C1) which transcomplements the vir-1::Tn5 mutation in B. pertussis 347. Introduction of pLAF-C1 into vir mutant and vir+ B. pertussis strains also gives the Mod-1 constitutive phenotype, indicating that mod-1 is a dominant allele. These data suggest that the mod gene product could have sensory functions for the environmental signals that affect the expression of vir-regulated genes of B. pertussis. The mod constitutive strains and plasmids described here also have applications in pertussis vaccine development.     

Construction of the genetically attenuated bacteria Bordetella pertussis devoid of dermonecrotic toxin activity and producing modified nontoxic pertussis toxin form

The recombinant modified (attenuated) bacteria A. pertussis were constructed. These bacteria contained knockout mutation of the dnt gene and produced nontoxic pertussis toxin derivative. The immunological properties of the mutant bacteria B. pertussis strain KS were studied. The recombinant bacteria B. pertussis strain KS were found to be devoid of dermonecrotic toxin activity, conserved the structure of the mutant dnt gene in condition of cultivation on selective growth media, and long-term survival in laboratory animal organism. Intranasal immunization of mice with living bacteria B. pertussis, attenuated strain KS provided protection of animals from virulent strains of the pertussis. The efficiency of the protection was comparable with protection efficiency provided by standard corpuscular pertussis vaccine OSO-3.