Multidrug resistant (MDR) Escherichia coli strains are ideal species to monitor the antimicrobial resistance (AMR) gene flux from a One Health perspective. Slaughterhouses serve as a potential hotspot for environmental AMR dissemination, and the current study thus aimed to identify MDR E. coli contamination in slaughterhouses in comparison with their associated poultry farms. Comparative resistome analysis has identified both shared a similar resistance pattern with highest resistance to tetracycline (85%), followed by ciprofloxacin, ampicillin (>80% each), and cephalosporin (>70%). Here, 58.8% of the MDR strains were also found to be extended-spectrum beta-lactamase (ESBL) positive, and ESBL gene screening shows high genotype-to-phenotype correlation. A strong ESBL-producing F2.4 strain was further selected for whole-genome sequencing (WGS) and the result revealed it to have close similarity to the pathogenic E. coli O101:H9 serotype, which is least reported from India. The pathogen finder has further predicted the strain to have 85% potential possibility to be a human pathogen. The WGS-based analysis of F2.4 has also revealed it to have the presence of hlyE, which encodes for the avian E. coli alpha-hemolysin, along with other virulence factors such as fdeC, iss2, ibeC, csg, fim, and esp genes, which are of zoonotic potential. The genome also contained around seven beta-lactamase-encoding genes imparting resistance to penicillin and cephalosporin, whereas 74% of the antibiotic resistance genes predicted from F2.4 encoded for the efflux pump-related genes. Among them, genes such as emrK, emrY, acrD, evgS, evgA, and YojI were underreported, highlighting the importance and the need for geographical-based AMR risk analysis.
AbdelwahabGE, IshagHZA, Al HammadiZM, et al.Antibiotics resistance in Escherichia coli isolated from livestock in the emirate of Abu Dhabi, UAE, 2014–2019. Int J Microbiol, 2022; 2022(1):3411560; doi: 10.1155/2022/3411560
2.
AktherT, RanjaniS, HemalathaS. Nanoparticles engineered from endophytic fungi (Botryosphaeria rhodina) against ESBL-producing pathogenic multidrug-resistant E. coli. Environ Sci Eur, 2021; 33(1):83.
3.
BagheriM, GhanbarpourR, AlizadeH. Shiga toxin and beta-lactamases genes in Escherichia coli phylotypes isolated from carcasses of broiler chickens slaughtered in Iran. Int J Food Microbiol, 2014; 177:16–20; doi: 10.1016/j.ijfoodmicro.2014.02.003
4.
CarattoliA, ZankariE, García-FernándezA, et al.In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother, 2014; 58(7):3895–3903; doi: 10.1128/aac.02412-14
5.
ChenL, ZhengD, LiuB, et al.VFDB 2016: Hierarchical and refined dataset for big data analysis—10 years on. Nucleic Acids Res, 2016; 44(D1):D694–D7; doi: 10.1093/nar/gkv1239
6.
ChirilaF, TabaranA, FitN, et al.Concerning increase in antimicrobial resistance in Shiga toxin-producing Escherichia coli isolated from young animals during 1980–2016. Microbes Environ., 2017; 32(3):252–259; doi: 10.1264/jsme2.ME17023
7.
Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing—30th Edition. CLSI supplement M100. 2020.
8.
CosentinoS, Voldby LarsenM, Møller AarestrupF, et al.PathogenFinder-distinguishing friend from foe using bacterial whole genome sequence data. PLoS One, 2013; 8(10):e77302; doi: 10.1371/journal.pone.0077302
9.
DierikxCM, van der GootJA, SmithHE, et al.Presence of ESBL/AmpC-producing Escherichia coli in the broiler production pyramid: A descriptive study. PLoS One, 2013; 8(11):e79005; doi: 10.1371/journal.pone.0079005
10.
DosterE, LakinSM, DeanCJ, et al.MEGARes 2.0: A database for classification of antimicrobial drug, biocide and metal resistance determinants in metagenomic sequence data. Nucleic Acids Res, 2020; 48(D1):D561–D569; doi: 10.1093/nar/gkz1010
11.
DwiyantoJ, HorJW, ReidpathD, et al.Pan-genome and resistome analysis of extended-spectrum ß-lactamase-producing Escherichia coli: A multi-setting epidemiological surveillance study from Malaysia. PLoS One, 2022; 17(3):e0265142; doi: 10.1371/journal.pone.0265142
12.
EftekharF, RastegarM, GolalipoorM, et al.Detection of Extended Spectrum B-Lactamases in Urinary Isolates of Klebsiella pneumoniae in Relation to blaSHV, blaTEM and blaCTX-M gene carriage. Iran J Public Health, 2012; 41(3):127–132.
13.
FeldgardenM, BroverV, HaftDH, et al.Validating the AMRFinder tool and resistance gene database by using antimicrobial resistance genotype-phenotype correlations in a collection of isolates. Antimicrob Agents Chemother, 2019; 63(11):e00483–e00419; doi: 10.1128/aac.00483-19
14.
GrantJR, EnnsE, MarinierE, et al.Proksee: In-depth characterization and visualization of bacterial genomes. Nucleic Acids Res., 2023; 51(W1):W484–W492; doi: 10.1093/nar/gkad326
15.
GuptaSK, PadmanabhanBR, DieneSM, et al.ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes. Antimicrob Agents Chemother, 2014; 58(1):212–220; doi: 10.1128/aac.01310-13
16.
IngleDJ, ValcanisM, KuzevskiA, et al.In silico serotyping of E. coli from short read data identifies limited novel O-loci but extensive diversity of O: H serotype combinations within and between pathogenic lineages. Microb Genom, 2016; 2(7):e000064; doi: 10.1099/mgen.0.000064
17.
JainC, Rodriguez-RLM, PhillippyAM, et al.High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun, 2018; 9(1):5114; doi: 10.1038/s41467-018-07641-9
18.
JiaB, RaphenyaAR, AlcockB, et al.CARD 2017: Expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res, 2017; 45(D1):D566–D573; doi: 10.1093/nar/gkw1004gkw1004
19.
JoensenKG, TetzschnerAM, IguchiA, et al.Rapid and easy in silico serotyping of Escherichia coli isolates by use of whole-genome sequencing data. J. Clin. Microbiol., 2015; 53(8):2410–2426; doi: 10.1128/jcm.00008-15
20.
JolleyKA, BrayJE, MaidenMC. Open-access bacterial population genomics: BIGSdb software, the PubMLST. org website and their applications. Wellcome Open Res, 2018; 3:124; doi: 10.12688/wellcomeopenres.14826.1
21.
La RagioneRM, CooleyWA, WoodwardMJ. The role of fimbriae and flagella in the adherence of avian strains of Escherichia coli O78:K80 to tissue culture cells and tracheal and gut explants. J. Med. Microbiol., 2000; 49(4):327–338; doi: 10.1099/0022-1317-49-4-327
22.
LebertL, MartzSL, JaneckoN, et al.Prevalence and antimicrobial resistance among Escherichia coli and Salmonella in Ontario smallholder chicken flocks. Zoonoses Public Health, 2018; 65(1):134–141; doi: 10.1111/zph.12381
23.
MagiorakosAP, SrinivasanA, CareyRB, et al.Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect, 2012; 18(3):268–281; doi: 10.1111/j.1469-0691.2011.03570.x
24.
MehdiY, Létourneau-MontminyMP, GaucherML, et al.Use of antibiotics in broiler production: Global impacts and alternatives. Anim Nutr, 2018; 4(2):170–178; doi: 10.1016/j.aninu.2018.03.002
25.
MoriyaY, ItohM, OkudaS, et al.KAAS: An automatic genome annotation and pathway reconstruction server. Nucleic Acids Res, 2007; 35(Web Server issue):W182–W185; doi: 10.1093/nar/gkm321
26.
MuazK, RiazM, AkhtarS, et al.Antibiotic residues in chicken meat: Global prevalence, threats, and decontamination strategies: A review. J. Food Prot., 2018; 81(4):619–627; doi: 10.4315/0362-028X.JFP-17-086
27.
NayfachS, CamargoAP, SchulzF, et al.CheckV assesses the quality and completeness of metagenome-assembled viral genomes. Nat. Biotechnol., 2021; 39(5):578–585; doi: 10.1038/s41587-020-00774-7
28.
OloweOA, IdrisOJ, TaiwoSS. Prevalence of tet genes mediating tetracycline resistance in Escherichia coli clinical isolates in Osun State, Nigeria. Eur J Microbiol Immunol (Bp), 2013; 3(2):135–140; doi: 10.1556/eujmi.3.2013.2.7
29.
PoppeC, MartinLC, GylesCL, et al.Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poult intestinal tract. Appl. Environ. Microbiol., 2005; 71(3):1184–1192; doi: 10.1128/AEM.71.3.1184-1192.2005
30.
RamadanAA, AbdelazizNA, AminMA, et al.Novel bla CTX-M variants and genotype-phenotype correlations among clinical isolates of extended spectrum beta lactamase-producing Escherichia coli. Sci Rep, 2019; 9(1):4224; doi: 10.1038/s41598-019-39730-0
RoerL, JohannesenTB, HansenF, et al.CHTyper, a web tool for subtyping of extraintestinal pathogenic Escherichia coli based on the fumC and fimH alleles. J. Clin. Microbiol., 2018; 56(4):e00063–e00018; doi: 10.1128/jcm.00063-18
33.
Salgado-CaxitoM, BenavidesJA, AdellAD, et al.Global prevalence and molecular characterization of extended-spectrum β-lactamase producing-Escherichia coli in dogs and cats–a scoping review and meta-analysis. One Health, 2021; 12:100236; doi: 10.1016/j.onehlt.2021.100236
SharmaJ, KumarD, HussainS, et al.Prevalence, antimicrobial resistance and virulence genes characterization of nontyphoidal Salmonella isolated from retail chicken meat shops in Northern India. Food Control, 2019; 102:104–111; doi: 10.1016/j.foodcont.2019.01.021
36.
SreejithS, ShajahanS, PrathiushPR, et al.Healthy broilers disseminate antibiotic resistance in response to tetracycline input in feed concentrates. Microb. Pathog., 2020; 149:104562; doi: 10.1016/j.micpath.2020.104562
37.
SreekumaranS, PremnathM, PrathyushPR, et al.Predicting human risk with multidrug resistant Enterobacter hormaechei MS2 having MCR 9 gene isolated from the feces of healthy broiler through whole-genome sequence-based analysis. Curr Microbiol, 2024; 81(1):8; doi: 10.1007/s00284-023-03492-w
38.
SuzekBE, WangY, HuangH, et al.; UniProt Consortium. UniRef clusters: A comprehensive and scalable alternative for improving sequence similarity searches. Bioinformatics, 2015; 31(6):926–932; doi: 10.1093/bioinformatics/btu739
39.
TanTC, TangTX, ZhangZX, et al.Serotypes and virulence genes of extraintestinal pathogenic Escherichia coli isolates from diseased pigs in China. Vet J, 2012; 192(3):483–488; doi: 10.1016/j.tvjl.2011.06.038
40.
TitilawoY, SibandaT, ObiL, et al.Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of faecal contamination of water. Environ. Sci. Pollut. Res., 2015; 22(14):10969–10980; doi: 10.1007/s11356-014-3887-3
41.
UddinMB, AlamMN, HasanM, et al.Molecular detection of colistin resistance mcr-1 gene in multidrug-resistant Escherichia coli isolated from chicken. Antibiotics (Basel), 2022; 11(1):97; doi: 10.3390/antibiotics11010097
42.
WaghamareRN, PaturkarAM, VaidyaVM, et al.Screening of antimicrobial residues and confirmation of doxycycline in samples collected from chicken farms and processing units located around Mumbai, India. IJAR, 2020; 54(Of):1415–1421; doi: 10.18805/ijar.B-3899
43.
WeiB, ShangK, ChaSY, et al.Conjugative plasmid-mediated extended spectrum cephalosporin resistance in genetically diverse Escherichia coli from a chicken slaughterhouse. Animals (Basel), 2021; 11(9):2491; doi: 10.3390/ani11092491
44.
WickRR, JuddLM, GorrieCL, et al.Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol, 2017; 13(6):e1005595; doi: 10.1371/journal.pcbi.1005595
45.
WidodoA, KhairullahAR, EffendiMH, et al.Extended-spectrum β-lactamase-producing Escherichia coli from poultry: A review. Vet World, 2024; 17(9):2017–2027; doi: 10.14202/vetworld.2024.2017-2027
46.
WuS, ZhuZ, FuL, et al.WebMGA: A customizable web server for fast metagenomic sequence analysis. BMC Genomics, 2011; 12(1):444; doi: 10.1186/1471-2164-12-444
47.
YılmazM, AkÖ, HacıseyitoğluD, et al.A comparative study of VITEK-2, Double Disc Synergy and Combined Disc Methods for detection of ESBL (Extended Spectrum Beta-Lactamase) production in Escherichia coli and Klebsiella pneumoniae strains. J. Contemp. Med, 2022; 12(1):116–120; doi: 10.16899/jcm.954056
48.
ZankariE, HasmanH, CosentinoS, et al.Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother, 2012; 67(11):2640–2644; doi: 10.1093/jac/dks261
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