SNP-Based Typing: A Useful Tool to Study Bordetella pertussis Populations

To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). In this study, a single nucleotide polymorphism (SNP) typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in the Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis

Global population structure and evolution of Bordetella pertussis and their relationship with vaccination.

Bordetella pertussis causes pertussis, a respiratory disease that is most severe for infants. Vaccination was introduced in the 1950s, and in recent years, a resurgence of disease was observed worldwide, with significant mortality in infants. Possible causes for this include the switch from whole-cell vaccines (WCVs) to less effective acellular vaccines (ACVs), waning immunity, and pathogen adaptation. Pathogen adaptation is suggested by antigenic divergence between vaccine strains and circulating strains and by the emergence of strains with increased pertussis toxin production. We applied comparative genomics to a worldwide collection of 343 B. pertussis strains isolated between 1920 and 2010. The global phylogeny showed two deep branches; the largest of these contained 98% of all strains, and its expansion correlated temporally with the first descriptions of pertussis outbreaks in Europe in the 16th century. We found little evidence of recent geographical clustering of the strains within this lineage, suggesting rapid strain flow between countries. We observed that changes in genes encoding proteins implicated in protective immunity that are included in ACVs occurred after the introduction of WCVs but before the switch to ACVs. Furthermore, our analyses consistently suggested that virulence-associated genes and genes coding for surface-exposed proteins were involved in adaptation. However, many of the putative adaptive loci identified have a physiological role, and further studies of these loci may reveal less obvious ways in which B. pertussis and the host interact. This work provides insight into ways in which pathogens may adapt to vaccination and suggests ways to improve pertussis vaccines. IMPORTANCE Whooping cough is mainly caused by Bordetella pertussis, and current vaccines are targeted against this organism. Recently, there have been increasing outbreaks of whooping cough, even where vaccine coverage is high. Analysis of the genomes of 343 B. pertussis isolates from around the world over the last 100 years suggests that the organism has emerged within the last 500 years, consistent with historical records. We show that global transmission of new strains is very rapid and that the worldwide population of B. pertussis is evolving in response to vaccine introduction, potentially enabling vaccine escape

Shifts of Bordetella pertussis Variants in Sweden from 1970 to 2003, during Three Periods Marked by Different Vaccination Programs

The Swedish population of Bordetella pertussis strains was characterized from 1,247 isolates covering a whole-cell vaccine program up to 1979, a 17-year period without vaccination (1979 to 1996), and a period after the introduction of general vaccination among newborns with acellular pertussis vaccines (1997 to 2003). Strains were characterized by serotyping and genotyping of pertactin and ptxA and by means of pulsed-field gel electrophoresis (PFGE). With emphasis on vaccine-related markers, the vast majority of circulating strains were of nonvaccine type. There were shifts of serotype connected with shifts of vaccination program. Serotype Fim3 was most frequent during the periods with general vaccination schedules, whereas serotype Fim2 was predominant during the 17-year vaccine-free period. Pertactin 1 was predominant during the pertussis whole-cell (Pw) vaccine period but was thereafter replaced by prn2 and has not reappeared after the introduction of acellular pertussis (Pa) vaccines. ptxA (1) was predominant over all three decades. There was a significant difference in the distribution of serotypes between vaccinated and unvaccinated individuals, but not for pertactin. A few PFGE profiles were predominant over the years: BpSR25 (serotype Fim3 prn1/7) and BpSR18 (serotype Fim3 prn2) during the Pw period, BpSR1 (serotype Fim2 prn2) during the 17 years without general vaccination, and BpSR11 (serotype Fim3 prn2) after the reintroduction of general vaccination in 1996. Despite differences between the pertactin and toxin types of Pa vaccines and circulating strains, there is no evidence that there is a threat, i.e., the vaccination program so far has been effective against whooping cough, and there seems to be no impact on the effectiveness of the vaccination program from the bacterial polymorphism

Antibody Response Patterns to Bordetella pertussis Antigens in Vaccinated (Primed) and Unvaccinated (Unprimed) Young Children with Pertussis▿

In a previous study, it was found that the antibody response to a nonvaccine pertussis antigen in children who were vaccine failures was reduced compared with the response in nonvaccinated children who had pertussis. In two acellular pertussis vaccine efficacy trials in Sweden, we studied the convalescent-phase enzyme-linked immunosorbent assay (ELISA) geometric mean values (GMVs) in response to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM 2/3) in vaccine failures and controls with pertussis. In Germany, the antibody responses to Bordetella pertussis antigens PT, FHA, PRN, and FIM-2 were analyzed by ELISA according to time of serum collection after onset of illness in children with pertussis who were vaccine failures or who were previously unvaccinated. Antibody values were also compared by severity of clinical illness. In Sweden, infants who had received a PT toxoid vaccine and who were vaccine failures had a blunted response to the nonvaccine antigen FHA compared with the response in children who had received a PT/FHA vaccine. Similarly, infants who had pertussis and who had received a PT/FHA vaccine had a blunted response to the nonvaccine antigens PRN and FIM 2/3 compared with the response in children who were vaccine failures and who had received a PT, FHA, PRN, and FIM 2/3 vaccine. In Germany, in sera collected from 0 to 15 days after pertussis illness onset, the GMVs for all 4 antigens (PT, FHA, PRN, and FIM-2) were significantly lower in an unvaccinated group than in children who were diphtheria-tetanus-acellular pertussis (DTaP) vaccine failures. In the unvaccinated group, the GMV of the PT antibody rose rapidly over time so that it was similar to that of the DTaP vaccine recipients at the 16- to 30-day period. In contrast, the antibody responses to FHA, PRN, and FIM-2 at all time periods were lower in the diphtheria-tetanus vaccine (DT) recipients than in the DTaP vaccine failures. In both Sweden and Germany, children with less severe illness had lower antibody responses than children with typical pertussis. Our findings indicate that upon exposure and infection, previous vaccinees have more-robust antibody responses to the antigens contained in the vaccine they had received than to Bordetella antigens that were not in the vaccine they had received. In addition, over time the antibody responses to FHA, PRN, and FIM-2 were greater in children with vaccine failure (primed subjects) than in unvaccinated children (unprimed subjects) whereas the responses to PT were similar in the primed and unprimed children, as determined from sera collected after 15 days of illness. Our findings lend support to the idea that DTaP vaccines should contain multiple antigens