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

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.

Multidrug-resistant phenotypes among biofilm-producing staphylococcal isolates from farm-raised fish: a molecular scrutiny.

AIMS: To describe the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from farm-raised fish. METHODS AND RESULTS: Fish samples (n = 180) collected from aquaculture farms in three major districts of Kerala, yielded 45 methicillin-resistant staphylococci (MRS) (25%) isolates. All of the isolates were resistant to tested beta-lactams, and 19 (42.22%) were resistant to flouroquinolones. According to the resistance profile of each isolate, 33 of 45 (73.33%) are identified as multidrug-resistant (MDR) isolates, with an average multiple antibiotic resistance (MAR) index of 0.41 ± 0.09. Notably, virulence determinants such as classical enterotoxin (sea & seb), and SE-like toxin (seg & sei) genes co-existed with Antimicrobial Resistance Genes (ARGs) in isolates studied. Regardless of the isolates being strong biofilm producers, only 23 (51.1%) harbored icaA and icaD genes. The heterogeneity of MRSA (n = 17) clones were evident as the species belonged to three major spa types (t345, t2526, and t1309) and sequence types (STs), viz., ST772, ST88, and ST672. CONCLUSION: The current study on molecular characterization of the MRS isolates sheds light on the preventive measures that need to be implemented to limit the spread of AMR pathogens in aquaculture.

Resistance profiles and genotyping of extended-spectrum beta-lactamase (ESBL) -producing and non-ESBL-producing E. coli and Klebsiella from retail market fishes.

Studies on antimicrobial resistance (AMR) profiles and epidemiological affirmation for AMR transmission are limited in fisheries and aquaculture settings. Since 2015, based on Global Action Plan on AMR by World Health Organization (WHO) and World Organization for Animal Health (OIE), several initiatives have been under taken to enhance the knowledge, skills and capacity to establish AMR trends through surveillance and strengthening of epidemiological evidence. The focus of this study was to determine the prevalence of antimicrobial resistance (AMR), its resistance profiles and molecular characterization with respect to phylogroups, antimicrobial resistance genes (ARGs), virulence genes (VGs), quaternary ammonium compounds resistance (QAC) genes and plasmid typing in retail market fishes. Pulse field gel electrophoresis (PFGE) to understand the genetic lineage of the two most important Enterobacteriaceae members, E. coli and Klebsiella sp. was performed. 94 fish samples were collected from three different sites viz., Silagrant (S1), Garchuk (S2) and North Guwahati Town Committee (NGTC) Region (S3) in Guwahati, Assam. Out of the 113 microbial isolates from the fish samples, 45 (39.82%) were E. coli; 23 (20.35%) belonged to Klebsiella genus. Among E. coli, 48.88% (n = 22) of the isolates were alerted by the BD Phoenix M50 instrument as ESBL, 15.55% (n = 7) as PCP and 35.55% (n = 16) as non-ESBL. E. coli (39.82%) was the most prevalent pathogen among the Enterobacteriaceae members screened and showed resistance to ampicillin (69%) followed by cefazoline (64%), cefotaxime (49%) and piperacillin (49%). In the present study, 66.66% of E. coli and 30.43% of Klebsiella sp. were categorized as multi drug resistance (MDR) bacteria. CTX-M-gp-1, with CTX-M-15 variant (47%), was the most widely circulating beta-lactamase gene, while other ESBL genes blaTEM (7%), blaSHV (2%) and blaOXA-1-like (2%) were also identified in E. coli. Out of the 23 isolates of Klebsiella, 14(60.86%) were ampicillin (AM)-resistant (11(47.82%) K. oxytoca, 3(13.04%) K. aerogenes), whereas 8(34.78%) isolates of K. oxytoca showed intermediate resistance to AM. All Klebsiella isolates were susceptible to AN, SCP, MEM and TZP, although two K. aerogenes were resistant to imipenem. DHA and LAT genes were detected, respectively, in 7(16%) and 1(2%) of the E. coli strains while a single K. oxytoca (4.34%) isolate carried MOX, DHA and blaCMY-2 genes. The fluoroquinolone resistance genes identified in E. coli included qnrB (71%), qnrS (84%), oqxB (73%) and aac(6)-Ib-cr (27%); however, in Klebsiella, these genes, respectively, had a prevalence of 87%, 26%, 74% and 9%. The E. coli isolates belonged to phylogroup A(47%), B1(33%) and D(14%). All of the 22(100%) ESBL E. coli had chromosome-mediated disinfectant resistance genes viz., ydgE, ydgF, sugE(c), mdfA while 82% of ESBL E. coli had emrE. Among the non-ESBL E. coli isolates, 87% of them showed the presence of ydgE, ydgF and sugE(c) genes, while 78% of the isolates had mdfA and 39% had emrE genes respectively. 59% of the ESBL and 26% of the non-ESBL E. coli had showed the presence of qacEΔ1. The sugE(p) was present in 27% of the ESBL-producing E. coli and in 9% of non-ESBL isolates. Out of the 3 ESBL-producing Klebsiella isolates, 2(66.66%) K. oxytoca isolates were found harboring plasmid-mediated qacEΔ1 gene while one (33.33%) K. oxytoca isolate had sugE(p) gene. IncFI was the most prevalent plasmid type detected in the isolates studied, with A/C (18%), P (14%), X, Y (9% each) and I1-Iγ (14%, 4%). 50% (n = 11) of the ESBL and 17% (n = 4) of the non-ESBL E. coli isolates harboured IncFIB and 45% (n = 10) ESBL and one (4.34%) non-ESBL E. coli isolates harboured IncFIA. Dominance of E. coli over other Enterobacterales and diverse phylogenetic profiles of E. coli and Klebsiella sp. suggests the possibility of contamination and this may be due to compromised hygienic practices along the supply chain and contamination of aquatic ecosystem. Continuous surveillance in domestic markets must be a priority in addressing antimicrobial resistance in fishery settings and to identify any unwarranted epidemic clones of E. coli and Klebsiella that can challenge public health sector.

Genomic insights of beta-lactamase producing Klebsiella quasipneumoniae subsp. similipneumoniae belonging to sequence type 1699 from retail market fish, India.

Klebsiella quasipneumoniae is a recently described species and often misidentified as Klebsiella pneumoniae. Here, we report the genomic characterization of Klebsiella quasipneumoniae subsp. similipneumoniae (India238 strain) isolated from fish. The annotated genome acknowledged the presence of blaCTX-M-15, blaOKP-B-1, fosA5, oqxAB and virulence genes. The strain with ST1699 and serotypes KL52 and OL103 also harboured insertion sequences (ISs): ISKpn26 and ISEc9. Three complete phage genomes were identified in contigs 1 and 6 of the bacterial genome, enhancing the prospects of genome manipulation. The study highlights the pitfall of conventional microbiological identification methods to distinguish K. pneumoniae and K. quasipneumoniae. This is the first Indian study documenting the incidence of extended-spectrum beta-lactamase (ESBL)-producing K. quasipneumoniae subsp. similipneumoniae from a non-clinical environment, equipped with virulomes and associated mobile genetic elements. Given that fish can act as a potential vector for transmission of antimicrobial resistance genes, our findings have paramount importance on human health.