Genome Diversity of Clinical Bordetella pertussis Isolates Circulating in Iran

Background and Objectives: Whooping cough is a highly contagious illness, caused by Bordetella pertussis. Pathogen adaptation and antigenic divergence of circulating isolates from vaccine strains are the main causes of pertussis resurgence. This study aimed to investigate the genetic diversity and evolutionary characteristics of recent circulating Iranian pertussis by pulsed-field gel electrophoresis (PFGE) and genotyping of pathogenicity-associated genes, during 2015-2018. Finally, we used comparative genomics of predominant isolates selected from PFGE dendrogram by next generation genome sequencing. Materials and Methods: Real-time PCR was performed by targeting IS481 and ptxP (promoter of pertussis toxin gene) genes for species confirmation. The polymorphism of pathogenicity-associated genes was determined using PCR-based sequencing. PFGE profiles were obtained using the XbaI enzyme (Fermentas, ABI, and Germany). For next generation sequencing, genomic DNA was extracted and purified from pure culture using phenol-chloroform method. DNA libraries were constructed using Nextera XT kit, according to manufacturer’s protocol and sequenced on the Illumina NextSeq instrument using 2x150 bp paired-end protocol. Results: PFGE results showed, our isolates clustered into 19 PFGE profiles among 62 clinical isolates and vaccine and reference strains that indicate the higher level of heterogeneity in the population during 2015-2018. The predominant Bordetella pertussis genotype harbored pertussis toxin promoter allele, ptxP3, and the expansion of ptxA1 isolates, were also observed in our population. The comparative genomic analysis of predominant isolates provided new insights into the evolution of pertussis isolates in our population. Our findings indicate that changes in ptxP3 genome structure including 33 unique SNPs and four unique indels may have contributed to the expansion of the predominant clone. Conclusion: Epidemiological results of sequencing and PFGE is very helpful to control and prevention of pertussis. However, Iran has not experienced pertussis epidemic after 2013, but our findings suggest the fact that the presence of non-vaccine ptxP3/ptxA1, may be associated with rising trend of pertussis among populations since 2007. The comparative genomic analysis of the ptxP3 and ptxP1 isolates provided new insights into the evolution of pertussis isolates in Iran. Our findings suggest that changes in ptxP3 genome structure including 33 unique SNPs and four unique indels may have contributed to the expansion of the predominant clone. However, there is a need to perform more sequencing of ptxP3 isolates in order to obtain a better picture of the pathogen adaptation under vaccine pressure.