Predominance of genetically diverse ESBL Escherichia coli identified in resistance mapping of Largest fresh cum

26 Antimicrobial resistance (AMR) burden in Escherichia coli along the 90 km stretch of Vembanad Lake, 27 Kerala, India was assessed. Seventy-seven percent of water samples drawn from 35 different stations of the Lake 28 harbored E. coli. Antibiotic susceptibility test performed on 116 E. coli isolates revealed 81% were resistant to ≥ 29 one antibiotic with 39 AMR profiles, 30% multidrug resistant, 32% extended spectrum  lactamase (ESBL) 30 producers as per CLSI.The probability of isolating cefotaxime resistant E. coli was the highest 0.7 (P ≤ 0.05) in the 31 Lake. Genetically diverse ESBL types bla TEM-116, bla CTX-M -152 , bla CTX-M -27, bla CTX-M -55 , bla CTX-M-205, and bla SHV-27 were 32 identified. Molecular typing (ERIC PCR, MLST and PBRT) confirmed the diversity among E. coli between and 33 within the stations. ST11439 and Single and Double Loci Variants of ST443, ST4533 were identified in Multi locus 34 sequence typing (MLST) analysis. Inc plasmids (B/O, F, W, I1, FIIA, HI1, P-1  , K/B and N) identified in the Lake 35 evidences the transmission potential. Low multiple antibiotic resistance index (average < 0.2) indicating lower risk 36 to the human population albeit, an emerging concern of ESBL resistance in the Lake. The occurrence of genetically 37 variant ESBL E. coli in Vembanad Lake signals health hazards and necessitates pragmatizing strategic control 38 measures. 39


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The human and animal health care systems, off late have been witnessing the menace of Antimicrobial 51 Resistance (AMR) (  4 Sequence Typing (MLST), Plasmid Based Replicon Typing (PBRT) and phenotypic methods like antimicrobial 77 resistance profiling, carbon utilization, etc (Nemoy et al. 2005; Mohapatra et al. 2007). Of all these, PBRT and 78 ERIC PCR are very powerful and cost-effective tools next to MLST, and PFGE for the discrimination of E. coli 79 based on the genetic relatedness (Harwood et al. 2014; Kim et al. 2017). Implementation of these phenotypic and 80 genotyping tools in fecal indicator bacteria from water bodies remains an indispensable marker tool for microbial 81 source tracking using bacteria .

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The use of clinical breakpoints in determining AMR in health care may not be suitable for assessing the

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Vembanad Lake which spreads over three districts (Alappuzha, Kottayam, and Ernakulam) of Kerala, India 90 is considered to be the longest (96 km) in India (09˚00' -10˚40'N and 76˚00'-77˚30'E). It has an inflow of water 91 from six major rivers and is a complex wetland system (Haldar et al. 2019). Freshwater dominant southern zone and 92 a brackish water dominant northern zone separated by brackish water regulating barrage (bund) are the salient 93 features of the Lake. Livelihood activities in the Lake are agriculture, fishing, tourism, inland navigation, coir 94 retting, and lime shell collection. This biologically diverse Lake is facing threats due to industrialization and 95 urbanization (Selvam et al. 2012). Tourism, the major activity in this lake, is concentrated in the southern zone.

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The information on AMR in Vembanad Lake and their genetic characteristics is scant. The present study 97 planned to understand 1. The prevalence of E. coli in the Vembanad Lake at different stations with CLSI 98 breakpoints; 2. Determine the prevalent AMR patterns and multidrug resistance (MDR); 3. Estimate the risk 99 associated by multiple antibiotic resistance index (MAR) and 4. To link the genetic diversity of ESBL genes with 100 the ERIC PCR tool as a pilot study for microbial source tracking.

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The collected water samples were enriched in 3x sterile Presence-Absence (P-A) broth (1: 3 ratio) and after 114 overnight incubation at 35 ±1°C, streaked on pre-set Eosin Methylene Blue (EMB) agar for primary screening from 115 which 2-10 characteristic colonies were picked and secondary confirmation was carried out on Mac Conkey and 116 HiCrome ECC agars (Baird et al. 2017). Gram-negative rods with catalase production, oxidase non-production, and 117 IMVC test (++--) characteristics were subjected for molecular confirmation. DNA template was prepared with the 118 washed cell suspension from 1mL overnight culture in 1x TE Buffer pH 8.0 by heat shock method and stored at -119 80°C until further use. PCR reaction was carried out in thermocycler (Veriti, Applied BioSystem) using uidA 120 primers (Godambe et al. 2017), and E. coli that did not produce amplicons specific for uidAwere further tested for 121 phoAgene-specific primers (Murugadas et al. 2016) and PCR products were analyzed in 2% agarose gel in 1x TAE 122 buffer at 80V for 1h in horizontal gel electrophoresis system and visualized in the gel documentation system.

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In vitro antimicrobial susceptibility testing 124 Phenotypicantimicrobial susceptibility testing (AST) was carried out by Disc Diffusion Assay (DDA) 125 with15 antibiotics belonging to nine different classes (

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dendrogram was generated based on the similarity matrix in SPSS software version 16.

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Results and discussion

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Prevalence of E. coli in the lake

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The study is the first of its kind that established the AMR pattern in 35 stations of the Vembanad Lake,

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of the E. coli isolated from Kottayam, Alapuzha and Ernakulam regions of Vembanad Lake, respectively (Table S1).

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In the Indian health care system, the major variants of ESBL producers harboredblaTEM followed by blaCTX-  the Vembanad Lake but similarities also existed between and within the isolates from different geographical 313 locations of the Lake. Within the clusters, several clades were formed that indicated very closely related E

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.coliisolates from different sites of the Lake (Fig. 3).

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ERIC PCR analysis and clustering of the phenotypic AMR profile data revealed that the multidrug-resistant

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This clone ST155 has been recognized as the most important strain that has an intrinsic ability to acquire colistin 332 resistance (Matamoros et al.2017

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Mean MAR index for environmental E. coli was 0.14 (ranged from 0.0 to 0.6) and region-wise MAR index 356 for Alapuzha, Ernakulam, and Kottayam regions werealso below the risk criteriaof 0.2 (Table S1). The majority of inferred that the Kottayam region of the Vembanad Lake carried diverse and high-risk AMR isolates which is the 361 major tourist point of the state. High-risk areas identified in the study are marked in the resistance map (Fig. 2b).

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The

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Ecoff value determined for the E. coli isolates of the present study revealed that the majority of these 377 isolates were wild type in nature for the tested antibiotics; however, the presence of antibiotic resistance genes 378 cannot be disregarded. When applying Ecoff values, the decreased susceptibility to amoxicillin/clavulanic acid (45%

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With the total population of 35 million, density of more than 859 km -2 in the Lake vicinity that is three     The base data generated in this study is attached as a supplementary file as Table S1.