Molecular characterization of multidrug-resistant (MDR) Escherichia coli in the Greater Accra Region, Ghana: a ‘One Health’ approach.

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Michael A. Olu-Taiwo, Beverly Egyir, Christian Owusu-Nyantakyi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4480595/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 May, 2025 Read the published version in One Health Outlook → Version 1 posted 5 You are reading this latest preprint version Abstract Background : ‘One health’, a concept that highlights the need to bring on board multiple players and actors together to addressing major health problems, have been proposed to be effective in data gathering to mitigate the menace of antimicrobial drug resistance (AMR). Data on MDR and extended-spectrum-beta-lactamase-producing Escherichia coli (ESBL- EC ) across humans, animals and the environment are limited in low-and-middle-income-countries (LMICs) including Ghana. Objective : This study used one health approach to determine the prevalence, antibiogram and AMR genes of ESBL- EC from diverse sources. Methodology : A cross-sectional study conducted in the Accra and Tema metropolis, Ghana. We randomly collected 1500 non-duplicated specimens from healthy human, cattle, pigs, lettuce, spring onions, pork, beef and soil samples. Escherichia coli ( E. coli ) was isolated and confirmed by MALDI-TOF MS. E. coli isolates were screened for their susceptibility against 13 antibiotic agents and ESBL-production. ESBL- EC isolates were whole-genome sequenced (WGS) and in silico analysis was used to determine AMR genes, sequence types (STs) and plasmid replicon types. Result : Overall, E. coli was recovered from 140 of 1500 (9.3%) specimens processed. About one-third of these E. coli isolates 50 (35.7%) were resistant to three or more antibiotics, and 30 (21.5%) were ESBL- EC . The proportion of ESBL- EC identified in healthy humans, cattle, pig, beef and soil were 14 (20.0%), 9 (22.5%), 3 (15.0%), 1 (50.0%) and 3 (37.5%), respectively. No E. coli was isolated from lettuce, spring onions and pork. Overall, the ESBL- EC exhibited high levels of resistance to ampicillin (100%), cefuroxime (100%), ciprofloxacin (53.6%), and to tetracycline (58.2%). However, all ESBL- EC isolates were sensitive to meropenem. The prevalent AMR genes detected were bla TEM-1B (32.0%; n=8), tet A (48.0%; n=12) and sul2 (32.0%; n=8). The dominant STs were ST10 (12%; n=3), ST 9312 (12%; n=3), ST 206 (12%; n=3) and ST 4151 (12%; n=3). IncFIB(Apoo1918) (40.0%; n=10) and IncFII(pCoo) (36.0%; n=9) plasmid replicons were commonly detected. Conclusion : Within the metropolis surveyed, we identified MDR ESBL- EC harbouring various AMR genes and plasmid replicons with diverse E. coli STs in healthy humans, animals and the environment. This study finding of bla CTX-M-15 in agricultural soil isolate is worrisome, emphasizing the need for a one-health approach in combating AMR. Antimicrobial resistance (AMR) multi-drug resistant (MDR) Escherichia coli (E. coli) Extended-spectrum-beta-lactamase (ESBL) Ghana. Figures Figure 1 Figure 2 Figure 3 1. Introduction Among global health challenges, AMR is a typical illustration of a ‘One Health' crisis that affects humans, animals and the environment [ 1 – 2 ]. Of particular public health concern in the 21st century, is the steadily escalating prevalence of AMR among pathogenic and commensal bacteria [ 3 – 5 ]. In recent times, AMR has been recognized to be a major driver for the persistence, dissemination and distribution of MDR high-risk clones, including E. coli ST131, ST10, ST38, ST73 and ST206 often associated with intestinal and extra-intestinal diseases [ 6 – 7 ]. In 2019, it was estimated that of the 4.95 million human associated deaths, 1.27 million deaths was attributable to MDR bacterial infections [ 8 ]. This number has been projected to escalate to 10 million by 2050, if no measures are initiated to handle the AMR situation [ 8 ]. Thus, an integrated and multifaceted “One Health” approach is urgently needed to help mitigate and manage the AMR challenge. In a more recent systematic review, Ramatla and colleagues [ 9 ] showed that the pooled prevalence estimates (PPE) of E. coli AMR genes including bla TEM-1, amp C, tetA , and bla TEM were 36.3%, 34.4%, 32.9%, and 28.8%, respectively. The most prevalent E. coli AMR genes in animals were bla TEM (30%), sulI (36.2%), sulII (32.0%), tet A (31.5%), and strB (30.8%), while, among humans, bla TEM (28.8%), sulI (27.8%), sulII (42.2%), tetA (42.0%), and strB (34.9%) were the most dominant E. coli AMR genes as reported by Escher and coworkers [ 10 ]. In the last 20 years, fecal carriage prevalence of ESBL- EC in healthy individuals vary globally; the highest in Southeast Asia (27%), and the lowest in Europe (6%) [ 11 ]. Till date, data on AMR under a One Health lens in Africa is scanty [ 10 ]. In Africa, majority of (LMICs) lack the resource capacity to use WGS technology to determine the inter-connectedness of ESBL -EC strains recovered from humans, food-animals and the environment under a ‘One health' surveillance of AMR [ 12 ]. There is an urgent need to conduct a detailed molecular characterization of ESBL- EC isolates derived from humans, food producing animals and the environment to contribute baseline data on AMR genes, plasmid replicon types and STs of public health significance and relevance to the One health surveillance database [ 13 ]. According to a 2014 study finding by WHO, one major aspect to the global response to AMR is surveillance, however, the WHO Africa region possesses one of the highest gaps in data on the prevalence of AMR [ 14 ]. WGS and in silico analysis has shown to be a relevant and reliable molecular tool in AMR surveillance due to its sensitivity to detect AMR genes or mutations [ 15 ]. Therefore, this study aimed to use ‘One Health’ approach to determines the prevalence and patterns of AMR and to characterize AMR genes, STs and plasmid replicon types among ESBL- EC strains recovered from healthy humans, food-animals and environment sources in the Greater Accra Region, Ghana. 2. Methods 2.1 Study Design and Area This study was a cross-sectional study carried out in Accra and Tema metropolis in the Greater Accra Region of Ghana, between January, 2022-April, 2023. Greater Accra is one of the 16 administrative regions in Ghana. Though the smallest in terms of size it is highly populated, industrialized, and serves as the commercial nerve centre of Ghana [ 16 ]. Greater Accra encompasses a land mass size of 3,245 Km 2 or a 1.4% of the total area of Ghana. It has an estimated urban population of 4.6 million and accounted for 15.4% of Ghana’s total population in the year 2016 [ 17 ]. Accra metropolitan district and Tema metropolitan district are the only two districts with city status in the Greater Accra Region (Fig. 1.0 ). “Accra” usually referred to as the Accra metropolitan area, also serves as the capital city of Ghana [ 16 ]. In the Accra city, peri-urban communities such as James Town, Korle-Gonno, Abossey-Okai, and Agbogloshie were selected for sampling, while in Tema metropolitan area, peri-urban communities including Ashiaiman, Adjei-Kojo, Tema Newtown and community one, were also selected. These peri-urban communities were selected due to their dense population, inadequate sanitation facilities and the close proximity to domestic and farm animals. 2.1.1 Inclusion and exclusion criteria Only apparently healthy individuals (human) of all age group from the study area, who consented to participate was recruited. Also, only healthy animals from consenting owners were sampled. Individuals on antibiotics medication and unwell was excluded, likewise, sick animals and diseased animals were excluded from this study. 2.1.2 Sample size for apparently healthy human Sample size was determined based on the formula as described by Daniel and colleagues (1999). N = Z 2 Xp (1-p)/d 2 Where: N = required sample size Z = confidence Interval for 95% CI is 1.96 d = precision (margin of error at 5%), i.e. 0.05 p = expected prevalence of ESBL -EC in healthy human [Unknown 50%]. Using the formula above, an approximated total of 400 stool samples was sampled. 2.1.3 Sample size for cattle Expected sample size for cattle was calculated with 11.1% prevalence from a previous study conducted in Greater Kumasi in the Ashanti region of Ghana by Ohene Larbi et al, (2021), Using the formula above, an approximated total of 200 anal swabs from cattle was sampled. 2.1.4 Sample size for pig Expected sample size for pig was calculated with 7.1% prevalence from a previous study conducted by Ohene Larbi et al, (2021). Using the formula above, an approximated total of 100 anal swabs from pigs was sampled. 2.1.5 Sampling Overall, a total of 1500 non-duplicate specimens were collected from apparently healthy humans (stool samples n = 400), cattle (anal swabs n = 200), pigs (anal swabs n = 100), soil sample (n = 400), food samples (beef n = 100, pork n = 100, lettuce n = 100 and spring onions n = 100) into sterile containers or Cary Blair medium. All samples were labelled and transported on ice packs within 2 hr of collection to the Microbiology Laboratory at the School of Biomedical and Allied Health Sciences, University of Ghana for cultural analysis. 2.3 Bacteriological processing of samples 2.3.1 Stool and anal swabs In brief, 1 g of human stool sample was placed in 9 ml of EC enrichment broth (Oxoid, Basingstoke, UK) and homogenized for one minute and aerobically incubated at 37°C for 18–24 hr as described by Purushottam and colleagues [ 19 ]. Anal swabs from cattle and pigs were placed in 9 mls of EC broth for enrichment for 18–24 hr and aerobically incubated at 37°C overnight. An aliquot from both pre-enriched broths were streaked onto MacConkey agar medium plate (Oxoid, Basingstoke, UK) and aerobically incubated at 37°C for 18–24 hr as described by Purushottam and colleagues [ 19 ]. 2.3.2 Food samples In brief, 25 g portion of every food sample was aseptically placed in 225 ml of EC enrichment broth (Oxoid, Basingstoke, UK) and homogenized for one minute, then aerobically incubated at 37°C for 18–24 hr, followed by streaking an aliquot onto MacConkey agar medium plate (Oxoid, Basingstoke, UK) and incubated at 37°C and 44°C for 18–24 hr as described by Purushottam and colleagues [ 19 ]. 2.3.3 Soil samples In brief, 10 g of soil samples was placed in 90 ml of EC enrichment broth (Oxoid, Basingstoke, UK) and homogenized for one minute and incubated at 37°C for 18–24 hr. After serial dilution of 10 1 -10 5 , an aliquot was streaked onto MacConkey agar medium plate (Oxoid, Basingstoke, UK) and aerobically incubated at 37°C for 18–24 hr as described by Purushottam and colleagues [ 19 ]. 2.4. Identification and susceptibility testing of E. coli isolates Pink moist medium sized colonies, suggestive of E. coli isolates, were phenotypically characterized as per standard biochemical tests [ 20 ]. Further confirmation of E. coli isolates was done using the Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Germany). Susceptibility test of confirmed E. coli isolates were done using the Kirby-Bauer disc diffusion, and interpreted using the CLSI, [ 21 ] guidelines. In brief, one to two isolated colonies were taken from an overnight culture of confirmed E . coli and emulsified in sterile saline solution of approximately 5 ml with turbidity compared to a 0.5 McFarland standard. With the aid of a sterile cotton swab, the suspension was streaked onto a Mueller-Hinton agar medium plate (Oxoid, Basingstoke, UK). Commercially purchased antibiotic discs were then placed onto the Mueller-Hinton agar with the aid of sterile forceps. The following 13 antibiotic agents were tested: ampicillin (10 µg), cefotaxime (30 µg), ciprofloxacin (5 µg), cefuroxime (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), chloramphenicol (30 µg), gentamicin (10 µg), meropenem (10 µg), nitrofurantoin (300 µg), nalidixic acid (30 µg), tetracycline (30 µg) and trimethoprim (1.25/23.75 µg) (Oxoid, Basingstoke, UK). Due to the high number of antibiotics, two Mueller-Hinton agar medium plates were used for each isolate. This was followed by aerobic incubation at 37°C for 18–24 hr. After incubation, zone sizes of inhibition were measured with a caliper and interpreted as per sensitive, intermediate or resistant as recommended by CLSI [ 21 ]. E. coli ATCC 25922 was included in every batch as quality control. As stated by the international standard definition for acquired resistance, and relative to the panel of antibiotics screened, MDR phenotypic strains were described as in vitro non-susceptible to at least one agent in three or more class of antibiotics [ 22 ]. 2.5 Phenotypic Confirmation of ESBL -EC by Combination Disk Test ESBL - EC isolates was phenotypically confirmed by employing the combination disk diffusion test as briefly highlighted by (CLSI, [ 21 ] guidelines. Confirmation of ESBL- EC isolates was based on an increase of zone size diameter of ≥ 5 mm for either cephalosporin: cefotaxime or ceftazidime in combination with clavulanate, when compared to either of the cefotaxime or ceftazidime alone. Control strains of E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 served as negative and positive bacterial control strains [ 21 ]. 2.6 DNA Extraction and WGS of ESBL- EC strains Whole genome sequencing (WGS) based-method was conducted at the Noguchi Memorial Institute for Medical Research (NMIMR), Legon, Ghana, under the SEQAFRICA Project. In brief, following an overnight culture, all DNA of ESBL- EC isolates were extracted and purification was carried out with the Qiagen Kit as highlighted in the manufacturer’s protocol. With the aid of Qubit 4.0 Fluorometer Assay Kit (Thermo Fisher Scientific, MA, USA), quantification of DNA concentrations was done. WGS libraries were prepared with the aid of the Nextera Flex Kit as highlighted by the manufacturer’s guidelines and 2 × 300 paired-end whole genome sequencing was carried out on an Illumina Miseq platform (Illumina Inc., San Diego, CA, USA). Sequenced raw reads (fastq files) were subjected to quality filtration with Phred score ≥ 20, and was filtered for a minimum read length of 50 bp, and adaptor trimmed with the aid of Trimmomatic ( http://www.usadellab.org/cms/index.php?page=trimmomatic ). FastQC accessories were engaged to systematically retrieve the quality of the resultant sequenced reads ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Eventually, resultant high-quality sequence reads were engaged in de novo assemblage with the aid of a Unicycler assembler v0.4.9 [ 23 ]. 2.6.1 In silico prediction of AMR genes, STs and plasmid replicon types We carried out an in silico ResFinder 4.1 ( https://cge.cbs.dtu.dk/services/ResFinder/ (database version 2023-09-03) for the prediction of resistant genes with an identity threshold and minimal length set at 90% and 60%, respectively [ 24 ]. Resistant genes with the maximum sequence recognition and coverage were held, while generic traits that overlapped were filtered out. Plasmid-Finder 2.1 (database version 2022-07-01) was engaged for the prediction of plasmid replicon types of all ESBL- EC isolates [ 25 ]. MobileElementFinder (database version 2023-09-09) was used for the prediction of mobile genetic elements [ 26 ]. MLSTFinder (database version 2023-09-18) was used for MLST allelic sequence and profile data from PubMLST.org with representative STs on the basis of allelic variations to seven housekeeping genes ( adk , fumC , gyrB , icd , mdh , purA and recA ) that matched 100% identity [ 27 ]. 2.7 Data Analysis : Data were entered into Microsoft Excel 2019 and analyzed with online GraphPad Prism version 8.0. Descriptive statistics such as means, frequencies and percentages were used where applicable. Categorical variables were compared using Fishers exact test. Univariate analysis with Pearsons chi-square for association and p ≤ 0.05 was taken as statistical significance. 3. Result 3.1 Socio-demographic characteristics of apparently healthy humans. A total of 400 apparently healthy humans were sampled. The mean age was 33.13 years with an age range of 70 years. Majority of samples were obtained among age group 20-27 years (27.8%) and 30-39 years (20.0%) and most were female (58.8%) against male counterpart (41.72%) Table1. 3.2 Characteristics of animals sampled Table 2 shows the characteristics of animals sampled. A total of 300 animals were sampled, cattle (n=200) and pig (n=100). Animal age ranges between 5 months to 1½ years with a median age of 6 months. Majority of individual animals were more than 1 years (63.0%). More female animals (73.3% were sampled than male counterpart (26.7%). 3.3 Prevalence of E. coli and ESBL- EC from human, cattle, pig, beef and soil sources. An overall E. coli isolation rate of 9.3% (140/1500) was detected from all specimens. E. coli isolation rate among the various samples were humans 17.5% (70/400), cattle 20.0% (40/200) and pigs 20.0% (20/100). However, no E. coli was recovered from pork, lettuce and spring onions Table 3 . Overall, the prevalence of ESBL- EC isolates from all sampling sources was 21.5% (30/140). 3.4 Distribution of E . coli cultured from human stool samples by study location. The distribution of E. coli among human stool samples in Accra and Tema is shown in Table 4. While Agbobloshie had the least isolation rate of E. coli . Generally, there was no statistical difference in the isolation rate of E. coli among the different study locations in Accra and Tema ( p > 0.05). 3.5 Distribution of E. coli cultured from food sample, soil and fecal samples by study locations The distribution of E. coli among food sample, soil and fecal sample is shown in Table 5 . Generally, there was no statiscal difference between isolation rate of E. coli in Accra and Tema ( p >0.05). However, soil samples in Tema were more contaminated with E. coli than Accra ( p <0.05). 3.6 Resistant Patterns of E. coli isolates Resistance patterns of E. coli i solates from diverse sources is shown in Table 6 . E. coli exhibited high levels resistance to ampicillin (84.4%), ceftazidime (61.4%), cefotaxime (71.4%), and cefuroxime (96.4%). However, moderate levels of resistance to trimethoprim (31.2%) and tetracycline (25.7%) were observed. For ciprofloxacin, isolates recovered from humans exhibited a higher resistance levels compared to those from cattle ( p ≤ 0.05). Similarly for tetracycline, isolates from cattle exhibited a higher resistance compared to those from humans ( p ≤ 0.05). For ampicillin and nalidixic acid, isolates recovered from pigs showed a higher resistance levels compared to those from humans ( p ≤ 0.05) Table 6 . 3.6 Multi-drug Resistant (MDR) Patterns of E. coli isolates recovered from diverse sources. Overall, 35.7% (n=50) of all E. coli isolates were MDR, but resistance varied among the various sample types; human (28.5%; n=20), cattle (55.0%; n=22), pig (35.0%; n=7) and soil (12.5%; n=1) Table 7 . Also, 21.4% (n=30) of E. coli isolates were resistance to 1 antibiotic agent, 28.6% (n=40) to 2 antibiotics agents and 35.7% (n=50) to 3 or more antibiotic agents. Several MDR phenotypes were observed, and more than half of the MDR isolates, 56% (n=28) were co-resistant to ampicillin, cefuroxime, trimethoprim and tetracycline and usually in combination with other antibiotic agents like chloramphenicol. About one-quarter of MDR isolates, 24% (n=12) were resistant to the nalidixic acid and ciprofloxacin Table 7 . Table 7 is at the end of the article. 3.4 Resistance Patterns of ESBL- EC from diverse sources Overall, ESBL- EC isolates exhibited high levels resistance of 100%, 100%, 75.8%, 78.0%, and 58.2% to ampicillin, cefuroxime, ceftazidime, cefotaxime and tetracycline. However, low resistance of 17.2% and 0.0% to ceftriaxone and meropenem. ESBL -EC isolates from human, cattle, pig and soil-sourced exhibited resistant prevalence of 100% to ampicillin, 42.9%, 55.6%, 100% and 33.3% resistance to tetracycline ( Figure 2 ). Among human sourced isolates, there was no statistical significant difference between resistant pattern of ESBL -EC isolates and non-ESBL- EC isolates, with the exception of ampicillin, ciprofloxacin, gentamicin and nitrofurantoin ( p ≤ 0.05) Table 8 . Among cattle sourced isolates, with the exception of an ESBL- EC isolate that exhibited a higher resistance of (20.0% vrs 0 %, ) to ciprofloxacin as compared to non-ESBL- EC isolate ( p ≤ 0.05) Table 8. Overall, there was no statistical significant difference between antibiotic resistance of ESBL- EC isolates vrs non-ESBL- EC isolates Table 8. Among pig-sourced, there was no statistical significant difference in resistant pattern of ESBL- EC isolates as compared to non-ESBL- EC isolates. However, ESBL- EC isolates exhibited a higher resistance of (66.7% vrs 0%) and (100% vrs 5.9%) to ciprofloxacin and nalidixic acid ( p ≤ 0.05) Table 9 . Overall, MDR prevalence of ESBL- EC isolates was 66.7% (n=20) and among sourced samples, human 71.4% (n=10), cattle 66.7% (n=6), pig 100% (n=3) and soil 33.3% (n=1). 3.6 Prevalence of AMR genes in ESBL- EC isolates A total of 16 different AMR genes were from major antibiotic class were observed in the ESBL- EC isolates Table 10. Across all ESBL- EC isolates, the most common AMR genes was bla TEM-1B (32.0%; n=8), ( tet A) (48.0%; n=12), ( sul 2) (36.0%; n=9), ( aph (3'')- Id ) (24.0%; n=6) and ( dfr A14) (16.0%; n=4). Also identified were plasmid-mediated quinolone resistant genes ( qn rS1) (12.0%; n=3) and quinolone resistant determining region at the gyr A (4.0%; n=1) and bla CTX-M-15 (4.0%; n=1) from soil sourced. Majority of AMR genes were observed from human and soil ESBL-EC isolates Table 10. 3.7 Prevalence of plasmid replicon types in ESBL- EC isolates from diverse sources A total of 21 different plasmid replicon types were identified from in silico analysis of ESBL- EC isolates Table 11 . The most prevalent IncF plasmid replicon types were IncFIB(Apoo1918) (40.0%; n=10), followed by IncFII(pCoo) (36.0%; n=9). Overall, diverse plasmid replicon types were observed in human ESBL- EC isolates, followed by soil ESBL- EC isolates. IncFIB(Apoo1918) (40.0%; n=5) was most dominant among cattle ESBL- EC isolates, followed by IncFII(pCoo) (40.0%; n=10) Table 11. 3.8 Distribution of sequence types in ESBL- EC isolates from diverse sources A total of 17 different sequence types were identified from in silico analysis of ESBL- EC isolates from diverse sources Figure 3 . The most prevalent sequence types were ST10 (12.0%; n=3), ST206 (12.0%; n=3), ST9312 ( 12.0%; n=3) and ST4151 (12.0%; n=3). However, 4.0% (n=1) was observed for each of the following; ST73, ST835, ST159, ST1237, ST6311, ST6237, ST5557, ST2061, ST8535, ST960, ST1684, ST999 and ST697, respectively ( Figure 3) . Overall, ST10 was observed in both cattle and pig ESBL- EC isolates, while ST206 was found in both human and cattle ESBL- EC isolates. Furthermore, ST73 was observed in cattle. 4. Discussion 4.1 Prevalence of E. coli isolates from diverse sources E. coli is considered a commensal bacterium of the gastrointestinal tract of humans and animals [28-29]. The genomic plasticity of E. coli facilitates their adaptation to diverse environments, thus, their wide association as an opportunistic pathogen in intestinal and extra-intestinal infections of humans and animals [7, 30]. In this study, E. coli isolation rate of (9.3%) from diverse sources is in agreement with (8.0%) isolation rate reported in India from diverse sources [31]. In contrast to this study findings, higher prevalence of (26.5%) has been reported in Nigeria [32]. Our study result of 2% (2/100) E. coli isolates from beef specimen and no detection in pork, lettuce and spring onion was similar to a study by Day and coworkers [33] in the United Kingdom which recovered E. coli in beef (2%), pork (3%), and fruits and vegetables (0%). Variation in the prevalence may be attributed to differences in sample size, cultural methods, and geographical locations. E. coli is a member of Enterobacteriaceae, ranked third in the WHO catalogue of antibiotic-resistant ‘priority pathogens’, and is currently, associated with the highest burden of AMR [8]. Commensal E. coli is often used as an indicator organism to study AMR-trends in food animals, the environment, and in human surveillance systems [34]. Usually, resistant rates in the sentinel bacterium E. coli are termed as "prevalence" of resistance [35]. In this study, MDR prevalence of (35.7%) in E. coli isolates was in contrast to the higher MDR prevalence reported in studies conducted in New Zealand (64.0%) and Nigeria (48.8%) [36-37]. The monitoring of MDR E. coli strains in food animals and food produce is necessary to evaluate its potential risk to humans [38]. Our study observation of E. coli MDR prevalence of (28.5%), (55.0%), (35%) and (12.5%) among human, cattle, pig and soil sourced isolates was contrary to the lower MDR prevalence of humans (22%), animal (5.7%), and environments (31.3%) reported in a systematic review by Pormohammad and colleagues [39]. Recently, a study in Italy on AMR surveillance from diverse sources reported MDR prevalence in human (28.0%) and swine (24.0%) [40]. In Ghana, an earlier study by Ohene Larbi and coworkers [12] reported MDR prevalence of (23%) in pig, contrary to this study findings. More recently, a study by Tawfick and colleagues [41] in Egypt with commensal E. coli from healthy humans reported MDR prevalence of (64.3%). In furtherance, varying MDR prevalence have been reported in cattle in Portugal (69%), France (56%), Egypt (44.4%); Mexico (72.7%) and Australia (32.4%) [42-46]. The irrational and indiscriminate use of antibiotic agents in human and animals has led to the rapid emergence and dissemination of MDR strains [47-49]. 4.2 Prevalence and Resistance Patterns of ESBL- EC isolates Escherichia coli is recognized as a potential putative reservoir for ESBL resistance and has been increasingly reported globally [50]. In this study, ESBL- EC prevalence of (21.5%) is in agreement with the prevalence of (21.7%) and (23.57%) reported in Nigeria and Egypt [32, 49]. Our study observation of ESBL- EC prevalence of (20%) in healthy human subjects was consistent with the (22.5%) prevalence reported in a recent study in healthy pregnant women in Benin [50]. In contrast to this study findings, lower prevalence of (4.9% to 6.3%) has been documented among healthy subjects in Japan, Netherland and Germany [51-53]. However, higher prevalence of (31.0%), (37.8%), (46.2%), (38.0%), (30.5%) and (71.4%) have been reported in previous studies in Vietnam, India, china; Chad; Gambia and Egypt [41, 51, 54-57]. In a systematic review and meta-analysis conducted by Bezabih and colleagues [11], the global pooled prevalence of ESBL- EC fecal carriage in the community was reported as (17.6%) which was close to our study findings among healthy human subjects. Another study in Japan has reported ESBL fecal carriage of (15.6%) among healthy human subjects [58]. Pig sourced ESBL- EC prevalence of (15%) was in concordance with the (13.4%) and (14.7%) prevalence reported in Switzerland and West Indies [59-60]. However, a previous study carried out in Kumasi in Ghana by Ohene Larbi and coworkers [12] reported (0%) ESBL- EC prevalence in pigs. Cattle sourced ESBL- E.C prevalence of (22.5%) was consistent with the (20.0%) prevalence reported in a recent study in Egypt [49]. However, lower prevalence of (4.35%), (15.3%), (7.5%) and (11.0%) have been reported in previous studies conducted in Switzerland, India, West Indies and Ghana [12, 31, 59-60]. The variation in ESBL prevalence may be attributed to little or no regulation on antibiotic usage in animal husbandry and unsanitary environments mostly encountered in the developing nations [61-62]. In this study, our observation of the high ESBL- EC isolates resistance to 2 nd and 3 rd generation cephalosporins cefuroxime, ceftazidime and cefotaxime was in contrast to the lower resistance of (8.3%), (8.3%), and (8.3%) reported in a similar “One Health” review study in Iran [39]. Thus, under a ‘One Health’ approach, AMR to cephalosporins category of beta-lactams is a typical example of how antibiotics play a significant role in the animal and human health. Our resistance level findings for gentamicin, chloramphenicol and nalidixic acid was similar to the (50%), (31.3%) and (41.7%) resistance prevalence reported in a previous study in Nigeria [63] Likewise, our findings of resistance prevalence to tetracycline was consistent with the (60%) reported in some earlier studies in Ghana [31] and Egypt [41]. It is noteworthy that resistance to ampicillin and tetracycline in this study is not unexpected, since ampicillin and tetracycline remains one of the most commonly used antimicrobials agents in livestock production in Ghana [64]. Worldwide, resistance to penicillin, tetracycline and sulfonamide is well documented in animal production [65] Furthermore, in this study, the high resistance to ampicillin and 2 nd and 3 rd cephalosporin (cefuroxime, ceftazidime and cefotaxime) may be due to the indiscriminate use of beta-lactams agents due to their broad spectra, high effectiveness and minimal side effects [66]. In this study, MDR and ESBL prevalence to different sourced ESBL- EC isolates of human, cattle, pig and soil was contrary to a systematic review study finding that reported MDR and ESBL prevalence of (13%), (26.3%), and (25%) to human, animal, and environmental/food [39]. Literature has shown that the overuse and misuse of antibiotic agents in the agricultural, veterinary, and human medical departments may initiate the development and dissemination of MDR bacteria and allow for the emergence of unique AMR mechanisms [47, 67-69]. 4.3 AMR genes, plasmid replicons and sequence types in ESBL- Ec isolates The emergence and rapid dissemination of MDR bacterial strains, particularly ESBL- EC strains are of great concern [70]. In this study, our findings of bla TEM-1B, tet A and sul 2 as the prevalent AMR genes is in agreement with a recent systematic review outcome of bla TEM-1 (36.3%) and tetA (32.9%) reported by Ramatla and coworkers [9]. Escher and colleagues [10] in an earlier systematic review also reported bla TEM-1, sul 2and tetA as the most prevalent AMR genes. More recently, studies in Gambia, USA, Mexico, Benin, China and Egypt have found bla TEM as the most dominant ESBL gene type [34, 41, 50, 57, 71-72]. In Nigeria, Aworh and colleagues [73] study also observed bla TEM-1 in MDR E. coli as the most prevalent AMR genes. In Ghana, a recent study by Dsani and coworkers [74] with raw meats found bla TEM (4%) with absence of bla CTX-M. The high bla TEM-1B prevalence observed in this study could be responsible for the high resistance prevalence in ampicillin and probably 2 nd and 3 rd cephalosporins. Globally, bla CTX-M-15 is the most prevalent ESBL gene type and ESBL bacteria harbouring plasmid-mediated enzymes with capacity to cause resistance to beta-lactam antibiotics such as ampicillin and cephalosporin as well as co-resistance to non-beta-lactams agents including quinolones and aminoglycosides, thus treatment options are limited [49. 70, 75]. When bacteria acquire AMR to antibiotics, they as well acquire a higher propensity to disseminate resistance in animals, humans, and the natural environment [2]. In this study, the only bla CTX-M-15 gene identified was from soil sourced isolate in conjunction with quinolone resistant gene ( qnr S1). This study finding is in agreement with previous studies outcome in Brazil that reported soil-sourced ESBL- E. coli carrying bla CTX-M-15 and quinolone resistant gene ( qnr B19) along with other AMR genes [76-77]. This is the first report of ESBL- EC harbouring bla CTX-M-15 and qnr S1 in agricultural soil from Ghana and this signifies a threat to food and environmental safety. In this study, soil samples from Ashiaman-Tema area were found to be more contaminated with ESBL-EC isolates than those of Accra metropolis. This may be attributed to the high level of open defecation by cattles and human particularly at order side of the motor way close to Ashiaiman area. The highest AMR genes were observed in human sourced ESBL- EC isolates in this study. Several studies have shown that fecal deposition of AMR genes and active antibiotic agents from humans and animals waste and their persistence in the environment are known to promote the spread of AMR genes in the environment [78-81]. The presence of MDR and ESBL- EC isolates exhibiting a wide range of resistors to antimicrobials in agricultural soil may lead to contamination of vegetable crops and, since these types of foods are often consumed raw as ready-to-eat foods (R-T-E), the risk of human exposure to antibiotic-resistant bacteria (ABR) and AMR genes with high clinical significance is worrisome [76-77, 84]. Mobile genetic elements (MGE), specifically, plasmids, are involved in the dissemination of ESBL determinants and co-resistance encoding genes and could result in the rapid escalation of ESBL-producing strains from diverse sources [82-83]. In this study, IncFIB(Apoo1918) and IncFII(pCoo) were the most dominant plasmid replicon types. Studies in Gambia, Germany; Italy, USA and Mexico have reported IncF plasmid replicon types as the most prevalent plasmid replicons involved in encoding acquired AMR genes and often termed epidemic plasmids [34, 40, 57, 71 84]. The incompatibility (Inc) group F (IncF) which belongs to the narrow-host-range plasmids is one of the most significant plasmids involved in AMR genes transmission and spread to other bacteria [85]. This study observed diverse sequence types (STs) among ESBL- EC isolates. Our findings is in concordance with study finding as reported in United Kingdom, Ghana, Nigeria; Germany, Canada and Italy [13, 33, 40, 86-87]. Our finding included the well-known ST such as ST10 in animal and the environment. Suggestive that a probable transmission might have taken place amongst these sources. It's noteworthy that ST10, a global one health clone with potential to disseminate ESBL determinants in association with other AMR genes has been identified in diverse sources [88]. Additionally, ST10 possesses broad-host range and mostly associated with extra-intestinal infections [6, 13, 40, 83-84, 87, 89]. Globally, pandemic and high-risk zoonotic MDR E. coli clones have disseminated into diverse niches including food-animals, humans and the environment [83, 90]. In this study, ST206 was detected in human and cattle. Qiu and coworkers [91] study in China reported ST206 in minks (fur animals). Another study by Ayeni and colleagues [92] in Nigeria also reported ST206 in poultry livestock. The presence of ESBL- EC ST206 isolates in animal and human sources is suggestive of their potential for spread and persistence in different hosts [93]. In this study, no ST131 was detected. ST131 are well established pandemic clone with propensity to spread ESBL genes and AMR genes globally [6]. However, ST73, a member of the pandemic high-risk clone was identified in cattle in this study. Studies in the United Kingdom and Australia have reported ST73 among human sourced isolates as one of the most prevalent ST associated with bacteremia and UTI [88, 94]. Our study finding might be due to a probable circulation of host-adapted lineage of ESBL- EC strains. Conclusion Within the metropolis surveyed, we identified MDR ESBL -EC isolatesharbouringvarious AMR genes and plasmid replicons with diverse E. col i STs in healthy humans, animals and the environment. This study finding of bla CTX-M-15 in agricultural soil isolate is worrisome, emphasizing the need for a one-health approach in combating AMR. Abbreviations AMR: Antimicrobial resistance, MDR: multi-drug resistant, extended-spectrum-beta-lactamase-producing- Escherichia coli (ESBL- EC ), Escherichia coli ( E. coli ), whole-genome sequencing (WGS), Sequence types (STs), Clinical and laboratory Standard Institute (CLSI). Declarations Acknowledgements The authors would like to express their gratitude to all the staff of the Department of Medical Laboratory Science, department of Medical Microbiology and the Department of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana. Special thanks go to SEQAFRICA Project, Noguchi Memorial Institute for Medical Research for whole genome sequencing of bacterial isolates. Also, thanks to animal owners and agricultural farmers for their permission to samples. Authors’ contributions MOT, JO, FOA and BE conceived and designed the study. MOT collected data and performed laboratory analysis; BE and CO generated the whole genome sequence data for all the ESBL isolates. MOT conducted bioinformatics analysis, interpreted the data and wrote the first draft of the manuscript. JO, FOA and BE supervised the study, reviewed and edited the manuscript article. All authors read and approved the final manuscript. Funding None Consent to publish not applicable Availability of data and materials The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request. All the ESBL- EC sequence data reads and genome assemblies generated in this research study have been submitted to GenBank under the BioProject PRJNA1077263. Ethical approval and consent to participate The study was carried out in accordance with the Declaration of Helsinki, and ethical approval was obtained from the Ethics Committee of the College of Health Science, University of Ghana (Ethics approval number: CHS-Et/M.1-P5.12/2022-2023). All methods and protocols were performed in accordance with relevant regulations and guidelines, on involving humans and animal subjects in the study. Formal permission was sought from all the farm animal owners and participants involved. 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Rozwandowicz M, Brouwer MSM, Fischer J, Wagenaar JA, Gonzalez-Zorn B, Guerra B, Mevius DJ, Hordijk J. Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. J Antimicrob Chemother. 2018;73(5):1121-1137. Silva, A.; Silva, V.; Pereira, J.E.; Maltez, L.; Igrejas, G.; Valentão, P.; Falco, V.; Poeta, P (2023). Antimicrobial Resistance and Clonal Lineages of Escherichia coli from Food-Producing Animals. Antibiotics 2023;12:1061. Reid CJ, Blau K, Jechalke S, Smalla K and Djordjevic SP. Whole Genome Sequencing of Escherichia coli From Store-Bought Produce. Front. Microbiol . 2020;10:3050. Partridge, S. R., Kwong, S. M., Firth, N. & Jensen, S. O. Mobile genetic elements associated with antimicrobial resistance. Clin. Microbiol. Rev . 2018;31, 1. Massé J. Vanier G, Fairbrother J.M, de Lagarde M, Arsenault J, Francoz D, Dufour S & Archambault M. Description of Antimicrobial-Resistant Escherichia coli and Their Dissemination Mechanisms on Dairy Farms. Vet. Sci , 2023;10, 242. Aworh MK, Ekeng E, Nilsson P, Egyir B, Owusu-Nyantakyi C and Hendriksen RS (2022) Extended-Spectrum ß-Lactamase-Producing Escherichia coli Among Humans, Beef Cattle, and Abattoir Environments in Nigeria. Front. Cell. Infect. Microbiol. 12:869314. doi: 10.3389/fcimb.2022.869314. Day MJ, Doumith M, Abernethy J, et al. Population structure of Escherichia coli causing bacteraemia in the UK and Ireland between 2001 and 2010. J Antimicrob Chemother 2016;71: 213942. Hojabri Z, Mirmohammadkhani M, Darabi N, Arab M, & Pajand O. Characterization of antibiotic-susceptibility patterns and virulence genes of five major sequence types of Escherichia coli isolates cultured from extraintestinal specimens: a 1-year surveillance study from Iran. Infect Drug Resist . 2019;12:893-903. Eger E, Domke M, Heiden S.E, Paditz M, Balau V, Huxdorff C, Zimmermann D, Homeier-Bachmann T & Schaufler K. Highly Virulent and Multidrug-Resistant Escherichia coli Sequence Type 58 from a Sausage in Germany. Antibiotics 2022;11:1006. Qiu J, Jiang Z, Ju Z, Zhao X, Yang J, Guo H & Sun S. Molecular and Phenotypic Characteristics of Escherichia coli Isolates from Farmed Minks in Zhucheng, China. Hindawi BioMed Research International 2019;3917841. Ayeni FA, Falgenhauer J, Schmiede J, Schwengers O, Chakraborty T & Falgenhauer L. Detection of bla CTX-M-27-encoding Escherichia coli ST206 in Nigerian poultry stocks. J Antimicrob Chemother ; 2020;75: 3070–3072. Zhou Z, Alikhan NF, Mohamed K et al., The EnteroBase user’s guide,with case studies on Salmonella transmissions, Yersinia pestis phylogeny and Escherichia core genomic diversity. Genome Res ; 2020;30:138–52. Li D, Elankumaran P, Kudinha T, Kidsley AK, Trott DJ, Jarocki VM, Djordjevic SP. Dominance of Escherichia coli sequence types ST73, ST95, ST127 and ST131 in Australian urine isolates: a genomic analysis of antimicrobial resistance and virulence linked to F plasmids. Microb Genom. 2023;9(7):mgen001068. doi: 10.1099/mgen.0.00 Tables Tables 1 to 11 are available in the Supplementary Files section Supplementary Files Tables.docx Cite Share Download PDF Status: Published Journal Publication published 26 May, 2025 Read the published version in One Health Outlook → Version 1 posted Reviewers agreed at journal 14 Jul, 2024 Reviewers invited by journal 02 Jul, 2024 Editor assigned by journal 21 Jun, 2024 First submitted to journal 20 Jun, 2024 Editorial decision: Major revision 10 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4480595","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":321919156,"identity":"e17ce088-98fd-4d81-8547-10e400ee05db","order_by":0,"name":"Michael A. Olu-Taiwo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFAC5gYQyQNkHAAxGIFcNgJaQGoSQFrYEkjTArLIgDgtuu2NbRKMP+xkDI73fJP8wmAju+EA+7UH+LSYnTnYJsGQkMxjcObsNmkZhjTjDQd4yg3warmR2Cb9J4GZx+xG7jZpCYbDiUAtaRKEtABtqQdqyXkG1PKfaC2HQVrYJD8wHABqYT+GX8uZg80WDGnHeezPHDO2ZjBINp55mIcNv5bjzQdvMNhU20u2Nz+8+aPCTrbvePszvFpQADM4aiAkkYDxB5hif0C8llEwCkbBKBgJAACDfkl63A6T8AAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8164-3080","institution":"University of Ghana","correspondingAuthor":true,"prefix":"","firstName":"Michael","middleName":"A.","lastName":"Olu-Taiwo","suffix":""},{"id":321919157,"identity":"265937a4-484c-4aa2-b97b-f6ad7f2543f5","order_by":1,"name":"Beverly Egyir","email":"","orcid":"","institution":"University of Ghana Noguchi Memorial Institute for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Beverly","middleName":"","lastName":"Egyir","suffix":""},{"id":321919158,"identity":"2d010112-96ce-4fdd-91a6-89f42b7261a9","order_by":2,"name":"Christian Owusu-Nyantakyi","email":"","orcid":"","institution":"University of Ghana Noguchi Memorial Institute for Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Owusu-Nyantakyi","suffix":""},{"id":321919159,"identity":"5a300d70-702f-4e95-9154-0ee2089a5a09","order_by":3,"name":"Akua Obeng Forson","email":"","orcid":"","institution":"University of Ghana","correspondingAuthor":false,"prefix":"","firstName":"Akua","middleName":"Obeng","lastName":"Forson","suffix":""},{"id":321919160,"identity":"0665c671-6671-4a99-9ed6-783c7bf409ab","order_by":4,"name":"Japheth A. Opintan,","email":"","orcid":"","institution":"UGMS: University of Ghana Medical School","correspondingAuthor":false,"prefix":"","firstName":"","middleName":"Japheth A.","lastName":"Opintan","suffix":""}],"badges":[],"createdAt":"2024-05-26 15:16:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4480595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4480595/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s42522-025-00154-8","type":"published","date":"2025-05-26T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61056245,"identity":"8b805ea4-18f9-4250-8380-3a962e569dc6","added_by":"auto","created_at":"2024-07-25 05:34:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":109282,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Ghana showing the study Area (Source: [18])\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4480595/v1/b430cce3a9bc0e87c54f9ad2.png"},{"id":61055424,"identity":"925633d2-1452-4aaa-8dcf-b5792da328f3","added_by":"auto","created_at":"2024-07-25 05:26:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResistant pattern of ESBL-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e from diverse sources\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4480595/v1/7b0be12757f9ed216a159920.png"},{"id":61055462,"identity":"601f4633-1cef-4865-9b8d-35afa26be3a9","added_by":"auto","created_at":"2024-07-25 05:26:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of sequence types (ST) among ESBL-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEC \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eisolates from diverse sources\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4480595/v1/16af48e143c9d3ba4a5b9596.png"},{"id":83782842,"identity":"bb0b0cf1-0ad2-40f7-aa81-7ffa167ec6d9","added_by":"auto","created_at":"2025-06-02 16:07:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1638507,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4480595/v1/f7ba56d6-d27f-4b99-bde9-df2d92f7baeb.pdf"},{"id":61055425,"identity":"5bb469ae-12e0-4f8b-a3d8-23429c00848e","added_by":"auto","created_at":"2024-07-25 05:26:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":65351,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4480595/v1/bdc362a6a2cfb3116c3d3905.docx"}],"financialInterests":"","formattedTitle":"Molecular characterization of multidrug-resistant (MDR) Escherichia coli in the Greater Accra Region, Ghana: a ‘One Health’ approach.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAmong global health challenges, AMR is a typical illustration of a \u0026lsquo;One Health' crisis that affects humans, animals and the environment [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Of particular public health concern in the 21st century, is the steadily escalating prevalence of AMR among pathogenic and commensal bacteria [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In recent times, AMR has been recognized to be a major driver for the persistence, dissemination and distribution of MDR high-risk clones, including \u003cem\u003eE. coli\u003c/em\u003e ST131, ST10, ST38, ST73 and ST206 often associated with intestinal and extra-intestinal diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In 2019, it was estimated that of the 4.95\u0026nbsp;million human associated deaths, 1.27\u0026nbsp;million deaths was attributable to MDR bacterial infections [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This number has been projected to escalate to 10\u0026nbsp;million by 2050, if no measures are initiated to handle the AMR situation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, an integrated and multifaceted \u0026ldquo;One Health\u0026rdquo; approach is urgently needed to help mitigate and manage the AMR challenge. In a more recent systematic review, Ramatla and colleagues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] showed that the pooled prevalence estimates (PPE) of \u003cem\u003eE. coli\u003c/em\u003e AMR genes including \u003cem\u003ebla\u003c/em\u003eTEM-1, \u003cem\u003eamp\u003c/em\u003eC, \u003cem\u003etetA\u003c/em\u003e, and \u003cem\u003ebla\u003c/em\u003eTEM were 36.3%, 34.4%, 32.9%, and 28.8%, respectively. The most prevalent \u003cem\u003eE. coli\u003c/em\u003e AMR genes in animals were \u003cem\u003ebla\u003c/em\u003eTEM (30%), \u003cem\u003esulI\u003c/em\u003e (36.2%), \u003cem\u003esulII\u003c/em\u003e (32.0%), \u003cem\u003etet A\u003c/em\u003e (31.5%), and \u003cem\u003estrB\u003c/em\u003e (30.8%), while, among humans, \u003cem\u003ebla\u003c/em\u003eTEM (28.8%), \u003cem\u003esulI\u003c/em\u003e (27.8%), \u003cem\u003esulII\u003c/em\u003e (42.2%), \u003cem\u003etetA\u003c/em\u003e (42.0%), and \u003cem\u003estrB\u003c/em\u003e (34.9%) were the most dominant \u003cem\u003eE. coli\u003c/em\u003e AMR genes as reported by Escher and coworkers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the last 20 years, fecal carriage prevalence of ESBL-\u003cem\u003eEC\u003c/em\u003e in healthy individuals vary globally; the highest in Southeast Asia (27%), and the lowest in Europe (6%) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Till date, data on AMR under a One Health lens in Africa is scanty [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In Africa, majority of (LMICs) lack the resource capacity to use WGS technology to determine the inter-connectedness of ESBL\u003cem\u003e-EC\u003c/em\u003e strains recovered from humans, food-animals and the environment under a \u0026lsquo;One health' surveillance of AMR [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. There is an urgent need to conduct a detailed molecular characterization of ESBL-\u003cem\u003eEC\u003c/em\u003e isolates derived from humans, food producing animals and the environment to contribute baseline data on AMR genes, plasmid replicon types and STs of public health significance and relevance to the One health surveillance database [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. According to a 2014 study finding by WHO, one major aspect to the global response to AMR is surveillance, however, the WHO Africa region possesses one of the highest gaps in data on the prevalence of AMR [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. WGS and \u003cem\u003ein silico\u003c/em\u003e analysis has shown to be a relevant and reliable molecular tool in AMR surveillance due to its sensitivity to detect AMR genes or mutations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, this study aimed to use \u0026lsquo;One Health\u0026rsquo; approach to determines the prevalence and patterns of AMR and to characterize AMR genes, STs and plasmid replicon types among ESBL-\u003cem\u003eEC\u003c/em\u003e strains recovered from healthy humans, food-animals and environment sources in the Greater Accra Region, Ghana.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Study Design and Area\u003c/h2\u003e\n \u003cp\u003eThis study was a cross-sectional study carried out in Accra and Tema metropolis in the Greater Accra Region of Ghana, between January, 2022-April, 2023. Greater Accra is one of the 16 administrative regions in Ghana. Though the smallest in terms of size it is highly populated, industrialized, and serves as the commercial nerve centre of Ghana [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. Greater Accra encompasses a land mass size of 3,245 Km\u003csup\u003e2\u003c/sup\u003e or a 1.4% of the total area of Ghana. It has an estimated urban population of 4.6 million and accounted for 15.4% of Ghana\u0026rsquo;s total population in the year 2016 [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Accra metropolitan district and Tema metropolitan district are the only two districts with city status in the Greater Accra Region (Fig. \u003cspan class=\"InternalRef\"\u003e1.0\u003c/span\u003e). \u0026ldquo;Accra\u0026rdquo; usually referred to as the Accra metropolitan area, also serves as the capital city of Ghana [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the Accra city, peri-urban communities such as James Town, Korle-Gonno, Abossey-Okai, and Agbogloshie were selected for sampling, while in Tema metropolitan area, peri-urban communities including Ashiaiman, Adjei-Kojo, Tema Newtown and community one, were also selected. These peri-urban communities were selected due to their dense population, inadequate sanitation facilities and the close proximity to domestic and farm animals.\u003c/p\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003e2.1.1 Inclusion and exclusion criteria\u003c/h2\u003e\n \u003cp\u003eOnly apparently healthy individuals (human) of all age group from the study area, who consented to participate was recruited. Also, only healthy animals from consenting owners were sampled. Individuals on antibiotics medication and unwell was excluded, likewise, sick animals and diseased animals were excluded from this study.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.1.2 Sample size for apparently healthy human\u003c/h2\u003e\n \u003cp\u003eSample size was determined based on the formula as described by Daniel and colleagues (1999).\u003c/p\u003e\n \u003cp\u003eN\u0026thinsp;=\u0026thinsp;Z\u003csup\u003e2\u003c/sup\u003eXp (1-p)/d\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003eWhere: N\u0026thinsp;=\u0026thinsp;required sample size\u003c/p\u003e\n \u003cp\u003eZ\u0026thinsp;=\u0026thinsp;confidence Interval for 95% CI is 1.96\u003c/p\u003e\n \u003cp\u003ed\u0026thinsp;=\u0026thinsp;precision (margin of error at 5%), i.e. 0.05\u003c/p\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;expected prevalence of ESBL\u003cem\u003e-EC\u003c/em\u003e in healthy human [Unknown 50%]. Using the formula above, an approximated total of 400 stool samples was sampled.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.1.3 Sample size for cattle\u003c/h2\u003e\n \u003cp\u003eExpected sample size for cattle was calculated with 11.1% prevalence from a previous study conducted in Greater Kumasi in the Ashanti region of Ghana by Ohene Larbi et al, (2021), Using the formula above, an approximated total of 200 anal swabs from cattle was sampled.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.1.4 Sample size for pig\u003c/h2\u003e\n \u003cp\u003eExpected sample size for pig was calculated with 7.1% prevalence from a previous study conducted by Ohene Larbi et al, (2021). Using the formula above, an approximated total of 100 anal swabs from pigs was sampled.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.1.5 Sampling\u003c/strong\u003e\u003cbr\u003eOverall, a total of 1500 non-duplicate specimens were collected from apparently healthy humans (stool samples n\u0026thinsp;=\u0026thinsp;400), cattle (anal swabs n\u0026thinsp;=\u0026thinsp;200), pigs (anal swabs n\u0026thinsp;=\u0026thinsp;100), soil sample (n\u0026thinsp;=\u0026thinsp;400), food samples (beef n\u0026thinsp;=\u0026thinsp;100, pork n\u0026thinsp;=\u0026thinsp;100, lettuce n\u0026thinsp;=\u0026thinsp;100 and spring onions n\u0026thinsp;=\u0026thinsp;100) into sterile containers or Cary Blair medium. All samples were labelled and transported on ice packs within 2 hr of collection to the Microbiology Laboratory at the School of Biomedical and Allied Health Sciences, University of Ghana for cultural analysis.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Bacteriological processing of samples\u003c/h2\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.1 Stool and anal swabs\u003c/h2\u003e\n \u003cp\u003eIn brief, 1 g of human stool sample was placed in 9 ml of EC enrichment broth (Oxoid, Basingstoke, UK) and homogenized for one minute and aerobically incubated at 37\u0026deg;C for 18\u0026ndash;24 hr as described by Purushottam and colleagues [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Anal swabs from cattle and pigs were placed in 9 mls of EC broth for enrichment for 18\u0026ndash;24 hr and aerobically incubated at 37\u0026deg;C overnight. An aliquot from both pre-enriched broths were streaked onto MacConkey agar medium plate (Oxoid, Basingstoke, UK) and aerobically incubated at 37\u0026deg;C for 18\u0026ndash;24 hr as described by Purushottam and colleagues [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.2 Food samples\u003c/h2\u003e\n \u003cp\u003eIn brief, 25 g portion of every food sample was aseptically placed in 225 ml of EC enrichment broth (Oxoid, Basingstoke, UK) and homogenized for one minute, then aerobically incubated at 37\u0026deg;C for 18\u0026ndash;24 hr, followed by streaking an aliquot onto MacConkey agar medium plate (Oxoid, Basingstoke, UK) and incubated at 37\u0026deg;C and 44\u0026deg;C for 18\u0026ndash;24 hr as described by Purushottam and colleagues [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\n \u003ch2\u003e2.3.3 Soil samples\u003c/h2\u003e\n \u003cp\u003eIn brief, 10 g of soil samples was placed in 90 ml of EC enrichment broth (Oxoid, Basingstoke, UK) and homogenized for one minute and incubated at 37\u0026deg;C for 18\u0026ndash;24 hr. After serial dilution of 10\u003csup\u003e1\u003c/sup\u003e-10\u003csup\u003e5\u003c/sup\u003e, an aliquot was streaked onto MacConkey agar medium plate (Oxoid, Basingstoke, UK) and aerobically incubated at 37\u0026deg;C for 18\u0026ndash;24 hr as described by Purushottam and colleagues [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Identification and susceptibility testing of \u003cem\u003eE. coli\u003c/em\u003e isolates\u003c/h2\u003e\n \u003cp\u003ePink moist medium sized colonies, suggestive of \u003cem\u003eE. coli\u003c/em\u003e isolates, were phenotypically characterized as per standard biochemical tests [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Further confirmation of \u003cem\u003eE. coli\u003c/em\u003e isolates was done using the Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Germany). Susceptibility test of confirmed \u003cem\u003eE. coli\u003c/em\u003e isolates were done using the Kirby-Bauer disc diffusion, and interpreted using the CLSI, [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e] guidelines. In brief, one to two isolated colonies were taken from an overnight culture of confirmed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e and emulsified in sterile saline solution of approximately 5 ml with turbidity compared to a 0.5 McFarland standard. With the aid of a sterile cotton swab, the suspension was streaked onto a Mueller-Hinton agar medium plate (Oxoid, Basingstoke, UK). Commercially purchased antibiotic discs were then placed onto the Mueller-Hinton agar with the aid of sterile forceps. The following 13 antibiotic agents were tested: ampicillin (10 \u0026micro;g), cefotaxime (30 \u0026micro;g), ciprofloxacin (5 \u0026micro;g), cefuroxime (30 \u0026micro;g), ceftazidime (30 \u0026micro;g), ceftriaxone (30 \u0026micro;g), chloramphenicol (30 \u0026micro;g), gentamicin (10 \u0026micro;g), meropenem (10 \u0026micro;g), nitrofurantoin (300 \u0026micro;g), nalidixic acid (30 \u0026micro;g), tetracycline (30 \u0026micro;g) and trimethoprim (1.25/23.75 \u0026micro;g) (Oxoid, Basingstoke, UK). Due to the high number of antibiotics, two Mueller-Hinton agar medium plates were used for each isolate. This was followed by aerobic incubation at 37\u0026deg;C for 18\u0026ndash;24 hr. After incubation, zone sizes of inhibition were measured with a caliper and interpreted as per sensitive, intermediate or resistant as recommended by CLSI [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 was included in every batch as quality control. As stated by the international standard definition for acquired resistance, and relative to the panel of antibiotics screened, MDR phenotypic strains were described as \u003cem\u003ein vitro\u003c/em\u003e non-susceptible to at least one agent in three or more class of antibiotics [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Phenotypic Confirmation of ESBL\u003cem\u003e-EC\u003c/em\u003e by Combination Disk Test\u003c/h2\u003e\n \u003cp\u003eESBL\u003cstrong\u003e-\u003c/strong\u003e\u003cem\u003eEC\u003c/em\u003e isolates was phenotypically confirmed by employing the combination disk diffusion test as briefly highlighted by (CLSI, [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e] guidelines. Confirmation of ESBL-\u003cem\u003eEC\u003c/em\u003e isolates was based on an increase of zone size diameter of \u0026ge;\u0026thinsp;5 mm for either cephalosporin: cefotaxime or ceftazidime in combination with clavulanate, when compared to either of the cefotaxime or ceftazidime alone. Control strains of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ATCC 700603 served as negative and positive bacterial control strains [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 DNA Extraction and WGS of ESBL-\u003cem\u003eEC\u003c/em\u003e strains\u003c/h2\u003e\n \u003cp\u003eWhole genome sequencing (WGS) based-method was conducted at the Noguchi Memorial Institute for Medical Research (NMIMR), Legon, Ghana, under the SEQAFRICA Project. In brief, following an overnight culture, all DNA of ESBL-\u003cem\u003eEC\u003c/em\u003e isolates were extracted and purification was carried out with the Qiagen Kit as highlighted in the manufacturer\u0026rsquo;s protocol. With the aid of Qubit 4.0 Fluorometer Assay Kit (Thermo Fisher Scientific, MA, USA), quantification of DNA concentrations was done. WGS libraries were prepared with the aid of the Nextera Flex Kit as highlighted by the manufacturer\u0026rsquo;s guidelines and 2 \u0026times; 300 paired-end whole genome sequencing was carried out on an Illumina Miseq platform (Illumina Inc., San Diego, CA, USA). Sequenced raw reads (fastq files) were subjected to quality filtration with Phred score\u0026thinsp;\u0026ge;\u0026thinsp;20, and was filtered for a minimum read length of 50 bp, and adaptor trimmed with the aid of Trimmomatic (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.usadellab.org/cms/index.php?page=trimmomatic\u003c/span\u003e\u003c/span\u003e). FastQC accessories were engaged to systematically retrieve the quality of the resultant sequenced reads (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e Eventually, resultant high-quality sequence reads were engaged in \u003cem\u003ede novo\u003c/em\u003e assemblage with the aid of a Unicycler assembler v0.4.9 [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e2.6.1 \u003cem\u003eIn silico\u003c/em\u003e prediction of AMR genes, STs and plasmid replicon types\u003c/h2\u003e\n \u003cp\u003eWe carried out an \u003cem\u003ein silico\u003c/em\u003e ResFinder 4.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.cbs.dtu.dk/services/ResFinder/\u003c/span\u003e\u003c/span\u003e (database version 2023-09-03) for the prediction of resistant genes with an identity threshold and minimal length set at 90% and 60%, respectively [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Resistant genes with the maximum sequence recognition and coverage were held, while generic traits that overlapped were filtered out. Plasmid-Finder 2.1 (database version 2022-07-01) was engaged for the prediction of plasmid replicon types of all ESBL-\u003cem\u003eEC\u003c/em\u003e isolates [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. MobileElementFinder (database version 2023-09-09) was used for the prediction of mobile genetic elements [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. MLSTFinder (database version 2023-09-18) was used for MLST allelic sequence and profile data from PubMLST.org with representative STs on the basis of allelic variations to seven housekeeping genes (\u003cem\u003eadk\u003c/em\u003e, \u003cem\u003efumC\u003c/em\u003e, \u003cem\u003egyrB\u003c/em\u003e, \u003cem\u003eicd\u003c/em\u003e, \u003cem\u003emdh\u003c/em\u003e, \u003cem\u003epurA\u003c/em\u003e and \u003cem\u003erecA\u003c/em\u003e) that matched 100% identity [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.7 Data Analysis\u003c/strong\u003e: Data were entered into Microsoft Excel 2019 and analyzed with online GraphPad Prism version 8.0. Descriptive statistics such as means, frequencies and percentages were used where applicable. Categorical variables were compared using Fishers exact test. Univariate analysis with Pearsons chi-square for association and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 was taken as statistical significance.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Result","content":"\u003cp\u003e\u003cstrong\u003e3.1 Socio-demographic characteristics of apparently healthy humans.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 400 apparently healthy humans were sampled. The mean age was 33.13 years with an age range of \u0026lt; 1 year to \u0026gt; 70 years. \u0026nbsp; Majority of samples were obtained among age group 20-27 years (27.8%) and 30-39 years (20.0%) and most were female (58.8%) against male counterpart (41.72%) \u003cstrong\u003eTable1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Characteristics of animals sampled\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eshows the characteristics of animals sampled. A total of 300 animals were sampled, cattle (n=200) and pig (n=100). Animal age ranges between 5 months to 1\u0026frac12; years with a median age of 6 months. Majority of individual animals were more than 1 years (63.0%). More female animals (73.3% were sampled than male counterpart (26.7%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Prevalence of \u003cem\u003eE. coli\u003c/em\u003e and ESBL-\u003cem\u003eEC\u003c/em\u003e from human, cattle, pig, beef and soil sources.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn overall \u003cem\u003eE. coli\u003c/em\u003e isolation rate of 9.3% (140/1500) was detected from all specimens. \u003cem\u003eE. coli\u003c/em\u003e isolation rate among the various samples were humans 17.5% (70/400), cattle 20.0% (40/200) and pigs 20.0% (20/100). However, no \u003cem\u003eE. coli\u003c/em\u003e was recovered from pork, lettuce and spring onions \u003cstrong\u003eTable 3\u003c/strong\u003e. Overall, the prevalence of ESBL-\u003cem\u003eEC\u003c/em\u003e isolates from all sampling sources was 21.5% (30/140).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Distribution of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e cultured from human stool samples by study location.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe distribution of \u003cem\u003eE. coli\u003c/em\u003e among human stool samples in Accra and Tema is shown in \u003cstrong\u003eTable 4.\u0026nbsp;\u003c/strong\u003eWhile Agbobloshie had the least isolation rate of \u003cem\u003eE. coli\u003c/em\u003e. Generally, there was no statistical difference in the isolation rate of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eamong the different study locations in Accra and Tema (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Distribution of \u003cem\u003eE. coli\u003c/em\u003e cultured from food sample, soil and fecal samples by study locations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe distribution of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eamong food sample, soil and fecal sample is shown in \u003cstrong\u003eTable 5\u003c/strong\u003e. Generally, there was no statiscal difference between isolation rate of \u003cem\u003eE. coli\u003c/em\u003e in Accra and Tema (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05). However, soil samples in Tema were more contaminated with \u003cem\u003eE. coli\u003c/em\u003e than Accra (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Resistant Patterns of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eisolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResistance patterns of \u003cem\u003eE. coli i\u003c/em\u003esolates from diverse sources is shown in \u003cstrong\u003eTable 6\u003c/strong\u003e. \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eexhibited high levels resistance to ampicillin (84.4%), ceftazidime (61.4%), cefotaxime (71.4%), and cefuroxime (96.4%). However, moderate levels of resistance to trimethoprim (31.2%) and tetracycline (25.7%) were observed. For ciprofloxacin, isolates recovered from humans exhibited a higher resistance levels compared to those from cattle (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.05). Similarly for tetracycline, isolates from cattle exhibited a higher resistance compared to those from humans (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.05). For ampicillin and nalidixic acid, isolates recovered from pigs showed a higher resistance levels compared to those from humans (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.05) \u003cstrong\u003eTable 6\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Multi-drug Resistant (MDR) Patterns of \u003cem\u003eE. coli\u003c/em\u003e isolates recovered from diverse sources.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall, 35.7% (n=50) of all \u003cem\u003eE. coli\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eisolates were MDR, but resistance varied among the various sample types; human (28.5%; n=20), cattle (55.0%; n=22), pig (35.0%; n=7) and soil (12.5%; n=1) \u003cstrong\u003eTable 7\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Also, 21.4% (n=30) of \u003cem\u003eE. coli\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eisolates were resistance to 1 antibiotic agent,\u0026nbsp;28.6% (n=40) to 2 antibiotics agents and\u0026nbsp;35.7% (n=50) to 3 or more antibiotic agents. Several MDR phenotypes were observed, and\u0026nbsp;more than half of the MDR isolates, 56% (n=28) were co-resistant to ampicillin, cefuroxime, trimethoprim and tetracycline and usually in combination with other antibiotic agents like chloramphenicol. About one-quarter of MDR isolates, 24% (n=12) were resistant to the nalidixic acid and ciprofloxacin\u003cstrong\u003e\u0026nbsp;Table 7\u003c/strong\u003e. Table 7 is at the end of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Resistance Patterns of ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003efrom diverse sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall,\u0026nbsp;ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates\u003cem\u003e\u0026nbsp;\u003c/em\u003eexhibited\u0026nbsp;high levels resistance of 100%, 100%, 75.8%, 78.0%, and 58.2% to ampicillin, cefuroxime, ceftazidime, cefotaxime and tetracycline. However, low resistance of 17.2% and 0.0% to ceftriaxone and meropenem. ESBL\u003cem\u003e-EC\u0026nbsp;\u003c/em\u003eisolates from human, cattle, pig and soil-sourced exhibited resistant prevalence of 100% to ampicillin, 42.9%, 55.6%, 100% and 33.3% resistance to tetracycline\u0026nbsp;(\u003cstrong\u003eFigure 2\u003c/strong\u003e).\u0026nbsp;Among human sourced isolates, there was no statistical significant difference between resistant pattern of ESBL\u003cem\u003e-EC\u0026nbsp;\u003c/em\u003eisolates\u0026nbsp;and\u003cem\u003e\u0026nbsp;\u003c/em\u003enon-ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates, with the exception of ampicillin, ciprofloxacin, gentamicin and nitrofurantoin\u0026nbsp;(\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026le; 0.05) \u003cstrong\u003eTable\u003c/strong\u003e \u003cstrong\u003e8\u003c/strong\u003e.\u0026nbsp;Among cattle sourced isolates, with the exception of an ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolate\u003cem\u003e\u0026nbsp;\u003c/em\u003ethat\u003cem\u003e\u0026nbsp;\u003c/em\u003eexhibited a higher resistance of (20.0% vrs 0\u003cem\u003e%,\u003c/em\u003e) to ciprofloxacin as compared to non-ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolate\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026le; 0.05) \u003cstrong\u003eTable 8.\u0026nbsp;\u003c/strong\u003eOverall,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethere was no statistical significant difference between antibiotic resistance of\u0026nbsp;ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates vrs\u0026nbsp;non-ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates \u003cstrong\u003eTable 8.\u003c/strong\u003e Among pig-sourced, there was no statistical significant difference in resistant pattern of ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates\u003cem\u003e\u0026nbsp;\u003c/em\u003eas compared to non-ESBL-\u003cem\u003eEC\u003c/em\u003e isolates. However, ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates\u0026nbsp;exhibited a higher resistance of (66.7% vrs 0%) and (100% vrs 5.9%) to ciprofloxacin and nalidixic acid (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026le; 0.05) \u003cstrong\u003eTable 9\u003c/strong\u003e. Overall, MDR prevalence of ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates was\u003cem\u003e\u0026nbsp;\u003c/em\u003e66.7% (n=20) and among sourced samples, human 71.4% (n=10), cattle 66.7% (n=6), pig 100% (n=3) and soil 33.3% (n=1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Prevalence of AMR genes in ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 16 different AMR genes were from major antibiotic class were observed in the\u0026nbsp;ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates \u003cstrong\u003eTable 10.\u003c/strong\u003e Across all ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates, the most common AMR genes was \u003cem\u003ebla\u003c/em\u003eTEM-1B (32.0%; n=8), (\u003cem\u003etet\u003c/em\u003eA) (48.0%; n=12), (\u003cem\u003esul\u003c/em\u003e2) (36.0%; n=9), (\u003cem\u003eaph\u003c/em\u003e(3\u0026apos;\u0026apos;)-\u003cem\u003eId\u003c/em\u003e) (24.0%; n=6) and (\u003cem\u003edfr\u003c/em\u003eA14) (16.0%; n=4). Also identified were plasmid-mediated\u0026nbsp;quinolone\u0026nbsp;resistant genes (\u003cem\u003eqn\u003c/em\u003erS1) (12.0%; n=3) and quinolone resistant determining region at the \u003cem\u003egyr\u003c/em\u003eA (4.0%; n=1) and \u003cem\u003ebla\u003c/em\u003eCTX-M-15 (4.0%; n=1) from soil sourced. Majority of AMR genes were observed from human and soil \u003cem\u003eESBL-EC\u0026nbsp;\u003c/em\u003eisolates \u003cstrong\u003eTable 10.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Prevalence of plasmid replicon types in ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates from diverse sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 21 different plasmid replicon types were identified from \u003cem\u003ein silico\u0026nbsp;\u003c/em\u003eanalysis of ESBL-\u003cem\u003eEC\u003c/em\u003e isolates \u003cstrong\u003eTable 11\u003c/strong\u003e. The most prevalent IncF plasmid replicon types were\u0026nbsp;IncFIB(Apoo1918)\u0026nbsp;(40.0%; n=10), followed by\u0026nbsp;IncFII(pCoo)\u0026nbsp;(36.0%; n=9). Overall, diverse plasmid replicon types were observed in human\u0026nbsp;ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates, followed by soil ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates.\u0026nbsp;IncFIB(Apoo1918)\u0026nbsp;(40.0%; n=5) was most dominant among cattle\u0026nbsp;ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates, followed by IncFII(pCoo) (40.0%; n=10) \u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e11.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDistribution\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of sequence types in ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates from diverse sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 17 different sequence types were identified from \u003cem\u003ein silico\u003c/em\u003e analysis of ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates from diverse sources \u003cstrong\u003eFigure 3\u003c/strong\u003e. The most prevalent sequence types were ST10 (12.0%; n=3), ST206 (12.0%; n=3), ST9312 \u003cstrong\u003e(\u003c/strong\u003e12.0%; n=3) and ST4151 (12.0%; n=3). However, 4.0% (n=1) was observed for each of the following; ST73, ST835, ST159, ST1237, ST6311, ST6237, ST5557, ST2061, ST8535, ST960, ST1684, ST999 and ST697, respectively (\u003cstrong\u003eFigure 3)\u003c/strong\u003e.\u0026nbsp;Overall, ST10 was observed in both cattle and pig ESBL-\u003cem\u003eEC\u003c/em\u003e isolates, while ST206 was found in both human and cattle ESBL-\u003cem\u003eEC\u003c/em\u003e isolates. Furthermore, ST73 was observed in cattle.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cstrong\u003e4.1 Prevalence of \u003cem\u003eE. coli\u003c/em\u003e isolates from diverse sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e is considered a commensal bacterium of the gastrointestinal tract of humans and animals [28-29]. The genomic plasticity of \u003cem\u003eE. coli\u003c/em\u003e facilitates their adaptation to diverse environments, thus, their wide association as an opportunistic pathogen in intestinal and extra-intestinal infections of humans and animals [7, 30]. In this study, \u003cem\u003eE. coli\u003c/em\u003e isolation rate of (9.3%) from diverse sources is in agreement with (8.0%) isolation rate reported in India from diverse sources [31]. In contrast to this study findings, higher prevalence of (26.5%) has been reported in Nigeria [32]. Our study result of 2% (2/100) \u003cem\u003eE. coli\u003c/em\u003e isolates from beef specimen and no detection in pork, lettuce and spring onion was similar to a study by Day and coworkers [33] in the United Kingdom which recovered \u003cem\u003eE. coli\u003c/em\u003e in beef (2%), pork (3%), and fruits and vegetables (0%). Variation in the prevalence may be attributed to differences in sample size, cultural methods, and geographical locations. \u003cem\u003eE. coli\u003c/em\u003e is a member of Enterobacteriaceae, ranked third in the WHO catalogue of antibiotic-resistant ‘priority pathogens’, and is currently, associated with the highest burden of AMR [8]. Commensal \u003cem\u003eE.\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e is often used as an indicator organism to study AMR-trends in food animals, the environment, and in human surveillance systems [34]. Usually, resistant rates in the sentinel bacterium \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eare termed as \"prevalence\" of resistance [35]. In\u0026nbsp;this study, MDR prevalence of (35.7%) in \u003cem\u003eE. coli\u003c/em\u003e isolates was in contrast to the higher MDR prevalence reported in studies conducted in New Zealand (64.0%) and Nigeria (48.8%) [36-37].\u0026nbsp;The monitoring of MDR \u003cem\u003eE. coli\u003c/em\u003e strains in food animals and food produce is necessary to evaluate its potential risk to humans [38]. Our study observation of \u003cem\u003eE. coli\u003c/em\u003e MDR prevalence of (28.5%), (55.0%), (35%) and (12.5%) among human, cattle, pig and soil sourced isolates was contrary to the\u0026nbsp;lower MDR prevalence of humans\u0026nbsp;(22%),\u0026nbsp;animal (5.7%), and environments\u0026nbsp;(31.3%)\u0026nbsp;reported in a systematic review by\u0026nbsp;Pormohammad and colleagues\u0026nbsp;[39]. Recently,\u0026nbsp;a study in Italy on AMR surveillance from diverse sources reported MDR prevalence in human (28.0%) and swine (24.0%) [40]. In Ghana, an earlier study by\u0026nbsp;Ohene\u0026nbsp;Larbi and coworkers [12] reported MDR prevalence of (23%) in pig, contrary to this study findings. More recently, a study by Tawfick and colleagues [41] in Egypt with commensal \u003cem\u003eE. coli\u003c/em\u003e from healthy humans reported MDR prevalence of (64.3%). In furtherance, varying MDR prevalence have been reported in cattle in Portugal (69%), France (56%), Egypt (44.4%); Mexico (72.7%) and Australia (32.4%) [42-46]. The irrational and indiscriminate use of antibiotic agents in human and animals has led to the rapid emergence and dissemination of MDR strains [47-49].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Prevalence and Resistance Patterns of ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e is recognized as a potential putative reservoir for ESBL resistance and has been increasingly reported globally [50]. In this study, ESBL-\u003cem\u003eEC\u003c/em\u003e prevalence of (21.5%) is in agreement with the prevalence of (21.7%) and (23.57%) reported in Nigeria and Egypt [32, 49]. Our study observation of ESBL-\u003cem\u003eEC\u003c/em\u003e prevalence of (20%) in healthy human subjects was consistent with the (22.5%) prevalence reported in a recent study in healthy pregnant women in Benin [50]. In contrast to this study findings, lower prevalence of (4.9% to 6.3%) has been documented among healthy subjects in Japan, Netherland and Germany [51-53].\u0026nbsp;However, higher prevalence of\u0026nbsp;(31.0%), (37.8%), (46.2%), (38.0%), (30.5%) and (71.4%)\u0026nbsp;have been reported in previous studies in\u0026nbsp;Vietnam, India, china; Chad; Gambia and Egypt [41, 51, 54-57]. In a systematic review and meta-analysis conducted by Bezabih and colleagues [11], the global pooled prevalence of ESBL-\u003cem\u003eEC\u003c/em\u003e fecal carriage in the community was reported as (17.6%) which was close to our study findings among healthy human subjects. Another study in Japan has reported ESBL fecal carriage of (15.6%) among healthy human subjects [58]. Pig sourced ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eprevalence of (15%) was in concordance with the (13.4%) and (14.7%) prevalence reported in Switzerland and West Indies [59-60]. However, a previous study carried out in Kumasi in Ghana by\u0026nbsp;Ohene\u0026nbsp;Larbi and coworkers [12] reported (0%) ESBL-\u003cem\u003eEC\u003c/em\u003e prevalence in pigs. Cattle sourced ESBL-\u003cem\u003eE.C\u0026nbsp;\u003c/em\u003eprevalence of (22.5%) was consistent with the (20.0%) prevalence reported in a recent study in Egypt [49]. However, lower prevalence of (4.35%), (15.3%), (7.5%) and (11.0%) have been reported in previous studies conducted in Switzerland, India, West Indies and Ghana [12, 31, 59-60]. The variation in ESBL prevalence may be attributed to little or no regulation on antibiotic usage in animal husbandry and unsanitary environments mostly encountered in the developing nations [61-62]. In this study, our observation of the high ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003eisolates resistance to 2\u003csup\u003end\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e generation cephalosporins cefuroxime, ceftazidime and cefotaxime was in contrast to the lower resistance of (8.3%), (8.3%), and (8.3%) reported in a similar “One Health” review study in Iran [39]. Thus, under a ‘One Health’ approach, AMR to cephalosporins category of beta-lactams is a typical example of how antibiotics play a significant role in the animal and human health. Our resistance level findings for gentamicin, chloramphenicol and nalidixic acid was\u0026nbsp;similar to the\u0026nbsp;(50%), (31.3%) and (41.7%) resistance prevalence\u0026nbsp;reported in a previous study in Nigeria [63]\u0026nbsp;Likewise, our findings of resistance prevalence to tetracycline was consistent with the (60%) reported in some earlier studies in Ghana [31] and Egypt [41]. It is noteworthy that resistance to ampicillin and tetracycline in this study is not unexpected, since ampicillin and tetracycline remains one of the most commonly used antimicrobials agents in livestock production in Ghana [64]. Worldwide, resistance to penicillin, tetracycline and sulfonamide is well documented in animal production [65] Furthermore, in this study, the high resistance to ampicillin and 2\u003csup\u003end\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e cephalosporin (cefuroxime, ceftazidime and cefotaxime) may be due to the indiscriminate use of beta-lactams agents due to their broad spectra, high effectiveness and minimal side effects [66]. In this study, MDR and ESBL prevalence to different sourced ESBL-\u003cem\u003eEC\u003c/em\u003e isolates of\u0026nbsp;human, cattle,\u0026nbsp;pig\u0026nbsp;and soil was\u0026nbsp;contrary to a systematic review study finding that reported MDR and ESBL prevalence\u0026nbsp;of (13%), (26.3%), and (25%) to human, animal, and environmental/food [39].\u0026nbsp;Literature has shown that the overuse and misuse of antibiotic agents in the agricultural, veterinary, and human medical departments may initiate the development and dissemination of MDR bacteria and allow for the emergence of unique AMR mechanisms [47, 67-69].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 AMR genes, plasmid replicons and sequence types in ESBL-\u003cem\u003eEc\u0026nbsp;\u003c/em\u003eisolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe emergence and rapid dissemination of MDR bacterial strains, particularly ESBL-\u003cem\u003eEC\u003c/em\u003e strains are of great concern [70]. In this study, our findings of \u003cem\u003ebla\u003c/em\u003eTEM-1B, \u003cem\u003etet\u003c/em\u003eA and \u003cem\u003esul\u003c/em\u003e2 as the prevalent AMR genes is in agreement with a recent systematic review outcome of\u0026nbsp;\u003cem\u003ebla\u003c/em\u003eTEM-1\u0026nbsp;(36.3%) and \u003cem\u003etetA\u003c/em\u003e (32.9%) reported by Ramatla and coworkers [9]. Escher and colleagues [10] in an earlier systematic review also reported \u003cem\u003ebla\u003c/em\u003eTEM-1,\u0026nbsp;\u003cem\u003esul\u003c/em\u003e2and\u003cem\u003e\u0026nbsp;tetA\u003c/em\u003e as the most prevalent AMR genes. More recently, studies in Gambia, USA, Mexico, Benin, China and Egypt have found \u003cem\u003ebla\u003c/em\u003eTEM as the most dominant ESBL gene type [34, 41, 50, 57, 71-72].\u0026nbsp;In Nigeria, Aworh and colleagues [73] study also observed \u003cem\u003ebla\u003c/em\u003eTEM-1 in MDR \u003cem\u003eE. coli\u003c/em\u003e as the most prevalent AMR genes. \u0026nbsp;In Ghana, a recent study by\u0026nbsp;Dsani\u0026nbsp;and coworkers [74] with raw meats found \u003cem\u003ebla\u003c/em\u003eTEM (4%) with absence of \u003cem\u003ebla\u003c/em\u003eCTX-M. The high \u003cem\u003ebla\u003c/em\u003eTEM-1B prevalence observed in this study could be responsible for the high resistance prevalence in ampicillin and probably 2\u003csup\u003end\u003c/sup\u003e and 3\u003csup\u003erd\u003c/sup\u003e cephalosporins.\u0026nbsp;Globally, \u003cem\u003ebla\u003c/em\u003eCTX-M-15 is the most prevalent ESBL gene type and\u0026nbsp;ESBL bacteria harbouring\u0026nbsp;plasmid-mediated enzymes with capacity to cause resistance to beta-lactam antibiotics such as ampicillin and cephalosporin as well as co-resistance to non-beta-lactams agents including quinolones and aminoglycosides, thus treatment options are limited [49. 70, 75]. When bacteria acquire AMR to antibiotics, they as well acquire a higher propensity to disseminate resistance in animals, humans, and the natural environment [2]. In this study, the only \u003cem\u003ebla\u003c/em\u003eCTX-M-15 gene identified was from soil sourced isolate in conjunction with quinolone resistant gene (\u003cem\u003eqnr\u003c/em\u003eS1). This study finding is in agreement with previous studies outcome in Brazil that reported soil-sourced ESBL-\u003cem\u003eE. coli\u003c/em\u003e carrying \u003cem\u003ebla\u003c/em\u003eCTX-M-15 and quinolone resistant gene (\u003cem\u003eqnr\u003c/em\u003eB19) along with other AMR genes [76-77]. This is the first report of ESBL-\u003cem\u003eEC\u003c/em\u003e harbouring \u003cem\u003ebla\u003c/em\u003eCTX-M-15 and \u003cem\u003eqnr\u003c/em\u003eS1 in agricultural soil from Ghana and this signifies a threat to food and environmental safety. In this study, soil samples from Ashiaman-Tema area were found to be more contaminated with \u003cem\u003eESBL-EC\u0026nbsp;\u003c/em\u003eisolates than those of Accra metropolis. This may be attributed to the high level of open defecation by cattles and human particularly at order side of the motor way close to Ashiaiman area. The highest AMR genes were observed in human sourced ESBL-\u003cem\u003eEC\u003c/em\u003e isolates in this study. Several studies have shown that fecal deposition of AMR genes and active antibiotic agents from humans and animals waste and their persistence in the environment are known to promote the spread of AMR genes in the environment [78-81]. The presence of MDR\u0026nbsp;and ESBL-\u003cem\u003eEC\u003c/em\u003e isolates exhibiting a wide range of resistors to antimicrobials in agricultural soil may lead to contamination of vegetable crops and, since these types of foods are often consumed raw as ready-to-eat foods (R-T-E), the risk of human exposure to antibiotic-resistant bacteria (ABR) and AMR genes with high clinical significance is worrisome [76-77, 84].\u0026nbsp;Mobile genetic elements (MGE), specifically, plasmids, are involved in the dissemination of ESBL determinants and co-resistance encoding genes and could result in the rapid escalation of ESBL-producing strains from diverse sources [82-83].\u0026nbsp;In this study, IncFIB(Apoo1918) and IncFII(pCoo) were the most dominant plasmid replicon types. Studies in Gambia, Germany; Italy, USA and Mexico have reported IncF plasmid replicon types as the most prevalent plasmid replicons\u0026nbsp;involved in encoding acquired AMR genes and often termed epidemic plasmids\u0026nbsp;[34, 40, 57, 71 84]. The incompatibility (Inc) group F (IncF) which belongs to the narrow-host-range plasmids is one of the most significant plasmids involved in AMR genes transmission and spread to other bacteria [85].\u0026nbsp;This study observed diverse sequence types (STs) among ESBL-\u003cem\u003eEC\u003c/em\u003e isolates. Our findings is in concordance with study finding as reported in United Kingdom, Ghana, Nigeria; Germany, Canada and Italy [13, 33, 40, 86-87]. Our finding included the well-known ST such as ST10 in animal and the environment. Suggestive that a probable transmission might have taken place amongst these sources. It's noteworthy that ST10, a global one health clone with potential to disseminate ESBL determinants in association with other AMR genes has been identified in diverse sources\u0026nbsp;[88].\u0026nbsp;Additionally, ST10 possesses broad-host range and mostly associated with extra-intestinal infections [6, 13, 40, 83-84, 87, 89].\u0026nbsp;Globally, pandemic and high-risk zoonotic MDR \u003cem\u003eE. coli\u003c/em\u003e clones have disseminated into diverse niches including food-animals, humans and the environment [83, 90].\u0026nbsp;In this study, ST206 was detected in human and cattle.\u0026nbsp;Qiu and coworkers [91]\u0026nbsp;study in China reported ST206 in minks (fur animals). Another study by\u0026nbsp;Ayeni\u0026nbsp;and colleagues [92] in Nigeria also reported ST206 in poultry livestock.\u0026nbsp;The\u0026nbsp;presence of ESBL-\u003cem\u003eEC\u003c/em\u003e ST206 isolates in animal and human sources is suggestive of their potential for spread and persistence in different hosts\u0026nbsp;[93].\u0026nbsp;In this study, no ST131 was detected. ST131 are well established pandemic clone with propensity to spread ESBL genes and AMR genes globally [6]. However, ST73, a member of the pandemic high-risk clone was identified in cattle in this study. Studies in the United Kingdom and Australia have reported ST73 among human sourced isolates as one of the most prevalent ST associated with bacteremia and UTI [88, 94]. Our study finding might be due to a probable circulation of host-adapted lineage of ESBL-\u003cem\u003eEC\u0026nbsp;\u003c/em\u003estrains.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion ","content":"\u003cp\u003eWithin the metropolis surveyed, we identified MDR ESBL\u003cem\u003e-EC\u0026nbsp;\u003c/em\u003eisolatesharbouringvarious AMR genes and plasmid replicons with diverse \u003cem\u003eE. col\u003c/em\u003e\u003cem\u003ei\u003c/em\u003e STs in healthy humans, animals and the environment.\u0026nbsp;This study finding of \u003cem\u003ebla\u003c/em\u003eCTX-M-15 in agricultural soil isolate is worrisome, emphasizing the need for a one-health approach in combating AMR.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAMR: Antimicrobial resistance, MDR: multi-drug resistant, extended-spectrum-beta-lactamase-producing-\u003cem\u003eEscherichia coli\u003c/em\u003e (ESBL-\u003cem\u003eEC\u003c/em\u003e), \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e), whole-genome sequencing (WGS), Sequence types (STs), Clinical and laboratory Standard Institute (CLSI).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their gratitude to all the staff of the Department of Medical Laboratory Science, department of Medical Microbiology and the Department of Bacteriology, Noguchi Memorial Institute for Medical Research, University of Ghana. Special thanks go to\u0026nbsp;SEQAFRICA Project,\u0026nbsp;Noguchi Memorial Institute for Medical Research\u0026nbsp;for whole genome sequencing of bacterial isolates. Also, thanks to animal owners and agricultural farmers for their permission to samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMOT, JO, FOA and BE conceived and designed the study. MOT collected data and performed laboratory analysis; BE and CO generated the whole genome sequence data for all the ESBL isolates. MOT conducted bioinformatics analysis, interpreted the data and wrote the first draft of the manuscript. JO, FOA and BE supervised the study, reviewed and edited the manuscript article. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish not applicable\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.\u0026nbsp;All the ESBL-\u003cem\u003eEC\u003c/em\u003e sequence data reads\u0026nbsp;and genome assemblies generated in this research study have been submitted to GenBank under the BioProject PRJNA1077263.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was carried out in accordance with the Declaration of Helsinki, and ethical approval was obtained from the Ethics Committee of the College of Health Science, University of Ghana \u0026nbsp; (Ethics approval number: CHS-Et/M.1-P5.12/2022-2023). All methods and protocols were performed in accordance with relevant regulations and guidelines, on \u0026nbsp;involving humans and animal subjects in the study. Formal permission was sought from all the farm animal owners and participants involved. Participation was voluntary and written consent was obtained from each participant following the ethical committee’s guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAslam B, Khurshid M, Muzammil S, Rasool M, Arshad MI, Yasmeen N, Shah T, Chaudhry TH, Shahid A, Rasool MH, Xueshan X and Baloch Z. Antibiotic Resistance: One Health One World Outlook. Front. Cell. Infect. 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G., Yambao, J. C., \u0026amp; Qui\u0026ntilde;ones, B. \u0026nbsp;Isolation, genotyping and antimicrobial resistance of Shiga toxin-producing \u003cem\u003eEscherichia coli. J. Microbiol. Immunol. Infect\u003c/em\u003e. 2018;51:425\u0026ndash;434.\u003c/li\u003e\n \u003cli\u003eGraham, D. W., Bergeron, G., Bourassa, M. W., Dickson, J., Gomes, F., Howe, A., et al., Complexities in understanding antimicrobial resistance across domesticated animal, human, and environmental systems. Ann. N. Y. \u003cem\u003eAcad. Sci\u003c/em\u003e. 2019;1441: 17\u0026ndash;30.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSobur, M. A., Sabuj, A. A. M., Sarker, R., Rahman, A. T., Kabir, S. L., \u0026amp; Rahman, M. T. Antibiotic-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e spp. associated with dairy cattle and farm environment having public health significance. Veterinary world 2019;12:984\u0026ndash;993.\u003c/li\u003e\n \u003cli\u003eRozwandowicz M, Brouwer MSM, Fischer J, Wagenaar JA, Gonzalez-Zorn B, Guerra B, Mevius DJ, Hordijk J. Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. \u003cem\u003eJ Antimicrob Chemother.\u003c/em\u003e 2018;73(5):1121-1137.\u003c/li\u003e\n \u003cli\u003eSilva, A.; Silva, V.; Pereira, J.E.; Maltez, L.; Igrejas, G.; Valent\u0026atilde;o, P.; Falco, V.; Poeta, P (2023). Antimicrobial Resistance and Clonal Lineages of \u003cem\u003eEscherichia coli\u003c/em\u003e from Food-Producing Animals. \u003cem\u003eAntibiotics\u003c/em\u003e 2023;12:1061.\u003c/li\u003e\n \u003cli\u003eReid CJ, Blau K, Jechalke S, Smalla K and Djordjevic SP. Whole Genome Sequencing of \u003cem\u003eEscherichia coli\u003c/em\u003e From Store-Bought Produce. \u003cem\u003eFront. Microbiol\u003c/em\u003e. 2020;10:3050.\u003c/li\u003e\n \u003cli\u003ePartridge, S. R., Kwong, S. M., Firth, N. \u0026amp; Jensen, S. O. Mobile genetic elements associated with antimicrobial resistance. \u003cem\u003eClin. Microbiol. Rev\u003c/em\u003e. 2018;31, 1.\u003c/li\u003e\n \u003cli\u003eMass\u0026eacute; J. Vanier G, Fairbrother J.M, de Lagarde M, Arsenault J, Francoz D, Dufour S \u0026amp; Archambault M. Description of Antimicrobial-Resistant \u003cem\u003eEscherichia coli\u003c/em\u003e and Their Dissemination Mechanisms on Dairy Farms. \u003cem\u003eVet. Sci\u003c/em\u003e, 2023;10, 242.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAworh MK, Ekeng E, Nilsson P, Egyir B, Owusu-Nyantakyi C and Hendriksen RS (2022) Extended-Spectrum \u0026szlig;-Lactamase-Producing \u003cem\u003eEscherichia coli\u003c/em\u003e Among Humans, Beef Cattle, and Abattoir Environments in Nigeria. Front. Cell. Infect. Microbiol. 12:869314. doi: 10.3389/fcimb.2022.869314.\u003c/li\u003e\n \u003cli\u003eDay MJ, Doumith M, Abernethy J, et al. Population structure of Escherichia coli causing bacteraemia in the UK and Ireland between 2001 and 2010. \u003cem\u003eJ Antimicrob Chemother\u003c/em\u003e 2016;71: 213942.\u003c/li\u003e\n \u003cli\u003eHojabri Z, Mirmohammadkhani M, Darabi N, Arab M, \u0026amp; Pajand O. Characterization of antibiotic-susceptibility patterns and virulence genes of five major sequence types of \u003cem\u003eEscherichia coli\u003c/em\u003e isolates cultured from extraintestinal specimens: a 1-year surveillance study from Iran. \u003cem\u003eInfect Drug Resist\u003c/em\u003e. 2019;12:893-903.\u003c/li\u003e\n \u003cli\u003eEger E, Domke M, Heiden S.E, Paditz M, Balau V, Huxdorff C, Zimmermann D, Homeier-Bachmann T \u0026amp; Schaufler K. Highly Virulent and Multidrug-Resistant \u003cem\u003eEscherichia coli\u003c/em\u003e Sequence Type 58 from a Sausage in Germany. \u003cem\u003eAntibiotics\u003c/em\u003e 2022;11:1006.\u003c/li\u003e\n \u003cli\u003eQiu J, Jiang Z, Ju Z, Zhao X, Yang J, Guo H \u0026amp; Sun S.\u0026nbsp;Molecular and Phenotypic Characteristics of \u003cem\u003eEscherichia coli\u003c/em\u003e Isolates from Farmed Minks in Zhucheng, China. \u003cem\u003eHindawi BioMed Research International\u003c/em\u003e 2019;3917841.\u003c/li\u003e\n \u003cli\u003eAyeni FA, Falgenhauer J, Schmiede J, Schwengers O, Chakraborty T \u0026amp; Falgenhauer L. Detection of \u003cem\u003ebla\u003c/em\u003eCTX-M-27-encoding \u003cem\u003eEscherichia coli\u003c/em\u003e ST206 in Nigerian poultry stocks. \u003cem\u003eJ Antimicrob Chemother\u003c/em\u003e; 2020;75: 3070\u0026ndash;3072.\u003c/li\u003e\n \u003cli\u003eZhou Z, Alikhan NF, Mohamed K et al., The EnteroBase user\u0026rsquo;s guide,with case studies on Salmonella transmissions, Yersinia pestis phylogeny and \u003cem\u003eEscherichia\u003c/em\u003e core genomic diversity. \u003cem\u003eGenome Res\u003c/em\u003e; 2020;30:138\u0026ndash;52.\u003c/li\u003e\n \u003cli\u003eLi D, Elankumaran P, Kudinha T, Kidsley AK, Trott DJ, Jarocki VM, Djordjevic SP. Dominance of \u003cem\u003eEscherichia coli\u003c/em\u003e sequence types ST73, ST95, ST127 and ST131 in Australian urine isolates: a genomic analysis of antimicrobial resistance and virulence linked to F plasmids. Microb Genom. 2023;9(7):mgen001068. doi: 10.1099/mgen.0.00\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 11 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"one-health-outlook","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oneh","sideBox":"Learn more about [One Health Outlook](https://onehealthoutlook.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/oneh/default.aspx","title":"One Health Outlook","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial resistance (AMR), multi-drug resistant (MDR), Escherichia coli (E. coli), Extended-spectrum-beta-lactamase (ESBL), Ghana.","lastPublishedDoi":"10.21203/rs.3.rs-4480595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4480595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: ‘One health’, a concept that highlights the need to bring on board multiple players and actors together to addressing major health problems, have been proposed to be effective in data gathering to mitigate the menace of antimicrobial drug resistance (AMR). Data on MDR and extended-spectrum-beta-lactamase-producing \u003cem\u003eEscherichia coli\u003c/em\u003e (ESBL-\u003cem\u003eEC\u003c/em\u003e) across humans, animals and the environment are limited in low-and-middle-income-countries (LMICs) including Ghana. \u003cstrong\u003eObjective\u003c/strong\u003e: This study used one health approach to determine the prevalence, antibiogram and AMR genes of ESBL-\u003cem\u003eEC\u003c/em\u003e from diverse sources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology\u003c/strong\u003e: A cross-sectional study conducted in the Accra and Tema metropolis, Ghana. We randomly collected 1500 non-duplicated specimens from healthy human, cattle, pigs, lettuce, spring onions, pork, beef and soil samples. \u003cem\u003eEscherichia coli\u003c/em\u003e(\u003cem\u003eE. coli\u003c/em\u003e)\u003cem\u003e \u003c/em\u003ewas isolated and confirmed by MALDI-TOF MS. \u003cem\u003eE. coli\u003c/em\u003e isolates were screened for their susceptibility against 13 antibiotic agents and ESBL-production. ESBL-\u003cem\u003eEC\u003c/em\u003eisolates were whole-genome sequenced (WGS) and \u003cem\u003ein silico\u003c/em\u003e analysis was used to determine AMR genes, sequence types (STs) and plasmid replicon types.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult\u003c/strong\u003e: Overall, \u003cem\u003eE. coli\u003c/em\u003e was recovered from 140 of 1500 (9.3%) specimens processed. About one-third of these \u003cem\u003eE. coli\u003c/em\u003e isolates 50 (35.7%) were resistant to three or more antibiotics, and 30 (21.5%) were ESBL-\u003cem\u003eEC\u003c/em\u003e. The proportion of ESBL-\u003cem\u003eEC \u003c/em\u003eidentified in healthy humans, cattle, pig, beef and soil were 14 (20.0%), 9 (22.5%), 3 (15.0%), 1 (50.0%) and 3 (37.5%), respectively. No \u003cem\u003eE. coli\u003c/em\u003e was isolated from lettuce, spring onions and pork. Overall, the ESBL-\u003cem\u003eEC \u003c/em\u003eexhibited high levels of resistance to ampicillin (100%), cefuroxime (100%), ciprofloxacin (53.6%), and to tetracycline (58.2%). However, all ESBL-\u003cem\u003eEC \u003c/em\u003eisolates were sensitive to meropenem. The prevalent AMR genes detected were \u003cem\u003ebla\u003c/em\u003eTEM-1B (32.0%; n=8), \u003cem\u003etet\u003c/em\u003eA (48.0%; n=12) and \u003cem\u003esul2\u003c/em\u003e (32.0%; n=8). The dominant STs were ST10 (12%; n=3), ST 9312 (12%; n=3), ST 206 (12%; n=3) and ST 4151 (12%; n=3). IncFIB(Apoo1918) (40.0%; n=10) and IncFII(pCoo) (36.0%; n=9) plasmid replicons were commonly detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eWithin the metropolis surveyed, we identified MDR ESBL-\u003cem\u003eEC\u003c/em\u003eharbouring various AMR genes and plasmid replicons with diverse \u003cem\u003eE. coli\u003c/em\u003eSTs in healthy humans, animals and the environment. This study finding of \u003cem\u003ebla\u003c/em\u003eCTX-M-15 in agricultural soil isolate is worrisome, emphasizing the need for a one-health approach in combating AMR.\u003c/p\u003e","manuscriptTitle":"Molecular characterization of multidrug-resistant (MDR) Escherichia coli in the Greater Accra Region, Ghana: a ‘One Health’ approach.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-25 05:26:05","doi":"10.21203/rs.3.rs-4480595/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-07-14T21:13:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-02T21:38:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-21T06:00:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"One Health Outlook","date":"2024-06-20T08:42:21+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revision","date":"2024-06-10T17:18:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"one-health-outlook","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"oneh","sideBox":"Learn more about [One Health Outlook](https://onehealthoutlook.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/oneh/default.aspx","title":"One Health Outlook","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"972ac69b-d141-45b8-99bb-ef5e0a729ca5","owner":[],"postedDate":"July 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:00:17+00:00","versionOfRecord":{"articleIdentity":"rs-4480595","link":"https://doi.org/10.1186/s42522-025-00154-8","journal":{"identity":"one-health-outlook","isVorOnly":false,"title":"One Health Outlook"},"publishedOn":"2025-05-26 15:57:15","publishedOnDateReadable":"May 26th, 2025"},"versionCreatedAt":"2024-07-25 05:26:05","video":"","vorDoi":"10.1186/s42522-025-00154-8","vorDoiUrl":"https://doi.org/10.1186/s42522-025-00154-8","workflowStages":[]},"version":"v1","identity":"rs-4480595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4480595","identity":"rs-4480595","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}