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

Marjolein Van Gent,Hans O Hallander,Koen Janssens,Kees J Heuvelman,Abdolreza Advani,Qiushui He,Jussi Mertsola,Peter W Hermans,Han G J Van Der Heide,Marieke J Bart,Teemu Kallonen,Frits R Mooi

doi:10.1371/journal.pone.0020340

Abstract

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.

Highlights

Whooping cough or pertussis, caused by the Gram-negative bacterium Bordetella pertussis, is an infection of the human respiratory tract and most severe for young, unvaccinated, infants [1,2]
B. pertussis strain 18323, which harbors the ptxP4 allele, was used to root the tree as we found that this strain was most closely related to B. bronchiseptica in this set of strains, consistent with previous studies using multi-locus enzyme electrophoresis and comparative genomic hybridization [31,32,33]
In 125 strains, 14 different single nucleotide polymorphism (SNP) types (STs) could be distinguished which resulted in a discriminatory index (DI) of 0.85 (Table 1)

Summary

Introduction

Whooping cough or pertussis, caused by the Gram-negative bacterium Bordetella pertussis, is an infection of the human respiratory tract and most severe for young, unvaccinated, infants [1,2]. Vaccination against whooping cough, introduced in most countries in the 1950s and 1960s, has been highly successful in controlling the disease [2]. Several causes for the increase in whooping cough have been proposed, most prominently waning immunity and pathogen adaptation [2,4,5,6]. Pathogen adaptation involved antigenic divergence between clinical isolates and vaccines strains [7,8,9,10,11,12, 13,14,15]. More recently strains emerged with increased pertussis toxin (Ptx) production which harbored a novel allele for the Ptx promoter, ptxP3 [16]. The ptxP3 strains have been found in several European countries, Japan and the Americas, suggesting a worldwide distribution [16]

Methods

Results

Conclusion