ABSTRACT: Resurgence of pertussis has been observed in many countries with high vaccination coverage and clonal expansion of certain Bordetella pertussis strains has been associated with recent epidemics in Europe. It is known that vaccinations have selected strains which are different from those used for vaccine production. However, little is known about the differences in genomic content of strains circulating before the vaccination was introduced. In this study, we compared the genomes of 39 vaccine strains and old clinical isolates collected from Finland (N=5), Poland (N=14), Serbia (N=10) and the UK (N=10). The analysis included genotyping, pulsed-field gel electrophoresis (PFGE) and comparative genomic hybridisation (CGH). Compared to the strain Tohama I, the European strains analyzed have lost three major regions of difference (RD3, 5 and 29). However, difference in frequency of the absent RDs 3, 5 or 29 was observed among the four countries. Of the strains with absent RD5, half had also a duplicated region in the genome. All four RDs (RD22, 23, 24 and 26) absent in Tohama I were present in majority of the tested strains. Results obtained from PFGE analysis correlated well with those of CGH. Recently a novel pertussis toxin promoter allele (ptxP3) was described. Strains with ptxP3 have replaced resident ptxP1 strains. When the recent strains (N=22) selected from the four countries were examined, the ptxP3 allele was found in all countries except Poland. Our result indicates that at least three clusters of B. pertussis circulated in Europe in pre-vaccine era and their genomes were distinct from that of the reference strain Tohama I. Although progressive gene loss occurs in B. pertussis population with time, difference in frequency of the lost genes were observed among the countries. The observed differences in genomic content might be vaccine-driven. The strains were serotyped for the fimbriae, genotyped with LightCycler RT-PCR for pertussis toxin subunit 1 (ptxA), pertussis toxin promoter (ptxP) (Kallonen et al 2011, unpublished data, submitted) and pertactin (prn). The vaccine and old strains were assigned to profiles according to the PFGE results. From every PFGE profile from all countries at least 1 strain was selected and tested for CGH. Altogether 29 strains were analysed with CGH from Poland, Serbia and the United Kingdom, 12, 7 and 10, respectively. In addition 5 old previously published Finnish strains were included in the analysis as well as the reference strain Tohama I (Heikkinen et al., 2007). The hybridisations were performed as previously described (Heikkinen et al., 2007). Microarray data analysis was performed with R/Bioconductor (R Development Core Team, 2008). Print-tip-loess normalization was performed with default parameters and the gains/losses were filtered with Limma package. The regions of difference (RDs) found in this study are presented with the nomenclature defined by Brinig et al. (2006). The RDs which were not described by Brinig et al are indicated by RDs and the consequent unused number.
