Transmissible antimicrobial resistance in Escherichia coli isolated from household drinking water in Ibadan, Nigeria

Transmissible antimicrobial resistance in Escherichia coli isolated from household drinking water in Ibadan, Nigeria | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Confirmatory Results Transmissible antimicrobial resistance in Escherichia coli isolated from household drinking water in Ibadan, Nigeria View ORCID Profile Akeem G. Rabiu , Olutayo I. Falodun , View ORCID Profile Rotimi A. Dada , View ORCID Profile Ayorinde O. Afolayan , Olabisi C. Akinlabi , Elizabeth T. Akande , View ORCID Profile Iruka N. Okeke doi: https://doi.org/10.1101/2025.01.28.635236 Akeem G. Rabiu 1 Department of Microbiology, Federal University of Health Sciences , Ila-Orangun, Osun State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Akeem G. Rabiu Olutayo I. Falodun 2 Department of Microbiology, University of Ibadan , Ibadan, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: falod2013{at}gmail.com Rotimi A. Dada 3 College of Health Sciences, Bowen University , Iwo, Osun State, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rotimi A. Dada Ayorinde O. Afolayan 4 Institute for Infection Prevention and Hospital Epidemiology, Medical Centre-University of Freiburg , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ayorinde O. Afolayan Olabisi C. Akinlabi 5 Department of Pharmaceutical Microbiology, University of Ibadan , Ibadan, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elizabeth T. Akande 5 Department of Pharmaceutical Microbiology, University of Ibadan , Ibadan, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site Iruka N. Okeke 5 Department of Pharmaceutical Microbiology, University of Ibadan , Ibadan, Nigeria Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Iruka N. Okeke Abstract Full Text Info/History Metrics Preview PDF Abstract Contaminated household water in peri-urban urban communities is a reservoir for virulent Escherichia coli , but its role in the environmental transmission of antibiotic resistance genes (ARGs) remains poorly understood. This study characterized E. coli from household water and additionally aimed to investigate the transmissibility of ARGs from drug-resistant isolates. Twenty-five E. coli from thirteen household well water sources were tested for resistance to 14 antibiotics by disc diffusion and whole-genome sequenced using the Illumina platform. The ARGs and plasmid replicon types were respectively predicted using ResFinder and PlasmidFinder. Multidrug-resistant strains carrying plasmid replicons found in unrelated strains were conjugated with nalidixic acid-resistant (NAL R ) E. coli C 600 using the solid plate method. Fifteen isolates displayed a multi-drug resistance (MDR) phenotype with 18 possessing ARGs that confer resistance to trimethoprim-sulfamethoxazole, macrolide, sulphonamide, aminoglycoside, chloramphenicol, β-lactams, and tetracycline. Fifteen of the 25 isolates belonged to sequence type detected more than once, and fourteen of these were multidrug resistant. Through solid plate mating, beta-lactam-resistant qnrS1-tet-dfrA14- positive strains bearing IncFI-, IncHI2, and IncHI2A successfully transferred ampicillin resistance to a nalidixic acid-resistant derivative of E. coli - C600 . This research highlights the urgent need to safeguard household water sources against fecal contamination to curb the dissemination of ARGs among bacterial populations in this environment. Introduction The spread of antimicrobial resistance (AMR) threatens the efficacy of the antimicrobials administered in clinical practice. In 2019, 4.95 million individuals were estimated to have died globally due to drug-resistant bacterial infections [ 1 ]. AMR surveillance gives greater attention to human clinical isolates and less is known about transmission within and across other One Health sectors (animals and environment). This biased interest misses the opportunity to understand and interrupt the transmission of resistant organisms and/or resistance genes outside the clinic, especially in low- and middle-income (LMIC) countries [ 2 , 3 ]. Household water used for drinking and domestic purposes, particularly in informal urban settlements is an under-surveilled niche with the potential to impact human health [ 2 ]. Examining the role of contaminated water in the environmental transmission of AMR could assist in estimating the degree to which this niche contributes to AMR in settings where quality water is difficult to access and AMR data are scarce [ 1 ]. Moreover, the risk associated with contaminated drinking water as a medium for spreading resistance genes globally is yet to be investigated in detail [ 4 ]. Recent model estimates that improvements in Water Sanitation and Hygiene (WASH) could reduce AMR-related deaths by 247,800 [ 5 ]. However, the estimates did not include the possibility of non-pathogens in water serving as a reservoir for resistance genes because of a dearth of evidence. In Nigeria, waterborne diseases are common due to an inadequate supply of clean water, poor sanitation, and, in some cases, consumption of contaminated household water [ 6 - 8 ]. Alternate water sources to deficient public municipal water infrastructure [ 9 ], such as wells and boreholes, particularly when unprotected, used by householders are prone to faecal contamination. Recent research has shown that these sources harbour virulent E. coli and other enteric pathogens [ 8 , 10 , 11 ]. Still, little is known about the contribution of unprotected household water to the bacterial resistance reservoir and the potential of organisms in it to horizontally transfer antimicrobial resistance genes (ARGs). In southwestern Nigeria, one of the leading antimicrobial-resistant bacterial species found in clinical settings, and recovered from contaminated household water, is Escherichia coli [ 8 , 10 , 11 ]. Reports demonstrate that extended-spectrum-lactamase (ESBLs) and certain dfrA alleles are highly prevalent in African studies, including Nigeria [ 12 , 13 ]. We have recently reported proximal wells of different households can contain genetically indistinguishable strains such that household water is a vehicle for the clonal expansion of resistant bacteria. We theorize that bacteria in households carrying mobile elements may transmit them to other bacteria, particularly in our setting. Therefore, this study was designed to determine whether E. coli isolates recovered from contaminated household water are multidrug-resistant and can disseminate the ARGs they carry. We performed antibiotic susceptibility testing (AST) and whole genome sequencing of E. coli isolates, inspected the sequences of the strains for ARGs and plasmid replicons, and conjugated them with a plasmid-free E. coli -strain to explore the transfer of resistance phenotype and genotype. Materials and Methods Strain isolation and identification Twenty-five isolates were retrieved from our previous study in Ibadan notably Akinyele East (n=17), Akinyele South (n=5), and Lagelu (n=3) municipal area [ 10 ]. These two regions [Akinyele and Lagelu] have been each re-demarcated into three municipal districts due to urban renewal: Akinyele East LCDA (AELCDA, Akinyele South LCDA (ASLCDA), Akinyele LGA (ALGA), Lagelu West LCDA (LWLCDA), Lagelu North LCDA (LNLCDA) and Lagelu LGA (LLGA). The water sources were previously assessed for microbiological quality including isolation and characterisation of E. coli [ 10 ] and strains used in this study were from sites with very high coliform and E. coli counts ( Table 1 ). Figure 1 shows where the isolates were recovered in each local government area and Table 1 lists all the strains used in the study [ 10 ]. View this table: View inline View popup Download powerpoint Table 1. List of sample sources of E. coli used in this study Download figure Open in new tab Fig. 1: Whole genome SNP phylogeny and antimicrobial resistance of scherichia coli (n=25) isolated from 13 household drinking water in Ibadan, Nigeria. Red/blue colours respectively depict the presence/absence of the corresponding resistance phenotype, according to CLSI standards. Intermediate phenotypes are coloured yellow and green implies Not Applicable. The ARGs and plasmid replicon markers present are shown in black. Of the six municipal areas, isolates carrying plasmid replicons were retrieved from three municipalities: ASLCDA (yellow), AELCDA (black), and LLGA (pink). The colours white/black respectively illustrate the absence/presence of the corresponding ARGs. The sequence types were colour-annotated such that ST10=blue; ST2541=green; ST9987=yellow; ST48=cream; novel ST13028=brown; ST2008=pink; ST196=red; ST155=purple, novel ST11400=black; ST69=orange; ST4981=indigo; ST156=peach; ST414=violet; ST11=grape and ST9005=grey. The isolates are represented on the Microreact Map based on MLST colour coding. The majority of the isolates (n=18/25) carried plasmid replicons whereas most of the plasmid-bearers (n=16/18) were found in isolates obtained from the north-eastern part of Ibadan, especially the Akinyele East municipal area (AELCDA). The data can be visualised interactively on Microreact at https://microreact.org/project/bsddEhN1iRsb6rdGvmA5Yk-genomic-data-of-e-coli-isolated-from-well-water-sources-in-ibadan-nigeria . Distribution of isolates in the sampling regions Twenty-five E. coli were obtained from 13 households in Akinyele South (n=2), Akinyele East (n=8), and Lagelu (n=3) municipalities in the peri-urban Ibadan regions. No isolate was retrieved from Akinyele, Lagelu North, and Lagelu West districts. No isolate was also recovered from a borehole: all 25 isolates were retrieved from well water. A median of two E. coli isolates per E. coli -positive household well water samples was recorded. We previously indicated that fecal contamination of household well water sources in the examined water sources is exacerbated by rainfall [ 10 ]. AEW (1-3, 6-9, 11), AS (3, 5), and LAW (2-4) correspond to well water sources in the respective sampling points in Akinyele East and Akinyele South LCDA as well as Lagelu LGA. TCC and TEC were the sample points’ total coliform and total E. coli counts, the control strain was E. coli ATCC 25922 while the conjugative recipient was nalidixic acid-resistant E. coli ( E. coli NAL R )- C600 . Antibiotic Susceptibility Testing (AST) The isolates were subjected to AST using the Kirby-Bauer disc diffusion method and results were interpreted following the guidelines of the Clinical and Laboratory Standards Institute [ 15 ]. Fourteen antibiotics (Oxoid, United Kingdom) were used: ampicillin, AMP (10 μg), amoxicillin/clavullanate, AMC (10/20 μg), cefuroxime, CFX (30 μg), cefotaxime, CTX (30 μg), ceftazidime, CAZ (30 μg), aztreonam, ATM (30 μg), tetracycline, TE (30 μg), kanamycin, KAN (30 μg), chloramphenicol, C (30 μg), trimethoprim-sulfamethoxazole, SXT (1.25/23.75 μg), sulfonamide, S (250/300 μg), ciprofloxacin, CIP (5 μg), meropenem, MEP (10 μg) and imipenem, IMP (10 μg). Escherichia coli American Type Culture Collection (ATCC) 25922 was used as a control. Whole genome sequencing Genomic DNA extraction was performed as outlined previously [ 10 ]. Briefly, gDNA was extracted using the Promega Wizard DNA extraction kit and quantified by a dsDNA Broad Range fluorometric quantification assay (Invitrogen). Double-stranded DNA libraries were fragmented, tagged, and sequenced using the HiSeq X10 with 150 bp paired-ends Illumina technology. Genome assembly, antibiotic resistance, and plasmid gene identification The genomes were assembled de novo using Spades v3.9.0 [ 16 ] and annotated using Prokka v1.12 [ 17 ]. The complete sequence of E. coli _042 (accession no. FN554766 ) was used to deduce a whole-genome alignment of the sequence reads, and the SNP positions were determined using the SNP sites [ 18 ]. An identity point (90-100%), minimum gene length (60%), and threshold (90.0%) were used to match individual genes for each isolate to the reference database. The prediction of plasmid replicons was done using the PlasmidFinder 2.1 tool on the CGE website [ 19 ]. Multilocus sequence typing (MLST) was performed by Achtman’s MLST scheme through the website: https://cge.cbs.dtu.dk/services/MLST/ . Novel Sequence Types (STs) were assigned numbers by uploading the raw sequences to Enterobase at https://enterobase.warwick.ac.uk/species/ecoli/upload_reads . The ARGs were predicted using the ResFinder 4.1 tool available on the Center for Genomics Epidemiology (CGE) server ( https://cge.food.dtu.dk/services/ResFinder/ ) [ 20 ]. To determine mutation in the quinolone resistance determining region (QRDR) of qnrS -carrying isolates; gyrA, gyrB, parC , and parE regions encoding fluoroquinolone resistance were called from the raw fastq sequence files using ARIBA [ 21 ]. Mutational changes in the called sequences were assessed using PointFinder [ 22 ]. The presence of fluoroquinolone resistance-conferring variants was determined by mapping the gyrA, gyrB, parC , and parE sequences of the isolates to reference sequence [ 22 ]. The PointFinder database was queried to yield (i) point mutation specific to the species, and (ii) a summary of point mutations in the sequences. Conjugation on solid media The in vitro bacterial conjugation was performed using the solid plate mating method. Briefly, the donors – the isolates carrying bla TEM and the recipient – nalidixic acid-resistant E. coli - C600 (NAL R E. coli ) were grown overnight in Luria Bertani (LB) broth with appropriate antibiotic selection. The donors were selected with ampicillin or trimethoprim while nalidixic acid was used to select the recipient. Then, a ten-fold dilution of overnight donor and recipient culture was separately grown in LB at 37°C for 1 hr with no antibiotic selection. The donor and recipient cultures, 0.5 mL each, were dispensed into a fresh Eppendorf tube, spun at 6,000rpm for 5 minutes, and re-suspended in 10 µl LB without antibiotic. The suspension was spotted on LB plates, dried at room temperature for 15 minutes, and incubated at 37°C overnight. The mating culture was later suspended in 1 ml LB, vortexed and the conjugation was stopped on ice; the conjugation was performed in triplicate. A ten-fold dilution of the ended conjugation was prepared in cold phosphate-buffered saline and 10 µl of the reaction culture spread-plated on plates containing the antibiotic combination (i) ampicillin (100 mg/L), trimethoprim (50 mg/L) and nalidixic acid (100 mg/L); (ii) nalidixic acid (100 mg/L) and ampicillin (100 mg/L); (iii) nalidixic acid (100 mg/L) and trimethoprim (50 mg/L). Transconjugants and donors were verified by phenotype on MacConkey and Eosin Methylene Blue agars (Sumrall et al ., 2014) and re-tested for sensitivity to ampicillin, tetracycline, nalidixic acid, ciprofloxacin, cefotaxime, trimethoprim-sulfamethoxazole, and chloramphenicol. The donor and transconjugant colony-forming units were determined through serial dilutions and results were expressed as transconjugants per donor cell input to calculate the plasmid transfer efficiency [ 23 ]. Availability of whole genome sequence data The raw reads datasets produced in this study were deposited at the European Nucleotide Archive under bio project number PRJEB8667 ( https://www.ebi.ac.uk/ena/browser/view/PRJEB8667 ). Accessions are available in the supplementary file ( Table 1S ). RESULTS Phenotypic and Genotypic resistance of 25 Escherichia coli isolated from household water Antimicrobial susceptibility testing of 25 E. coli isolates from household water revealed that the majority were resistant to erythromycin (n=25, 100%) and ampicillin (n=19/25, 76%). Twelve isolates resisted sulphonamides. Resistance to tetracycline, kanamycin, and trimethoprim was seen in at least five strains. Multidrug resistance (MDR) was observed in 17 isolates, with 15 strains resisting antibiotics that belonged to at least three different classes. One isolate was non-susceptible to ampicillin, amoxicillin-clavullanate, sulphonamide, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole while two strains, in addition to the latter, resisted kanamycin ( Fig. 1 ). Genotypic analysis unveiled prevalent resistance genes, including aminoglycoside [ aph-6-ld, aph-3-lb (n=15), aac-3-IId (n=6), aadA1 (n=4) aadA5 (n=2)], β-lactam [ bla TEM-1B (n=13), tetracycline [ tetA (n=12)], chloramphenicol [ catA1 (n=8)], trimethoprim dfrA14 (n=7), dfrA1 (n=5), dfrA17 (n=2), and dfrA7 (n=1)]. Two variants of ampC plasmid-mediated class C β-lactamase genes ( bla CMY-75 and bla CMH-3 ) and a bla MOX gene were found in a bla TEM-1B - positive strain. Extended-spectrum-lactamase (ESBL) genes bla CTX-M-15 and bla CTX-M-65 co-existed in the genome of one strain, while two additional separate strains carried one of these ESBL genes: bla CTX-M-8 and bla CTX-M-15 . sul1, sul2 , and sul3 were respectively borne by six, 14, and one isolate(s). Isolates bearing sul1 bore sul2 but not vice versa while the sul3 was not detected with any other sul genes. No isolate carried aac-6-lb-cr , which confers ciprofloxacin and aminoglycoside resistance ( Fig. 1 ). Although no carbapenemase genes were detected, nor was resistance to carbapenems, one multidrug-resistant strain carrying mobile colistin resistance gene mcr-1 was found. Resistance genotypes detected in the isolates are likely responsible for their β-lactams, sulphonamide, and trimethoprim-sulfamethoxazole resistance. There was, however, discordance in aminoglycoside, tetracycline, and chloramphenicol phenotype-genotype. For instance, seven out of 12 isolates carrying tet , and five out of nine catA1- positives showed resistance ( Fig. 1 ). The topoisomerase IV subunit A parC genes of the isolates have about 99% percentage identity with the reference sequence [ 22 ]. Strains [182EC, 183EC, 184EC; SNP=15] and [172EC and 173EC; SNP=23] tested sensitive to quinolones even though they bore the qnrS1 PMQR gene. While 18/25 E. coli carried at least one plasmid replicon; isolates that showed resistance to more antimicrobials, particularly those harbouring ESBL genes, carried more plasmid replicons. For instance, E. coli (191EC) isolate carrying mcr-1 and other ARGs [ aph-6-Id, aph-3-Ib, aac-3-Iid, bla CTX-M-15 , bla CTX-M-65 , floR, qnrS1, tetA ] bore six replicons – ColMG828, IncFI-, IncFIB, IncX1, IncX1(1), IncX4(1) and IncI. It was observed that isolates (n=16/18) carrying plasmids were isolated from north-eastern Ibadan ( Fig 1 ). Three plasmid replicon types (Col-, IncF-, and IncHI-) were predominant and accounted for more than 60% of all the replicons. Five major STs were detected in this study -ST10 (5EC, 108EC, 172EC), ST2541 (13EC, 14EC, 15EC, 16EC), ST2008 (171EC, 172EC1, 173EC, 175EC), ST156 (69EC, 70EC, 70EC1), ST48 (166EC, 184EC) and novel ST13028 (167EC, 167EC1). Other strains individually belonged to STs-58, 69, 155, 196, 4981, 11400, and 9987*. Except for four ST2541 strains, which were near pan-sensitive, and three ST156 multidrug-resistant isolates with remarkably similar resistance gene ( aadA1, aph-3-Ib, aph-6-Id, bla TEM-1B , catA1, dfrA1, qnrB19, sul1, sul2 ) and IncF plasmid replicon, closely related strains belonging to the same ST typically had different resistance gene and plasmid replicon profiles. However, the diversity of the strain set was considerable and there were only six such clusters. Certain resistance gene combinations were seen repeatedly. Six of the seven strains with dfrA 14 , which carried a range of plasmid replicons (n=8) or no detected plasmid replicon (n=1), also bore tetA and qnrS1 , whilst four of five strains bearing dfrA1 carried qnrB19, sul1 and sul2 and carried IncF plasmid replicons. Furthermore, the strains were screened for a few bacteriocin genes common among E. coli . These include cia, cba, cma, cvaC -, cib, mchF, mchB, mchC . No colicin/ bacteriocin was detected more than once per isolate, and 22 of the isolates did not possess any of these genes. Spatial overview of isolate lineages resistance genes and plasmids A total of 15 multilocus sequence types were found among the 25 isolates with six STs recovered more than once. There were four isolates each belonging to STs 2008 (all from AELCDA), and 2541 (all recovered in ASLCDA), three ST156 isolates (from AELCDA). All isolates belonging to the same ST that had identical or near-identical resistance gene profiles were isolated from wells in the same LGA located less than 1 km apart. The three ST156 isolates recovered in AELCDA carried [ aadA1, dfrA1 ], and two carried qnr1b . These strains carried IncF [IncFI- and IncFIB-], which were also found in multidrug-resistant isolates from proximal sites in AELCDA ( Fig. 1 ). The four ST2008 isolates carried ( bla T EM-1B , bla CTX-M-8 , catA1, dfrA14, mph-A, qnrS1, sul2 , and tetA which were associated with IncF and IncH plasmid replicons. Two non-clonal ST10 isolates carrying IncF and IncH plasmid replicons and similar resistance gene profiles were also isolated from AELCDA, at locations proximal to the multidrug-resistant ST2008 strains. In addition to the IncH replicon-bearing strains, there were clusters of different STs that bore similar plasmid replicon and resistance gene repertoires. Most notable among these were IncX-bearing strains from STs 58, 196, and 4981, which carried among other ARGs ( aph-6-Id, aph-3-Ib, aac-3-Iid, tetA, qnrS1, bla TEM-1B , dfrA14 ) ESBL bla CTX-M-15 , bla CTX-M-65 , floR and plasmid-mediated colistin resistance gene, mcr-1 . Incidence of Escherichia coli across and within households The clonality of the strains was investigated through Single Nucleotide Polymorphism (SNP) analysis to understand isolate transmission within and across the 13 households where the organisms were found. There were two ST48 strains and four ST10 isolates but these had different resistance and plasmid replicon profiles and differed by >10,000 SNPS. As shown in Figure 2 , From ASLCDA, there was a cluster of four, predominantly sensitive ST2541 isolates from the same household (13EC, 14EC, 15EC, and 16EC), which differed by only 2-4 SNP differences. Three ST2008 (171EC, 173EC, 175EC) recovered from two neighboring AELCDA households carried identical ARGs [ dfrA14, aac-3(Iid), aph-3(Ib), aph_6(Id), bla TEM_1B , catA1, mphA, qnrS1, sul2, tetA ] and IncH replicons, and shared zero SNPs. Elsewhere in AELCDA, ST156 strains 69EC, 70EC, and 70EC1, harbouring identical ARGs were isolated from two households in the same neighbourhood. These strains had no SNP differences among them and, like the ST2008 isolates were therefore likely derived from a common ancestor. Download figure Open in new tab Figure 2: Matrix showing pairwise Single Nucleotide Difference (SNP) differences between Escherichia coli isolates obtained from household well water sources in Ibadan, Nigeria. Pairs with 10 SNPs are shaded light blue. STs and municipal Local Government Areas (LGA) are shaded as for Figure 1 . LLGA, AEL* and ASL* connote Lagelu local government area, Akinyele East and Akinyele South LCDA respectively. Conjugation of Escherichia coli strains with E. coli- C600 To explore the possibility of ARGs horizontal transmission, six isolates multidrug-resistant isolates not showing phenotypic quinolone resistance, were mated with a nalidixic acid-resistant variant of strain E. coli -C600. As shown in Table 2 ampicillin resistance, but not other resistances, were transferred in vitro from two of the strains. The efficiency of ampicillin resistance gene transfers for 171EC and 183EC respectively were 1.49 × 10 4 and 2.2 × 10 3 ( Table 2 ). Trimethoprim resistance was not transferred under the experimental conditions. View this table: View inline View popup Download powerpoint Table 2: Result of conjugation experiment and efficiency of plasmid transfer Discussion In this study, E. coli strains recovered from household well water were subjected to antimicrobial susceptibility testing and 15 (of 25) of these isolates were multidrug-resistant, carrying genes conferring resistance to three or more clinically significant antimicrobial classes. Resistance to commonly used ‘Access’ antimicrobials was frequently seen, but resistance to Watch/Reserve antimicrobials (cefotaxime, cefuroxime, ceftazidime, imipenem, meropenem, ciprofloxacin) was less common although plasmid-mediated quinolone resistance was often detected and mcr-1 , encoding colistin resistance was detected in one strain. This finding and the extent of the MDR E. coli (n=15, 60%) is alarming because the prevalence and types of resistance genes are similar to what is reported in clinical isolates in Nigeria (8,24,25). Antibiotic resistance phenotypes and genotypes for ampicillin, erythromycin, sulphonamide, and trimethoprim-sulfamethoxazole, but not for ciprofloxacin and tetracycline, were concordant. Most multiply-resistant isolates resisted tetracycline, ampicillin, trimethoprim-sulfamethoxazole, amoxicillin/clavulanic acid, and chloramphenicol. These Access antibiotics are cheap and orally active, commonly used at the primary care level in Nigeria [ 26 ]. The isolates were not resistant to ciprofloxacin, but PMQR genes [ qnrS 1 (n=6), qnrB81 (n=1), qnrB19 (n=3)] were common. These genes are insufficient to confer quinolone resistance, but can, however, contribute to ciprofloxacin resistance when present in combination with other quinolone resistance genes or mechanisms [ 27 ]. The absence of QRDR mutations in the strains carrying these genes accounts for why resistance was not seen but transfer of the PMQR genes can cause clinically significant quinolone resistance in a recipient strain that has them. In this study, qnrB81 and qnrB19 genes co-occurred with ColpHAD28 and Col440I plasmid replicons, which have been shown by other workers to mediate transfer and confer decreased susceptibility to quinolones [ 28 , 29 ]. Studies by Sumrall et al . [ 12 ] and Fortini et al . [ 30 ] in Nigeria indicated that the qnrS1 allele is associated with dfrA14 and tetA genes flanked by insertion sequences and carried on IncX plasmids. In this study, six isolates were qnrS1 -positive but none carried an IncX replicon: the gene repertoire co-occurred with IncFI, IncHI2, IncHI2A, and IncY replicons. Inspection of the genome sequences for dfrA-tet-qnrS1 revealed that qnrS1 was commonly seen in the genomes of multidrug-resistant strains while dfrA14 and tetA were co-founded with qnrS1 in four out of the six qnrS1 -positive MDR isolates. Of the three strains harbouring the qnrB19 gene: one bore tetA while the others carried dfrA1 . It is noteworthy that none of the strains carrying qnrB19 simultaneously bore tet or drfA genes. Previous studies in Akinyele that examined animal fecal matter reported isolates resistance to β-lactam (penicillin and ampicillin) and β-lactam inhibitor antibiotics (amoxicillin/clavulanic acid) [ 31 ]. Clinical human E. coli isolates from the same setting have also shown phenotypic resistance to similar (β-lactam – ampicillin) and other antibiotics – trimethoprim/sulfamethoxazole and ciprofloxacin [ 8 , 25 , 32 ]. Furthermore, the current study reports bla TEM-1B , ESBL bla CTX-M-8 , bla CTX-M-15, and bla CTX-M-65 and trimethoprim [ dfrA1, dfrA7, dfrA14 , and dfrA17 ] all of which have been detected in recent clinical studies [ 8 , 25 , 32 ] suggesting that these strains likely oscillate between humans and the environmental settings. A wide range of multilocus sequence types was detected among the isolates but there were four ST2008 and two ST48 strains isolated from the same local government area that were multidrug-resistant and other STs that showed within ST similarity in resistance gene profiles. In the case of two ST2008 and the ST156 isolates, the plasmid replicons were also similar, suggesting that these clusters, comprised of strains from different households may be derived from a point source even though a distance of about 500 metres lay between the wells from which the different isolates were recovered. SNP difference evaluations in Figure 2 show that, ST2008, ST 2541 (n=4), ST 156 (n=3), and ST 13028 (n=2) circulating in AELCDA and ASLDA, where most of the isolates were recovered, were clonal sharing identical ARGs and plasmids ( Fig. 1 ). In the case of ST2008 and ST156 clusters, clonal isolates were recovered from water sources of different but neighboring households within the Akinyele sampling region [Akinyele East and Ikereku]. In addition to pointing to the possible clonal transmission of multidrug-resistant E. coli strains, the finding of plasmid and resistance gene profiles in resistant clones that were seen in unrelated strains from other STs pointed to possible mobility of key resistance genes, something that has been previously reported for fecally derived isolates in Nigeria [ 12 ]. Our study included 11 isolates bearing the resistance gene combination dfrA-tet-qnrS1 . As stated above, these strains did not bear IncX plasmid replicons as found by Sumrall et al [ 12 ] but instead carried IncF and IncH replicons and all carried ESBL genes. When we mated six of these isolates with a nalidixic acid-resistant plasmid-free E. coli - C600 strain, resistance to trimethoprim, tetracycline, or the quinolones was not transferred but beta-lactam resistance was in two cases. The observed conjugation ESBL frequencies of 1.49 × 10 −4 and 2.2 × 10 3 were high enough to suggest that horizontal gene transfer could be possible outside the laboratory, and even in the household water ecosystem, at least for ESBLs. This study unveils that without preventing fecal contamination of drinking water, the spread of fecally-derived bacteria can support the horizontal spread of ARGs in household water by clonal expansion of resistant strains and transmission of mobile genetic elements. Further studies are suggested to determine whether household drinking water can lead to the human acquisition of environmentally disseminated ARGs. This would help to evaluate the extent of the threat to public health posed by consuming drinking water contaminated with bacteria carrying transmissible genes. In the interim, Household Water Treatment before consumption is recommended to forestall the spread of microbes carrying mobile and transmissible resistance traits. Conclusion Escherichia coli isolated from different household water sources in Ibadan carry a range of antimicrobial resistance genes, some of which can be transmitted horizontally, constituting a health threat and risk of infection with fecal-oral transmitted diseases. Improvements in Water, Sanitation, and Hygiene can potentially impact the transmission of antimicrobial resistance and enteric infections. Supporting Information 1S Table. Information on the isolates’ bioproject, sample, and sequence read archive accession Acknowledgments This work was supported by the African Research Leader’s Award MR/L00464X/1 to INO which was jointly funded by the United Kingdom Medical Research Council (MRC) and the United Kingdom Department for International Development (DFID) under the MRC/DFID Concordat agreement and is also part of the EDCTP2 program supported by the European Union. INO is a Calestous Juma Fellow supported by the Bill and Melinda Gates Foundation (INV-036234). References 1. ↵ Antimicrobial Resistance Collaborators . Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis . Lancet . 2022 ; 399 ( 10325 ): 629 – 55 . doi: 10.1016/S0140-6736(21)02724-0 OpenUrl CrossRef PubMed 2. ↵ Nadimpalli ML , Marks SJ , Montealegre MC , Gilman RH , Pajuelo MJ , Saito M , et al. Urban informal settlements as hotspots of antimicrobial resistance and the need to curb environmental transmission . Nat Microbiol . 2020 ; 5 ( 6 ): 787 – 95 . doi: 10.1038/s41564-020-0722-0 OpenUrl CrossRef PubMed 3. ↵ Ikhimiukor OO , Odih EE , Donado-Godoy P , Okeke IN . A bottom-up view of antimicrobial resistance transmission in developing countries . Nat Microbiol . 2022 ; 7 ( 6 ): 757 – 65 . doi: 10.1038/s41564-022-01124-w OpenUrl CrossRef PubMed 4. ↵ Madec J-Y , Haenni M , Ponsin C , Kieffer N , Rion E , Gassilloud B. Sequence type 48 Escherichia coli carrying the blaCTX-M-1 IncI1/ST3 Plasmid in drinking water in France . Antimicrob Agents Chemother . 2016 ; 60 ( 10 ): 6430 – 2 . doi: 10.1128/AAC.01135-16 OpenUrl Abstract / FREE Full Text 5. ↵ Lewnard JA , Charani E , Gleason A , Hsu LY , Khan WA , Karkey A , et al. Burden of bacterial antimicrobial resistance in low-income and middle-income countries avertible by existing interventions: an evidence review and modelling analysis . Lancet . 2024 ; 403 ( 10442 ): 2439 – 54 . doi: 10.1016/S0140-6736(24)00862-6 OpenUrl CrossRef 6. ↵ Adesoji AT , Ogunjobi AA . Detection of extended Spectrum Beta-Lactamases resistance genes among bacteria isolated from selected drinking water distribution channels in southwestern Nigeria . Biomed Res Int . 2016 ; 2016 : 1 – 9 . doi: 10.1155/2016/7149295 OpenUrl CrossRef 7. Olowe B , Oluyege J , Famurewa O. Prevalence of waterborne diseases and microbial assessment of drinking water quality in ado-Ekiti and its environs, southwestern, Nigeria . Br Microbiol Res J . 2016 ; 12 ( 2 ): 1 – 13 . doi: 10.9734/bmrj/2016/22444 OpenUrl CrossRef 8. ↵ Akinlabi OC , Nwoko E-SQ , Dada RA , Ekpo S , Omotuyi A , Nwimo CC , et al. Epidemiology and risk factors for diarrheagenic Escherichia coli carriage among children in northern Ibadan, Nigeria . Am J Trop Med Hyg . 2023 ; 109 ( 6 ): 1223 – 32 . doi: 10.4269/ajtmh.22-0618 OpenUrl CrossRef PubMed 9. ↵ Ojelabi SA , Agbede OA , Wahab BA , Aiyelokun OA , Ojelabi OA . Water quality assessment of Eleyele Dam . Vol. 10 . Ibadan, South-Western ; 2018 . 10. ↵ Rabiu AG , Falodun OI , Fagade OE , Dada RA , Okeke IN . Potentially pathogenic Escherichia coli from household water in peri-urban Ibadan, Nigeria . J Water Health . 2022 ; 20 ( 7 ): 1137 – 49 . doi: 10.2166/wh.2022.117 OpenUrl CrossRef PubMed 11. ↵ Alabi OS , Akintayo I , Odeyemi JS , Oloche JJ , Babalola CM , Nwimo C , et al. Suboptimal bacteriological quality of household water in municipal Ibadan, Nigeria . Am J Trop Med Hyg . 2024 ; 110 ( 2 ): 346 – 55 . doi: 10.4269/ajtmh.23-0134 OpenUrl CrossRef PubMed 12. ↵ Sumrall ET , Gallo EB , Aboderin AO , Lamikanra A , Okeke IN . Dissemination of the transmissible quinolone-resistance gene qnrS1 by IncX plasmids in Nigeria . PLoS One . 2014 ; 9 ( 10 ): e110279 . doi: 10.1371/journal.pone.0110279 OpenUrl CrossRef PubMed 13. ↵ Tanko N , Bolaji RO , Olayinka AT , Olayinka BO . A systematic review on the prevalence of extended-spectrum beta lactamase-producing Gram-negative bacteria in Nigeria . J Glob Antimicrob Resist . 2020 ; 22 : 488 – 96 . doi: 10.1016/j.jgar.2020.04.010 OpenUrl CrossRef PubMed 14. Appleyard RK . Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12 . Genetics . 1954 ; 39 ( 4 ): 440 – 52 . doi: 10.1093/genetics/39.4.440 OpenUrl FREE Full Text 15. ↵ CLSI . M100: Performance Standards for Antimicrobial Susceptibility Testing. 31st ed. Clinical & Laboratory Standards Institute ; 2021 . 16. ↵ Bankevich A , Nurk S , Antipov D , Gurevich AA , Dvorkin M , Kulikov AS et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing . Journal of Computational Biology . 2012 ; 19 ( 5 ): 455 – 477 . doi: 10.1089/cmb.2012.0021 OpenUrl CrossRef PubMed 17. ↵ Seemann T. Prokka: rapid prokaryotic genome annotation . Bioinformatics . 2014 ; 30 ( 14 ): 2068 – 9 . doi: 10.1093/bioinformatics/btu153 OpenUrl CrossRef PubMed Web of Science 18. ↵ Page AJ , Taylor B , Delaney AJ , Soares J , Seemann T , Keane JA , et al. SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments . Microb Genom . 2016 ; 2 ( 4 ): e000056 . doi: 10.1099/mgen.0.000056 OpenUrl CrossRef PubMed 19. ↵ Carattoli A , Zankari E , García-Fernández A , Voldby Larsen M , Lund O , Villa L , et al. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing . Antimicrob Agents Chemother . 2014 ; 58 ( 7 ): 3895 – 903 . doi: 10.1128/AAC.02412-14 OpenUrl Abstract / FREE Full Text 20. ↵ Zankari E , Hasman H , Cosentino S , Vestergaard M , Rasmussen S , Lund O , et al. Identification of acquired antimicrobial resistance genes . J Antimicrob Chemother . 2012 ; 67 ( 11 ): 2640 – 4 . doi: 10.1093/jac/dks261 OpenUrl CrossRef PubMed Web of Science 21. ↵ Hunt M , Mather AE , Sánchez-Busó L , Page AJ , Parkhill J , Keane JA , et al. ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads . Microb Genom . 2017 ; 3 ( 10 ): e000131 . doi: 10.1099/mgen.0.000131 OpenUrl CrossRef PubMed 22. ↵ Zankari E , Allesøe R , Joensen KG , Cavaco LM , Lund O , Aarestrup FM . PointFinder: a novel web tool for WGS-based detection of antimicrobial resistance associated with chromosomal point mutations in bacterial pathogens . J Antimicrob Chemother . 2017 ; 72 ( 10 ): 2764 – 8 . doi: 10.1093/jac/dkx217 OpenUrl CrossRef PubMed 23. ↵ Lu J , Manchak J , Klimke W , Davidson C , Firth N , Skurray RA , et al. Analysis and characterisation of the IncFV plasmid pED208 transfer region . Plasmid . 2002 ; 48 : 24 – 37 . OpenUrl CrossRef PubMed Web of Science 24. Abayneh M , Tesfaw G , Abdissa A. Isolation of Extended-Spectrum Beta-Lactamase-(ESBL-) Producing Escherichia coli and Klebsiella pneumoniae from Patients with Community-Onset Urinary Tract Infections in Jimma University Specialized Hospital, Southwest Ethiopia . Can. J Infect Dis Med Microbiol . 2018 25. ↵ Afolayan AO , Aboderin AO , Oaikhena AO , Odih EE , Ogunleye VO , Adeyemo AT , et al. An ST131 clade and a phylogroup A clade bearing an O101-like O-antigen cluster predominate among bloodstream Escherichia coli isolates from South-West Nigeria hospitals . Microb Genom . 2022 ; 8 ( 12 ). doi: 10.1099/mgen.0.000863 OpenUrl CrossRef 26. ↵ Babalola C , Awoleye S , Akinyemi J , Kotila O. Evaluation of prescription pattern in Osun State (Southwest) Nigeria . J Public Health Epidemiol . 2011 ; 3 : 94 – 8 . OpenUrl 27. ↵ Hooper DC , Jacoby GA . Mechanisms of drug resistance: quinolone resistance: Mechanisms of quinolone resistance . Ann N Y Acad Sci . 2015 ; 1354 ( 1 ): 12 – 31 . doi: 10.1111/nyas.12830 OpenUrl CrossRef PubMed 28. ↵ Fiegen U , Klein G , de Jong A , Kehrenberg C. Detection of a novel qnrB19-carrying Plasmid variant mediating decreased fluoroquinolone susceptibility in Salmonella enterica serovar Hadar . Microb Drug Resist . 2017 ; 23 ( 3 ): 280 – 4 . doi: 10.1089/mdr.2016.0067 OpenUrl CrossRef PubMed 29. ↵ Juraschek K , Malekzadah J , Malorny B , Käsbohrer A , Schwarz S , Meemken D , et al. Characterization of qnrB-carrying plasmids from ESBL- and non-ESBL-producing Escherichia coli . BMC Genomics . 2022 ; 23 ( 1 ): 365 . doi: 10.1186/s12864-022-08564-y OpenUrl CrossRef PubMed 30. ↵ Fortini D , Fashae K , García-Fernández A , Villa L , Carattoli A. Plasmid-mediated quinolone resistance and β-lactamases in Escherichia coli from healthy animals from Nigeria . J Antimicrob Chemother . 2011 ; 66 ( 6 ): 1269 – 72 . doi: 10.1093/jac/dkr085 OpenUrl CrossRef PubMed Web of Science 31. ↵ Fasiku OS , Omotosho OO , Ajulo SO , Adesanwo EE , Oni OD , Awoyele AA , et al. Occurrence and antibiotic sensitivity patterns of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in pigs in Ibadan, Nigeria . In: ECA 2023. Basel Switzerland: MDPI ; 2023 . p. 8 . 32. ↵ Seni J , Peirano G , Okon KO , Jibrin YB , Mohammed A , Mshana SE , et al. The population structure of clinical extra-intestinal Escherichia coli in a teaching hospital from Nigeria . Diagn Microbiol Infect Dis . 2018 ; 92 ( 1 ): 46 – 49 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted January 30, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. 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