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

Vibrio cholerae is responsible for the cause of severe life-threatening infection known as cholera. The study aimed to analyze the genetic make-up of V. cholerae O139 isolates from India and compare its phylogeny with the global strains. The genome data revealed that all isolates were of same sequence type (ST69) which belongs to seventh pandemic clone, with same virulence gene profile and, antimicrobial resistance gene profile except for two isolates. No known CRISPR repeats were identified in any of these isolates. Three different phages were identified among the isolates. All the isolates were found to harbour int SXT and seventh pandemic-specific gene (VC2346). Results from this study enhance our understanding on the persistence of ST69 V. cholerae O139 over 20 years.


Introduction
Historically, only the O1 serogroup of V. cholerae has been the causative agent of epidemic cholera. In late 1992, in India and Bangladesh, a novel serogroup designated V. cholerae O139 emerged, replacing V. cholerae O1, and gave rise to major cholera outbreaks [1]. Based on the whole genome phylogenies of V. cholerae, the relation of isolates responsible for global cholera could be understood [2]. Nearly 141 draft genomes of V. cholerae O139 are accessible in the NCBI genome projects. Evolution and adaptation mechanism of this pathogen can be demonstrated based on genomic diversity of clinical isolates from various geographical regions and timescale [3]. The current study aims to perform and report the whole genome shotgun sequences of V. cholerae O139 isolates for the first time in India. The genomes reported here will help to better understand the evolution and endemicity of cholera in India.

Next generation sequencing
The whole genome shotgun sequencing for the V. cholerae O139 isolates was performed using Ion Torrent (PGM, Life Technologies) with 400-bp read chemistry (Life Technologies, Carlsbad, CA). Genomic DNA libraries were prepared using Ion Plus Fragment Library Kit; (Life Technologies) according to manufacturer's instructions. AMPure beads were used to purify the genomic

Downstream genome analysis
The whole genome data were analyzed using open access tools at Center for Genomic Epidemiology (CGE) webbased server. Sequence types for the study isolates were determined using MLST 1.  [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].

Phylogenetic analyses
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

Genome features, virulence and antimicrobial resistance determinants
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.

Repeat regions and insertional elements
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).
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.

Phylogenetic analyses
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 abovementioned sequence types (Fig. 3).

Accession numbers
The genome sequences of V. cholerae O139 strains have been deposited at DDBJ/ENA/Genbank under the accession numbers NGQQ00000000-NGQZ00000000. The version described in this manuscript is version one.

Conclusion
To the best of our knowledge, this is the first report of ST69 V. cholerae O139 in India. Though there are few reports of clinical V. cholerae isolates from India, our study adds new dimension to the earlier finding to show that the sequence type of V. cholerae O139 continues to persist in India for over 20 years is of ST69. This implies that clonal expansion of V. cholerae O1 seventh pandemic has resulted in ST69 strains. The isolates exhibited same virulence profile but different antimicrobial resistance gene profile. This study helps in understanding the epidemiology of V. cholerae and genomic relationship between these isolates with the strains from China and Thailand. Also a continuous surveillance of V. cholerae O139 serogroup can provide more insights into the evolution of V. cholerae.
Authors' contributions DRNK, MSDP, SA and BV designed the study. MSDP and TS performed the laboratory work. DRNK and MSDP analysed the data and wrote the manuscript. All authors read and approved the final manuscript.