Vibrio cholerae serogroup O1 is responsible for epidemic and pandemic cholera and remains a global public health threat. This organism has been well established as a resident flora of the aquatic environment that alters its phenotypic and genotypic attributes for better adaptation to the environment. To reveal the diversity of clinical isolates of V. cholerae O1 in the Bay of Bengal, we performed whole-genome sequencing of isolates from Kolkata, India, and Dhaka, Bangladesh, collected between 2009 and 2016. Comparison with global isolates by phylogenetic analysis placed the current isolates in two Asian lineages, with lineages 1 and 2 predominant in Dhaka and Kolkata, respectively. Each lineage possessed different genetic traits in the cholera toxin B subunit gene, Vibrio seventh pandemic island II, integrative and conjugative element, and antibiotic-resistant genes. Thus, although recent global transmission of V. cholerae O1 from South Asia has been attributed only to isolates of lineage 2, another distinct lineage exists in Bengal. IMPORTANCE Cholera continues to be a global concern, as large epidemics have occurred recently in Haiti, Yemen, and countries of sub-Saharan Africa. A single lineage of Vibrio cholerae O1 has been considered to be introduced into these regions from South Asia and to cause the spread of cholera. Using genomic epidemiology, we showed that two distinct lineages exist in Bengal, one of which is linked to the global lineage. The other lineage was found only in Iran, Iraq, and countries in Asia and differed from the global lineage regarding cholera toxin variant and drug resistance profile. Therefore, the potential transmission of this lineage to other regions would likely cause worldwide cholera spread and may result in this lineage replacing the current global lineage.

The self-transferring integrative and conjugative elements (ICEs) are large genomic segments carrying several bacterial adaptive functions including antimicrobial resistance (AMR). SXT/R391 family is one of the ICEs extensively studied in cholera-causing pathogen Vibrio cholerae. The genetic characteristics of ICE-SXT/R391 in V. cholerae are dynamic and region-specific. These ICEs in V. cholerae are strongly correlated with resistance to several antibiotics such as tetracycline, streptomycin and trimethoprim-sulfamethoxazole. We screened V. cholerae O1 strains isolated from cholera patients in Kolkata, India from 2008 to 2015 for antibiotic susceptibility and the presence of ICEs, and subsequently sequenced their conserved genes. Resistance to tetracycline, streptomycin and trimethoprim-sulfamethoxazole was detected in strains isolated during 2008–2010 and 2014–2015. The genes encoding resistance to tetracycline (tetA), trimethoprim-sulfamethoxazole (dfrA1 and sul2), streptomycin (strAB), and chloramphenicol (floR) were detected in the ICEs of these strains. There was a decrease in overall drug resistance in V. cholerae associated with the ICEs in 2011. DNA sequence analysis also showed that AMR in these strains was conferred mainly by two types of ICEs, i.e., ICETET (comprising tetA, strAB, sul2, and dfrA1) and ICEGEN (floR, strAB, sul2, and dfrA1). Based on the genetic structure, Kolkata strains of V. cholerae O1 had distinct genetic traits different from the ICEs reported in other cholera endemic regions. Transfer of AMR was confirmed by conjugation with sodium azide resistant Escherichia coli J53. In addition to the acquired resistance to streptomycin and trimethoprim-sulfamethoxazole, the conjugally transferred (CT) E. coli J53 with ICE showed higher resistance to chloramphenicol and tetracycline than the donor V. cholerae. Pulsed-field gel electrophoresis (PFGE) based clonal analysis revealed that the V. cholerae strains could be grouped based on their ICEs and AMR patterns. Our findings demonstrate the epidemiological importance of ICEs and their role in the emergence of multidrug resistance (MDR) in El Tor vibrios.

Non-O1/non-O139 nontoxigenic Vibrio cholerae associated with cholera-like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non-O1/non-O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole-genome ---

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Experimental equipment Analytical TLC were performed on silica gel 60 F254 (Merck) and RP-18 F254 (Merck). Column chromatography was carried out on silica gel 60 (70-230 and 40-50 mesh). The NMR spectra were recorded on a Bruker AVANCE III 500 spectrometer (1 H NMR, 500 MHz; 13 C NMR, 125 MHz) or a JEOL ECA-600 (1 H NMR, 600 MHz; 13 C NMR, 150 MHz). Chemical shifts for 1 H and 13 C NMR are given in parts per million (d) relative to tetramethylsilane (dH 0.00) and residual solvent signals (dC 77.0) for CDCl3, (dH 3.30, dC 49.0) for CD3OD and (dH 7.16, dC 128.4) for C6D6 as internal standards. Mass spectra were measured on Exactive Orbitrap Mass Spectrometer (Thermo Fischer Scientific). IR was measured on a JASCO FVS-6000 spectrometer. UV spectra were recorded on a JASCO-V-730 spectrophotometer. HPLC analysis was performed on a JASCO AS-1555-10 Intelligent Sampler, a JASCO PU-4180 RHPLC Pump, a JASCO MD-4017 Photo Diode Array Detector (JASCO), which equipped with a COSMOSIL Packed Column 5C18-MS-II (φ4.6 mm×150 mm) (Nacalai tesque). Fungal material Aspergillus kawachii IFO 4308 and Colletotrichum incanum MAFF 238704 were obtained from Biological Resource Center, NITE (NBRC) and Ministry of Agriculture, Forestry Fisheries (MAFF) respectively. Each fungus was cultured in a PDB liquid medium for 3 days and the cultured mycelium was ground to a fine powder in liquid N2. To the mycelial powder in 1.5 mL tube, 100 µL TE buffer and lysis solution (SDS 1g, 2.5 mL Tris-HCl buffer (1 M), EDTA•2Na•2H2O 186 mg, NaCl 292 mg, 50 mL mQ) were added, gently inverted and incubated 5 min on ice. After centrifugation at 15,000 rpm, 4ºC, for 20 min, 100 µL of the supernatants were transferred to a new 1.5 mL tube, followed by phenol extraction using Phenol: Chloroform: Isoamyl Alcohol 25:24:1 (pH 5.2, Nacalai tesque) and ethanol precipitation. After centrifugation again, TE buffer were added to the pellet of DNA. The DNA solutions of A. kawachii and C. incanum were used as a template for cloning of akmlA-D and cimlA-D respectively. Heterologous host strains Aspergillus oryzae NSAR1 (niaD − , sC − , DargB, adeA −) S1 was used as the host for fungal expression. Escherichia coli Dh5a (TAKARA) was used for the cloning of akmlA-D and cimlA-D. Culture medium Culture medium for A. kawachii IFO 4308 and C. incanum MAFF 238704 PDB (agar) medium: Potato-Dextrose Broth (DIFCO) 7.2 g (and agarose (Nacalai tesque) 4.5 g) in 300 mL distilled water. CPS medium (0.015% adenine): Czapek-Dox Broth (Difco) 1.05 (2.63) g, peptone* 0.3 (0.75) g, Soluble Starch (Nacalai tesque) 0.6 (1.5) g, Maltose Monohydrate (Nacalai tesque) 0.3 (0.75) g and adenine 9 (22.5) mg in 60 (150) mL distilled water. *peptone: mixture of 0.2 (0.5) g of soy peptone, casein peptone and meat peptone (Nacalai tesque). MYG medium: Bacto Peptone (DIFCO) 0.06 g, Bacto Malt Extract (DIFCO) 1.2 g, D-(+)glucose (Nacalai tesque) in 60 mL distilled water. Culture medium for A. oryzae NSAR1 PDB agar medium (adenine 0.05%, arginine 0.1%): Potato-Dextrose Broth (DIFCO) 7.2 g and agarose (Nacala...