Genetic approach toward linkage of Iran 2012-2016 cholera outbreaks with 7th pandemic Vibrio cholerae.

Vibrio cholerae, as a natural inhabitant of the marine environment is among the world-leading causes of diarrheal diseases. The present study aimed to investigate the genetic relatedness of Iran 2012-2016 V. cholerae outbreaks with 7th pandemic cholera and to further characterize the non-ST69/non-ST75 sequence types strains by whole-genome sequencing (WGS).Twenty V. cholerae isolates related to 2012, 2013, 2015 and 2016 cholera outbreaks were studied by two genotyping methods - Pulsed-field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST)-and by antimicrobial susceptibility testing. Seven sequence types (STs) and sixteen pulsotypes were detected. Sequence type 69 was the most abundant ST confirming that most (65%, 13/20) of the studied isolates collected in Iran between 2012 and 2016 belonged to the 7th pandemic clone. All these ST69 isolates (except two) exhibited similar pulsotypes. ST75 was the second most abundant ST. It was identified in 2015 and 2016. ST438, ST178, ST579 and STs of 983 and 984 (as newfound STs) each were only detected in one isolate. All strains collected in 2016 appeared as distinct STs and pulsotypes indicative of probable different originations. All ST69 strains were resistant to nalidixic acid. Moreover, resistance to nalidixic acid, trimethoprim-sulfamethoxazole and tetracycline was only observed in strains of ST69. These properties propose the ST69 as a unique genotype derived from a separate lineage with distinct resistance properties. The circulation of V. cholerae ST69 and its traits in recent years in Iran proposes the 7th pandemic strains as the ongoing causes of cholera outbreaks in this country, although the role of ST75 as the probable upcoming dominant ST should not be ignored.Genomic analysis of non-ST69/non-ST75 strains in this study showed ST579 is the most similar ST type to 7th pandemic sequence types, due to the presence of wild type-El Tor sequences of tcpA and VC-1319, VC-1320, VC-1577, VC-1578 genes (responsible for polymyxin resistance in El Tor biotype), the traits of rstC of RS1 phage in one strain of this ST type and the presence of VPI-1 and VSP-I islands in ST579 and ST178 strains. In silico analysis showed no significant presence of resistance genes/cassettes/plasmids within non-ST69/non-ST75 strains genomes. Overall, these data indicate the higher susceptibility of V. cholerae non-ST69/non-ST75 strains in comparison with more ubiquitous and more circulating ST69 and ST75 strains.In conclusion, the occurrence of small outbreaks and sporadic cholera cases due to V. cholerae ST69 in recent years in Iran shows the 7th pandemic strains as the persistent causes of cholera outbreaks in this country, although the role of ST75 as the second most contributed ST should not be ignored. The occurrence of non-ST69/non-ST75 sequence types with some virulence factors characteristics in border provinces in recent years is noteworthy, and further studies together with surveillance efforts are expected to determine their likely route of transport.