Prevailing clone (ST69) of Vibrio cholerae O139 in India over 10 years

Clinical strains of V. cholerae O139 obtained from the past 20 years (FC1105-2005, FC1225-2001, FC1341-2002, FC1384-2000, FC1817-1994, FC1877-1995, FC2271-1997, FC2273-1998, FC3611a-1999 and FC3611b-1997) were revived from the repository at the Department of Clinical Microbiology, Christian Medical College, Vellore, India. Isolates were identified with standard biochemical methods and were confirmed by agglutination with O139 antisera raised in-house. The genomic DNA was extracted using Qiagen automated DNA extraction method with QIAsymphony DSP Virus/Pathogen Mini Kit (QIAsymphony, Qiagen, Germany). All 10 V. cholerae O139 was confirmed based on the 16S rRNA gene, by amplification, sequencing, and BLAST against the NCBI database.

The generated whole genome data were assembled de novo using Assembler SPAdes v.5.0.0.0 embedded in Torrent suite server v.5.0.5. The genome sequence was annotated using PATRIC (Pathosystems Resource Integration Center), the bacterial bioinformatics database and analysis resource (http://​www.​patricbrc.​org) [4], and the NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) (http://​www.​ncbi.​nlm.​nih.​gov/​genomes/​static/​Pipeline.​html).

The whole genome data were analyzed using open access tools at Center for Genomic Epidemiology (CGE) web-based server. Sequence types for the study isolates were determined using MLST 1.8 (https://​cge.​cbs.​dtu.​dk/​/​services/​MLST/​), and antimicrobial resistance were identified using ResFinder 2.1 (https://​cge.​cbs.​dtu.​dk/​/​services/​ResFinder/​) with the 90% threshold for identity and with 60% of minimum length coverage. Genome data was screened for virulence genes through PATRIC database. The presence of plasmids was analyzed using PlasmidFinder 1.3 (https://​cge.​cbs.​dtu.​dk/​/​services/​PlasmidFinder/​) with 95% threshold for identity. The study isolates were further screened for the presence of prophage sequences within the genome using PHAST (PHAge Search Tool) (http://​phast.​wishartlab.​com) [5].

The web-based MyDbFinder 1.0 (https://​cge.​cbs.​dtu.​dk/​services/​MyDbFinder/​) was used, in silico to determine the SXT elements (int _SXT), specific integrase genes of class 1 integron (intI gene) and specific gene (VC2346) of the seventh pandemic strains with a selected threshold equal to 98% identity as previously described [6].

The phylogenetic analysis was performed based on MLST housekeeping genes (adk, gyrB, mdh, metE, pntA, purM, pyrC) using the MEGA7 software [7]. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Maximum Composite Likelihood method. goEBURST analysis for the V. cholerae O139 strains was performed using PHYLOViZ 1.1 software [8] to identify the relation between study isolates and the global strains with sequence types of V. cholerae (ST168-KC895170.1, ST169-KC895206.1, ST170-KJ657169.1, ST182-KC895159.1, ST429-KC895162.1, N16961-AE003852.1, M2140_ST70-CP013315.1, M802_ST71-LT907989.1, M1616_ST73-KC895170.1, M796_ST75-DQ316973.1).

Assembly of the raw reads of the isolates ranged from 69 to 196X coverage (Table 1). NGS revealed that all isolates belong to same sequence type ST69. Similarly in a study by Siriphap et al. [6], most of the seventh pandemic isolates belonged to ST69 and all the clinical strains of O1 and O139 were highly conserved to ST69. Literature study reveals that the Bay of Bengal as a major hub connecting the spread of cholera around the globe and has continually being evolved in South Asia, later spread around the globe in at least three independent but overlapping waves. Besides V. cholerae serogroup O1 is widespread throughout the globe, whereas serogroup O139 is almost solely confined to Asia [9].The probable transmission of V. cholerae O139 sequence types (ST69, ST187, ST161, ST162 and ST163) that reported elsewhere from South Asia was depicted in Fig. 1.

ResFinder 2.1 returned same antimicrobial resistance genes for all except two isolates (Table 1). catB9 gene responsible for chloramphenicol resistance was found common in all isolates. strA and strB (streptomycin resistance), floR (florfenicol/chloramphenicol resistance), and sul2 (sulphonamide resistance) genes were identified in all except FC1225 and FC1341 strains. The occurrence of these resistance genes were previously been reported in V. cholerae O139 serogroup. Number of antimicrobial resistance genes identified in these genomes as per comprehensive antibiotic resistance database, CARD and antibiotic resistance genes database, ARDB database matching were ~ 17 and ~ 6 genes. PlasmidFinder 1.3 resulted negative for all 10 isolates. Additionally, all 10 isolates were found to possess virulence genes including ctxA, ctxB, rtx, hlyA, zot, ace, tcpT, higB, doc, parE.

The clusters of regularly interspaced short palindromic repeats (CRISPR) and spacer sequences in the genome were screened using CRISPR finder (http://​crispr.​u-psud.​fr/​Server/​) [10] and PATRIC database which resulted in no CRISPR regions in these genomes (Table 1).

However, ISfinder (https://​www-is.​biotoul.​fr/​blast.​php) [11] resulted various IS elements that were listed in Table 1. Most of the IS elements such as IS3-ISVch4, IS200-IS1004, ISAs1-IS1358 (O139), IS481, Tn3-ISShfr9, IS66-ISPsy43, IS5-ISVch8, IS5-ISVch5, IS3-ISVch4, Tn3-ISPsy42 were previously reported from V. cholerae genomes. Of these, ISAs1-IS1358 was reported to be from V. cholerae O139. Prophage screening results showed that the isolates FC1105, FC1225, FC1341, FC1877, FC3611a and FC3611b were found to have V. cholerae phage CTX (NC_015209). The phage K139 (NC_003313) was detected in FC1817, whereas VFJ (NC_021562) was seen in FC2271 and FC2273 respectively.

All study isolates lacked the intI gene but were possessed the SXT element (int _SXT) with 99.9% identity to the reference sequence, which was mostly known to be, associated with the sulfamethoxazole and trimethoprim resistant strains [6]. All isolates harboured the seventh pandemic-specific gene (VC2346), suggesting that they belong to the same clonal linage and might originate from a common ancestor of the seventh pandemic strain.

The phylogenetic analysis based on MLST housekeeping genes revealed the relation between ST69 Indian strains and global strains (Fig. 2). All the study isolates are closely related to ST429 originated from M802 (ST71) global isolates. Also, M2140 (ST70), M796 (ST75), ST168, M1616 (ST73), ST169, ST170 and ST182 belong to the same clonal complex with ST71 as the founder type. goEBURST analysis of V. cholerae confirmed that ST69 belong to the clonal complex CC0 with the above-mentioned sequence types (Fig. 3).