Molecular Characterization Of Bordetella Pertussis Isolates In China

Objective: Pertussis is an acute respiratory infectious disease in human, which is particularly severe in infants. Since the introduction of whole-cell vaccine (WCVs) against pertussis from the 1950s, few studies focus on the molecular epidemiology of B. pertussis in China. The aims of the study were to investigate the molecular characterization of Bordetella pertussis circulating in China.Methods: In this study, ninety six B. pertussis isolated from the period of 1953 to 2005 in China were investigated. Serotype of fimbriae and PFGE analysis were used. In the meantime, sequencing analysis was done for five virulence-related genes such as pertactin (prn); pertussis toxin subunits S1 (ptxS1); tracheal colonization factor (tcfA) and fimbrial protein 2 (fim2) and 3(fim3). Three strains used in the China pertussis whole-cell vaccine (WCV) and the WHO reference strain of B. pertussis 18323 were also analyzed. The relationship of genotypes, serotype and PFGE profile were also analyzed. In addition, we also wanted to compare the divergence between the clinical isolates and vaccine strains.Results: Three serotypes as fim2, fim3, and fim2&3 were observed among the 96 B. pertussis isolates. The proportion of each type has been changing over time. Serotype fim2 and fim2&3 isolates predominate prior to 1990s, whereas serotype fim3 isolates are collected after 1990s.Nucleotide polymorphisms in five genes were identified in this study, which included four ptxS1 alleles (ptxS1A, ptxS1B, ptxS1D and ptxS1E), six prn alleles (prn1, prn2, prn3, prn7, prn10 and prn11), four tcfA alleles (tcfA1, tcfA2, tcfA5 and tcfA9), two fim2 alleles (fim2-1 and fm2-2) and three (fim3A fim3B and fim3A*), respectively.The prn1 type was the most common type which was identified in 81.3% in the study isolates including three Chinese vaccine strains. Before 2000s, no Prn2 or Prn3 genotype was observed, which were firstly appeared in the analyzed strains in 2000 and 2003, respectively. The ptxS1A type firstly appeared in the analyzed strains in 1963, which replaced the vaccine allele ptxS1B and ptxS1A, increased gradually and has been found in 100% in the isolates after 1990s.This study showed that five allele combinations (prn/ptxS1/tcfA/fim2/fim3) were found in 93.5% (90/96) of these studied isolates. The proportions of the combinations were 60.4%, 18.8%, 8.3%, 3.1% and 2.1% of the 96 isolates, respectively. The old allele combinations of prn1 /pxS1B /tcfA2 /fim2-1/fim3A and prn1/pxS1D/tcfA2 /fim2-1 /fim3A isolates were the most circulating combinations during 1950s. After that, prn1/pxS1A/tcfA2/fim2-1/fim3A isolates were increasing and becoming the predominant allele combination which was called transitional alleles. In 2000, new allele combinations of prn3/pxS1A/tcfA2/fim2-1/fim3A and prn2/pxS1A/tcfA2/ fim2-1 /fim3B isolates were emerged, which were the circulating alleles in many developed countries.Twenty-six pulsed-field gel electrophoresis (PFGE) profiles were identified and the profiles were designated BPCH1, BPCH2, and BPCH3, etc. Most profiles were represented by few isolates. The BPCH2 and BPCH3 profile were circulating among the isolates recovered from 1953 to 1964, and comprised 52% of the 25 isolates collected during the period of 1950s. And the vaccine strains of CS and P3S10 share the same PFGE profile of BPCH2.The PFGE patterns of vaccine strain 18530 (BpCH25) was not detected among the 96 clinical isolates. The BPCH1 profile was identified as a dominant pattern that included 49 isolates ranged from 1963 to 2003 comprised 51% of study isolates. The profile BPCH1 which was represented allele combination of ptxS1A/prn1 isolates is widely circulating in China. BPCH6 profile emerged in 1999 and has increased in frequency to account for 30.8%of the B. pertussis isolates from 1999 to 2003.The phylogenetic tree using arithmetic averages (UPGAMA) cluster method showed that there was a collection between the PFGE type and ptxS1 allele.This study revealed that a close relationship between PFGE profiles and allele combinations. Six most frequent profiles were determined, except for BPCH1 (93.9%), they had 100% matches between the PFGE profiles and allele combinations. The distribution of ptxS1 alleles and fimbrial serotypes was analysed with Chi-square calculations. Statistically, significant differences in ptxS1-alleles variability (P<0.0001) and the fimbrial serotypes (P=0.001) were found for strains isolated in two periods of 1953-1979 and 1980-2005.Conclusion: The B.pertussis population is dynamic and has continuously evolved. The specific genotype and PFGE profile strains tended to be restricted to isolates circulating during a given time period. In last 50 years, the frequency of serotype has changed in different periods, antigenic divergence between vaccine strains (CS, P3S10 and 18530) and circulating strains have been observed.