Study On Molecular Identification, Genomic Polymorphisms And Microevolution Of Bordetella Pertusiss

Whooping cough or pertussis is an acute respiratory infectious disease caused by the bacterium Bordetella pertussis. It is easily transmissible in humans, especially in infants and children. Although pertussis vaccines have been introduced for many years, pertussis is still one of the leading causes of vaccine-preventable deaths in the world today. Recently, a resurgence of pertussis has been observed in developed countries despite the high vaccination coverage. Most importantly, more pertussis cases have been found in adults and adolescents. It is reported that in adults and adolescents, symptoms of whooping cough can be mild, often not recognized as being pertussis, but the bacterium can be spread to vulnerable infants. Thus, more and more attentions have been paid to pertussis now. In this study, we developed a multiplex real-time PCR assay for detection and differentiation of B. pertussis. To understand the molecular characterization of B. pertussis vaccine and circulating strains from China where pertussis vaccine have been used for more than 50 years, 115 clinical isolates collected in China from 1950 to 2007 were analyzed together with three China vaccine strains using a combination of multiple loci variable number tandem repeats analysis (MLVA) and multilocus sequence typing (MLST) methods. Complete genome sequences of Chinese vaccine and circulating strains (CS and BP284) have been determined for finding genetic polymorphisms at whole genomic level. The distribution of these polymorphism sites (SNPs and indels) were investigated in some isolates from different periods and sources. These data will help us to gain insight into evolution of B. pertussis.Development of multiplex real-time PCR assay for detection of Bordetella pertussis A multiplex real-time PCR assay for detection and differentiation of Bordetella pertussis was successfully developed. Three targets were used for amplification in a single tube, the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single-target real-time PCR assays by using DNAs extracted from B. pertussis and B. parapertussis reference strains, indicating that the sensitivity of multiplex PCR assay was not affected. The minimum detection limit of the multiplex PCR was 1 to 5 colony-forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra- and inter-assay variation for each target in multiplex PCR assay were less than 7%. As two targets IS481 and ptxP in the PCR format were designed to detect B. pertussis, specificity of PCR assay had been improved. This method was used to analyze B. pertussis strains that collected many years ago in our laboratory and these strains were further confirmed by DNA-based microbial detection technology. These results will facilitate the study of genomic polymorphism of B. pertussis in the future.Genomic polymorphisms of Chinese B. pertussis population We analyzed 115 B. pertussis isolates from 1950 to 2007 and three vaccine strains by MLST method. In this study, two important surface protein genes (Prn and tcfA) and ptxP were used as molecular markers of MLST assay. Twelve MLST profiles were found in the strains analyzed. MLST-1/1/2 (Prn/ptxP/tcfA) profile was the main one and two Chinese vaccine strains (CS and P3S10) belonged to this profile. Another vaccine strain 18530 was MLST-1/2/2 profile. These results suggested that MLST profiles of the current circulating isolates were similar to vaccine strains. In contrast, MLST-2/3/2 profile was firstly found in 2000 and its frequency was only 19.04% in China. 115 isolates and three vaccine strains produced 46 different B. pertussis MLVA profiles, of which 13 new profiles were found. Changes in MLVA profiles were observed with time, of which three main MLVA profiles including MLVA- N1, 95 and 33 were revealed. Compared to isolates from prevaccination or early postvaccination eras, genetic diversity of strains had not been statistically changed after pertussis vaccines have been introduced for more than 50 years in China. Taken together, these results indicated that Chinese circulating isolates were different from European countries with long history of high vaccine coverage. Furthermore, three typing methods, pulsed-field gel electrophoresis (PFGE), MLST and MLVA were compared using the diversity index from typing results. The results of statistical analysis indicated that MLVA was found to be the most discriminatory among these methods. These works will allow us to gain more insights into the molecular epidemiological patterns of the B. pertussis strains, facilitating for a better control of pertussis spread and new vaccine prevention strategies in China.Complete genome sequence of B. pertussis CS, Chinese pertussis vaccine strain Whole-genome sequencing of B. pertussis strain CS was performed with a combined strategy of Sanger shotgun approach and 454 pyrosequencing technology. The complete genome of B. pertussis CS contained a circular 4,124,236 bp chromosome with an average G+C content of 67.73 %. There were 3,456 protein coding sequences (CDSs) and 357 pseudogenes with an average size of 327 amino acids, 51 tRNA genes and three ribosomal RNA operons in the genome. It was shown that strain CS was 38,050 bp larger than that of the Tohama I and 2 large fragments (>10kb) were exclusive present in strain CS, of which contained 18 (BPTD₂835-2852) and 21 (BPTD₀387-0407) genes, respectively. Homology of CDS between two strains is 99.53%. Sequencing errors included 25 single nucleotide polymorphisms (SNPs) and ten deletion events (indels) in the published Tohama I genome were corrected. It was proved that except for the two large insertions above, 341 polymorphic sites were found between CS and Tohama I, consisting 301 SNPs and 40 small indels. Based on our analyses, an estimated SNP density is 1 SNP per 13,701 bases. This places B. pertussis to be one of the known most monomorphic human pathogens. Compared with the distribution of IS and pseudogenes between genomes of two strains, IS was further demonstrated to play a role at formation of pseudogene and genomic polymorphism. Comparative genomics of B. pertussis and other sequenced Bordetella strains including B. bronchiseptica, B. parapertussis, B. avium and B. petri showed that 1,785 homology CDSs shared in those strains analyzed. The CDSs were defined as core genes of Bordetella species. Phylogenetic analysis by maximum likelihood (PAML) for these core genes showed that less than 1% of genes may be affected by positive selection, suggesting that rare positive selection contribute to evolution of B. pertussis. These data will help us understand the evolution of B. pertussis and other Bordetella strains.?Insight into the microevolution of B. pertussisWe screened the polymorphism sites found between CS and Tohama I in seven isolates from different time period in China and Japan. Some new polymorphism sites including 131 SNPs and 8 indels were found. As bases in the SNP site of B.brochiseptica were served as the root, the base substitution of SNPs was shown to be GC bias in B. pertussis genome. The analysis of compatibility matrix program for SNPs revealed that core genes existed rare incompatible site compared to pan genes in B. pertussis, suggesting that nucleotide of pan genes were possibly the hallmarks of past recombination events or mutational hotspots. This may be a mechanism of evolution that played an important role in the adaptation of B. pertussis. Resequencing of BP284 showed that no novel gene was acquired and accumulation of SNPs seem to be play a role in adaptation in comparison with CS and Tohama I. As plylogenetics analysis showed, current circulating strains in China formed a distinct and relative deep branch, which located on the exterior to two vaccine strains isolated from 1950s. These data indicated that genomic variations were not only caused by vaccination selection for adaptation, but also seem to be fundamental change at plylogenetics branch. The detailed analysis of indel events occurred in each strain revealed that distributions of indel events were consistent with SNPs-based phylogenetic analysis and suggested that indels were related to the source of isolates. These data will facilitate us the in-depth studies of microevolution of B. pertussis and improvement of pertussis vaccines.