Emergence of multidrug resistant, ctx negative seventh pandemic Vibrio cholerae O1 El Tor sequence type (ST) 69 in coastal water of Kerala, India

Abstract

Seventh pandemic Vibrio choleare O1 El Tor strain is responsible for the on-going pandemic outbreak of cholera globally. This strain evolved from non-pathogenic V. cholerae by acquiring seventh pandemic gene (VC 2346), pandemic Islands (VSP1 and VSP2), pathogenicity islands (VP1 and VP2) and CTX prophage region. The cholera toxin production is mainly attributed to the presence of ctx gene in these strains. However, several variants of this strain emerged as hybrid strains or atypical strains. The present study aimed to assess the aquatic environment of Cochin, India, over a period of 5 years for the emergence of multidrug resistant V. cholerae and its similarity with seventh pandemic strain. The continuous surveillance and monitoring resulted in the isolation of ctx negative, O1 positive V. cholerae isolate (VC6) from coastal water, Cochin, Kerala. The isolate possessed the biotype specific O1 El Tor tcpA gene and lacked other biotype specific ctx, zot, ace and rst genes. Whole genome analysis revealed the isolate belongs to pandemic sequence type (ST) 69 with the possession of pandemic VC2346 gene, pathogenic island VPI1, VPI2, and pandemic island VSP1 and VSP2. The isolate possessed several insertion sequences and the SXT/R391 family related Integrative Conjugative Elements (ICEs). In addition to this, the isolate genome carried virulence genes such as VgrG, mshA, ompT, toxR, ompU, rtxA, als, VasX, makA, and hlyA and antimicrobial resistance genes such as gyrA, dfrA1, strB, parE, sul2, parC, strA, VC1786ICE9-floR, and catB9. Moreover, the phylogenetic analysis suggests that the isolate genome is more closely related to seventh pandemic V. cholerae O1 N16961 strain. This study reports the first incidence of environmental ctx negative seventh pandemic V. choleare O1 El Tor isolate, globally and its presence in the aquatic system likely to induce toxicity in terms of public health point of view. The presence of this isolate in the aquatic environment warns the strict implementation of the epidemiological surveillance on the occurrence of emerging strains and the execution of flagship program for the judicious use of antibiotics in the aquatic ecosystem.

Figure 1

Multiple mauve alignment of ICEs of isolate (VC6) with the reference ICEs. From top to bottom: VC-isolate genome (VC6); GQ463142.1-V. cholerae Ind5 integrating conjugative element ICEVchind5 (Accession No: GQ463142.1); JN648379.1- Vibrio cholerae strain VC1786ICE (Accession No: JN648379.1). The putative ICE elements of reference ICEs shown as homologous block and are linked to the ICE of isolate genome connected by line. Image is generated by MegAlign Pro sequence alignment software.

Figure 2

(A) Circular genome view of the isolate VC6 depicting the ICE region. B ICE element of the VC6 genome. A, (B): ICE element of the isolate genome found in between 2906562 to 2947023 bp and 3346860 bp to 3358885 bp with a total length of 40,462 bp in region 1 and 12026 bp in region 2. Region 1 and 2 of the ICE elements contained 44 and 7 genes constituting a total of 51 genes predicting various phenotypes of genes found in the SXT/R391 Family of ICEs.

Figure 3

Multiple mauve genome alignment view of the isolate VC6 and five other reference genomes using progressive mauve algorithm using MegAlign Pro sequence alignment software. From top to bottom: VC- Isolate genome (VC6); 2012Env-9- V. cholerae strain 2012Env-9; M66-2- V. cholerae strain M66-2; MJ-1236- V. cholerae strain MJ-1236; V060002- V. cholerae strain V060002 strain. The genomic sequences were arranged into locally collinear blocks with homologous region are connected by lines. Homologous genomic blocks including inverted regions are differently positioned in each genome.

Figure 4

BLAST comparison of isolate genome with reference genomes using Blast Ring Image Generator (BRIG). Vibrio cholerae strain V060002 strain used as reference genome. From inside to outside Ring 1: GC content; Ring 2: GC Skew; Ring 3: BLAST comparison with V. cholerae O1 biovar El Tor strain N16961; Ring 4: BLAST comparison with V. cholerae strain 2012Env-9; Ring 5: BLAST comparison with V. cholerae strain M66-2; Ring 6: BLAST comparison with V. cholerae strain MJ-1236; Ring 7: BLAST comparison with Isolate genome (VC6).

Figure 5

Bayesian phylogenetic tree of the isolate genome (VC6) with reference genomes. Unrooted bayesian phylogenetic tree with strict clock model was used for assessing the relationship among the isolate VC6 and the representative reference strains. The concatenated sequences of the both the chromosomes of the isolate were used along with full genome and/or concatenated sequences of reference genomes available in NCBI database (V. cholerae M66-2 complete sequence NC_012578.1/NC-012580.1; V. cholerae O1 biovar El Tor strain N16961 complete sequence AE003852.1/AE003853.1; V. cholerae strain V060002 chromosome, complete genome NZ_AP018677.1; V. cholerae MJ-1236 complete sequence NC_012668.1/CP001486.1; and V. cholerae strain 2012Env-9 complete sequence CP012997.1/CP012998.1). Posterior probability values of ≥ 0.91 are indicated as dark nodes Bayesian analysis shows the possible lineage of VC6 genome to the nearest clade containing seventh pandemic O1 El Tor biovar N16961 strain and pre-pandemic M66-2 strain.

Figure 6

SNP based phylogenetic tree obtained from CSIPhylogeny-1.4. The concatenated sequences of both the chromosomes of Vibrio cholerae O1 biovar El Tor strain N16961 (AE003852.1/AE003853.1) was used as reference genome. The tree is visualized by using Fig.Tree v1.3. Default input parameters such as minimum Z score of 1.96, read mapping quality of 25, and SNP quality of 30 were used for the construction of SNP tree. Total number of positions and percentage similarity of the investigated genomes with reference genome in SNP tree is given in Table 5.

Figure 7

Phylogenetic tree derived as a part of PATRIC comprehensive genome analysis report of the isolate genome. The tree is generated based on global protein families in which PATRIC PGFams automatically selects reference and representative genomes based on k-mer based functional assignments followed by Markov cluster algorithm. Bootstrap values were calculated by standard bootstrapping method and nodes are labelled with bootstrap values.