Introduction
The usage of antimicrobial drugs in veterinary and human medicine has led to the extensive development of antimicrobial resistance (AMR) in animals, humans, and the environment [1–3]. A significant concern is how AMR disseminates rapidly globally [4,5]. Third-generation cephalosporins are essential in our defence against bacterial infections [6]. Exposure to 3rd generation cephalosporin-resistant Escherichia coli (E. coli) may occur through different routes, such as interaction with animal or human carriers, consuming contaminated food or interaction with contaminated environmental compartments [7]. It is pivotal to elucidate the relative involvement of probable routes and sources to institute preventive mechanisms.
Various market segments, from consumers to restaurants and institutional markets, are regularly supplied with fresh produce in Nigeria. An enormous quantity of ready-to-eat (RTE) vegetables is retailed, particularly street foods [8]. Several outbreaks have highlighted fresh vegetables as potential vehicles of foodborne disease [9]. Although not in outbreak proportions, E. coli illnesses have also been reported in Southern Nigeria [10–12]. Its heightened concern over the spread of antimicrobial-resistant pathogens made this research imperative. Nigeria currently does not have a functional foodborne disease reconnaissance system. Its direct healthcare consequence is projected at approximately $3 billion, responsible for 17–25% of the projected cost for all illnesses [13]. Nigeria’s Area Councils Health System profile showed foodborne infection (FBI) as the foremost cause of mortality, averaging 25%. In Nigeria, >200,000 people die annually from food poisoning caused by foodborne pathogens, as estimated by the World Health Organization (WHO) [2,8,14]. Mortalities result from contaminated foods via improper preservation, service and processing [15].
Fecal E. coli can occur in the soil, water and on plants. ESBL-producing pathotypes of E. coli have also been recovered in the environment [16–18]. Since vegetables are usually eaten raw, ingesting ESBL-producing E. coli that can reach the gastrointestinal tract poses a potential public health risk [6,14,19]. In recent decades, the prevalence of multidrug resistance (MDR) E. coli has been increasing globally [20,21]. Selective pressure from antibiotics, which culminates from their usage and abuse, contributes to AMR [3,22]. A key mechanism for resistance to third-generation cephalosporins is centred on the plasmid-mediated enzyme (β-lactamase) production, which inactivates these compounds by cleaving the β-lactam ring. CTX-M is one of the most predominantly documented ESBL β-lactamase types from clinical environments in E. coli isolates [23].
ESBL-producing E. coli from animals and humans can enter the environment through manure and sewage. Fresh produce may become contaminated with ESBL-producing E. coli from the farm environment by irrigation with contaminated water sources or when cultivated in manure-supplemented soil. ESBL-producing E. coli could persist on vegetable surfaces and sometimes advance to the interior and spread to animals or humans [24,25]. In recent years, interest has focused on the microbiological hazards that challenge the food industry [1,8,14]. It is imperative to pinpoint the crucial role of consuming uncooked or undercooked vegetables in bacterial gastroenteritis [26]. There are limited studies on spreading and transmitting ESBL-producing E. coli in agricultural environments and food vegetables [7,27]. The incidence of ESBL E. coli in food, vegetables, and the farm environment is detrimental to public health and food safety. The current study intended to evaluate the presence of ESBL-producing E. coli on food vegetable samples in Edo State, Nigeria. Ready-to-eat (RTE) salads and vegetables that are potentially consumed uncooked were selected for isolation and characterization of ESBL E. coli isolates, as were vegetables from farms and markets. Vegetables from farms were included to reflect the occurrence of ESBL-producing E. coli in the agricultural environment.
Materials and methods
Study area and sample collection
The sample size for this study was determined using the sample size determination formula below:
Z1-α/2 = Standard normal variant at 5% type I error (P < 0.05); P = Expected prevalence of 4.0–10.12% was based on previous studies [7,21,28–33]; d = Absolute error or precision (which is 5%). The expected minimum sample to be collected ranged from 59–140. Edo State was selected to pilot the study due to its strategic location in Southern Nigeria and its large-scale cultivation of vegetables due to its conducive climate and environmental conditions. There are eighteen Local Government Areas (LGA) in Edo State, and samples were collected in each LGA (Figs 1 and 2). A total of 254 samples were examined. Edo South is the most populated among the three senatorial districts and is also where the state capital (Benin City) is located (Fig 1). LGA are geographical entities within a state in Nigeria which largely depend on the land mass of the states, and Local Government Councils administer these. The senatorial districts in Nigeria consist of a combination of LGA within a state, with each state consisting of three senatorial districts. A single representative farm was surveyed from each LGA, totaling 18 farms. Samples from agricultural farms include soil (n = 25), manure (n = 25), irrigation water (n = 25), and vegetables (n = 78). Samples from vendors and open markets include ready-to-eat (RTE) salads (n = 60) and vegetables potentially consumed uncooked (n = 41). The fresh produce from vendors and open markets potentially consumed uncooked included: lettuce (n = 7), carrot (n = 7), tomato (n = 7), cabbage (n = 6), pepper (n = 7) and cucumber (n = 7). Ethical approval was not obtained before sampling collection since samples were not obtained directly from humans or food-producing animals. However, verbal informed consent and permission were obtained from the farm owners before sampling collection from respective farms.
[Figure omitted. See PDF.]
[Figure omitted. See PDF.]
ESBL enumeration, isolation and identification
The use of the CHROMagar ESBL media for ESBL-E. coli enumeration and isolation were in line with previous studies [34,35]. Samples were processed by homogenizing 25 g of soil or vegetable samples or mixing 25 ml of irrigation water with 225 ml of buffered peptone water, diluted serially, followed by incubation for 24 h at 37°C. After that, one ml was spread plated onto CHROMagar ESBL (75006 Paris, France) media and plates were incubated for 18–24 h at 37°C. ESBL production was determined by characteristic colonial morphology and growth on culture media. Dark pink to reddish colonies on CHROMagar ESBL plate designated ESBL-producing E. coli. The cell counts of ESBL E. coli from CHROMagar ESBL were expressed in CFU/g (for vegetable, soil, and animal manure samples) or CFU/100ml (for water samples). Two to three colonies were picked per positive sample and assayed for ESBL phenotypically using the double-disk test [36]. Ceftazidime (30 μg) and ceftazidime-clavulanate (30/10 μg), as well as cefotaxime (30 μg) and cefotaxime-clavulanate (30/10 μg), were used in a standard disk diffusion procedure and incubated at 37°C for 18 h. A ≥5-mm increase in inhibition diameter for either tested antimicrobial agent in combination with clavulanate vs the zone diameter of the agent when tested alone was considered ESBL [36]. Polymerase chain reaction (PCR) was used to confirm the identity of the isolates as E. coli by targeting the uidA gene via specific primers [37]. A total of 64 isolates were confirmed and stored for further characterization.
Antimicrobial susceptibility testing
The antimicrobial susceptibility of the isolates was determined using the Kirby-Bauer disk-diffusion method following recommendations outlined by the Clinical and Laboratory Standards Institute antimicrobial (CLSI) [36]. Briefly, the purified isolates were inoculated in 5.0 mL Mueller-Hinton Broth (MHB) (Lab M, Lancashire, United Kingdom) and incubated overnight. The optical density (OD) of the turbidity of the broth was adjusted to 0.5 McFarland standard, equivalent to 1.5×108 cfu/mL. The McFarland turbidity standard was prepared following an established procedure [36]. Using a sterile swab stick, respective broth cultures were aseptically swabbed on Mueller Hinton Agar (Lab M, Lancashire, United Kingdom). The antimicrobial disks used (Mast Diagnostics, UK) included: Cephems (cefpodoxime 10 μg, cefotaxime 30 μg, ceftazidime 30 μg, ceftriaxone 30 μg); monobactams (aztreonam 30 μg), penicillins (ampicillin 10 μg, piperacillin 100 μg); β-lactam combination agents (ampicillin-sulbactam 10/10 μg, amoxicillin-clavulanate 20/10 μg, piperacillin-tazobactam 100/10 μg, meropenem-vaborbactam 20/10 μg, ceftazidime-avibactam 30/20 μg); carbapenems (meropenem 10 μg, ertapenem 10 μg, imipenem 10 μg); aminoglycosides (gentamicin 10 μg, kanamycin 30 μg, streptomycin 10 μg), tetracyclines (tetracycline 30 μg, doxycycline 30 μg); quinolones and fluoroquinolones (levofloxacin 5 μg, ofloxacin 5 μg, ciprofloxacin 5 μg); folate pathway antagonists (trimethoprim-sulfamethoxazole 1.25/23.75 μg), nitrofurans (nitrofurantoin 300 μg) and phenicols (chloramphenicol 30 μg) [36]. Isolates were classified as showing a resistant, intermediate or susceptible phenotype according to CLSI standards [36]. The multiple antibiotic resistance index (MARI) was determined to be the total number of antimicrobials the organism is resistant to divided by the total number of antimicrobials the organism was subjected to [38].
Characterization of ESBL E. coli isolates regarding ESBL determinants and other antibiotic resistance genes
All CHROMagar positive, ESBL-producing presumptive E. coli isolates that were confirmed by PCR as E. coli, were screened for genetic element determinants of ESBL resistance, i.e., blaCTX−M−15, blaTEM, blaSHV and all CTX-M-groups (blaCTX−M−1−group, blaCTX−M−2−group, blaCTX−M−9−group, blaCTX−M−8−group, blaCTX−M−14−group), as well as the blaOXA−1−group. The blaOXA−47, blaNDM−1 (carbapenem-resistance genes), and blaCMY−2 genes (AmpC lactamase) were PCR amplified using conditions as described previously [39]. The presence of resistance determinants for tetracycline [tetA, tetB, tetM], sulfonamides [sul1, sul2 andsul3], aminoglycosides [ant(4´)-Ia, aacC(3)-1], quinolones [qnrA, qnrB, qnrC and qnrS] and chloramphenicol [cat1, cat2, cat3] resistance were also determined [40]. As reported before, intI2 and intI1 genes (encoding class 2 and class 1 integrases, respectively) were also investigated [40]. Single colonies of individual isolates were aerobically cultivated in Tryptone Soy Broth (TSB) (Lab M, Lancashire UK) in a shaker incubator at 200 rpm at 30°C for 24 h. The cells were pelleted via centrifugation (12,500 ×g for 10 min). Deoxyribonucleic acid (DNA) was extracted using the peqGOLD Bacterial DNA Kit (Peqlab Biotechnologie GmbH, Germany), following the manufacturer’s protocols. PCR was performed using a Peltier-based thermal cycler (MG963/Y, Hangzhou, Zhejiang, China) with thermocycling conditions and specific primers for each of the determinants as described in S1 Table.
Data analysis
The data from this study were analyzed using Microsoft Excel 2013 and SPSS 21.0. The prevalence and occurrence from this study were expressed in percentage (%) while the population density from the samples was expressed as mean ± standard deviation of the mean.
Results
Prevalence of ESBL E. coli from agricultural farms and open markets
A total of 64 E. coli isolates were identified using PCR and further characterized (Table 1). In total, 68% (17/25) of ESBL E. coli were isolated from agricultural farm soil, 84% (21/25) from manure, 28%(7/25) from irrigation water and 24.4% (19/78) from vegetables, while 20% (12/60) were obtained from RTE salads from vendors and open markets and 36.6% (15/41) from vegetables that can be consumed uncooked which were also obtained from vendors and open markets. Amongst the vegetables from vendors which can be consumed uncooked, the tomato had a higher prevalence of 100% (7/7) than cabbage 66.7% (4/6), lettuce 42.9% (3/7) and pepper 14.3% (1/7). Amongst the vegetables from farms, the fluted pumpkin had the highest prevalence at 26.3% (5/19), followed by Lagos spinach at 21.1% (4/19), African spinach at 21.1% (4/19), waterleaf at 15.8% (3/19), jute leaves 10.5% (2/19) and curry leaf 5.3% (1/19). The mean counts of ESBL E. coli based on the occurrence of reddish to dark pink colonies on the CHROMagar ESBL plate were 2.72 × 103 ± 0.76 CFU/g for soil, 7.68 × 103 ± 1.49 CFU/g for manure, 1.13 × 102 ± 0.35 CFU/mL for irrigation water, 1.25 × 102 ± 0.26 CFU/g for vegetables, 2.51 × 101 ± 0.31 CFU/g for RTE salads and 9.4 × 101 ± 0.22 CFU/g for vegetables potentially consumed uncooked.
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Antimicrobial susceptibility profile of the ESBL E. coli isolates
ESBL E. coli isolates showed the following incidences of resistance towards 26 antibiotics (Table 2): ceftazidime and cefotaxime 100% (64/64); cefpodoxime 96.9% (62/64); ceftriaxone 98.4% (63/64); amoxicillin-clavulanate 50% (32/64); ciprofloxacin and ampicillin 48.4% (31/64); doxycycline 45.3% (29/64); piperacillin 43.8% (28/64); kanamycin 42.2% (27/64); ofloxacin and gentamicin 37.5% (24/64); levofloxacin and trimethoprim-sulfamethoxazole 35.9% (23/64); chloramphenicol 26.6% (17/64); ampicillin-sulbactam 14.1% (9/64); ceftazidime-avibactam 6.3% (4/64). All the isolates were sensitive to meropenem-vaborbactam, nitrofurantoin, ertapenem, imipenem and meropenem.
[Figure omitted. See PDF.]
A total of 85.9% (55/64) isolates were resistant to ≥3 and ≤7 antimicrobial classes, which categorizes these as being multidrug-resistant (MDR). The multiple antibiotic resistance (MAR) index of the isolates ranged from 0.27 –to 0.62 for these 55 MDR isolates. However, all the isolates were multi-resistant (resistant to a minimum of 4 antibiotics) and displayed 52 resistance phenotypes (Table 3). A MAR index >0.5 was observed to be associated with isolates recovered from the agricultural environments. In addition, isolates from agricultural environments seemingly showed a higher resistance phenotype than isolates from vendors and open markets. The phenotypic resistance phenotype of the isolates was independent of the beta-lactamase determinants of the isolates (Table 3).
[Figure omitted. See PDF.]
Presence of resistance determinants in ESBL E. coli isolates
The proportions of ESBL E. coli isolates that harboured beta-lactamase genes in Fig 3 include blaTEM 50% (32/64), blaCTX−M−15 10.9% (7/64) and blaCTX−M−1 76.6% (49/64). The proportions of ESBL E. coli isolates harbouring other resistance determinants include tetA 14.1% (9/64), tetB 4.7% (3/64), tetM 32.8% (21/64), sul1 10.9% (7/64), sul2 18.8% (11/64), sul3 6.3% (4/64), ant(4´)-Ia 10.9% (7/64), aacC(3)-1 14.1% (9/64), qnrA 12.5% (8/64), qnrB 3.1% (2/64), qnrC 9.4% (6/64), qnrS 10.9% (7/64), cat::pC194 9.4% (6/64), cat::pC221 10.9% (7/64), cat::pC223 3.1% (2/64), intI1 20.3% (13/64), intI2 3.1% (2/64). The 55 MDR isolates harboured a minimum of 1 and a maximum of 5 AMR determinants. The 55 MDR isolates also harboured a minimum of 1 and a maximum of 3 beta-lactamase determinants. The detection of the beta-lactamase determinants of the isolates was independent of the detection of other antibiotic resistance genes. All isolates harbouring respective antibiotic resistance genes showed phenotypic resistance to ≥1 antibiotic from the antimicrobial class (Table 3).
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Discussion
Vegetables are highly prone to contamination by microorganisms through contact with soil, water and handling during harvest or after-harvest processing. They may therefore harbour a diverse range of bacterial pathotypes, including human and plant pathogens. Quarcoo et al. [21] reported that counts expressed in CFU/g ranged from 186 to 3000, with the highest counts found in lettuce, with all lettuce samples positive for E. coli. Though the population density from Quarcoo et al. [21] study is similar to ours, the occurrence rate is higher than ours. This could be attributed to the selective characteristics of the media used in our research. Reported microbial load range of <3 to >1100 MPN/g from Vegetables in Malaysia [41] and 2.31 ±0.86 log CFU/g to 5.50 ±0.7 log CFU/g in Spain [31] were also similar to those of our study. The total counts of ESBL-E. coli from the farm produce and environments were determined to evaluate their microbiological quality and safety. The potential sources of ESBL-E. coli contamination could result from faecal contamination, poor water quality, poor distribution and sanitary conditions, and poor handling practices. Lower ESBL-E. coli prevalence from farmed vegetables compared to those of our study have been reported from Finland, Spain, Germany, Southern Thailand, and the United States [31,42–46]. A higher prevalence of 62.11% (59/95) has been reported in Malaysia [41]. Zurfluh et al. [47] documented that 25.7% of retail store samples determined were positive for ESBL isolates, which was more comparable to the results of this study. Soré et al. [48] reported that 42.47% of salad samples were positive for ESBL-producing isolates. On the other hand, that prevalence was higher than the prevalence recovered from our study, and the differences may be explained by different soils, manure and agricultural practices of the other regions/countries. Variations in the prevalence of ESBL-producing E. coli isolates depend on the sample size and types of vegetables tested. ESBL strains have been occasionally found in human infections and foods [8,49,50]. In a recent population-based modelling study, 18.9% of human ESBL-E. coli carriage was attributed to a food source [51], highlighting the importance of AMR surveillance and hygiene measures for food products.
In this study, high heterogeneity in the presence of ESBL E. coli was observed both at the farm level and from open markets. Njage and Buys [52] documented an increased incidence of ESBL-producing E. coli in irrigation water (73%-64%) samples and lettuce at harvest (90%) in South Africa. A higher prevalence was recounted from the irrigation water in this study. In the Netherlands, irrigation water (100%) and harvested lettuce (14.7%) were also found to harbour beta-lactamase-producing Enterobacteriaceae [7]. The incidence in irrigation water was far higher than observed in this study. Using a discrete-time model by Costa et al. [53], it was reported that ESBL-E. coli prevalence reached an equilibrium prevalence of 0.65% in the open community, where the colonization of the open community could primarily be attributed to the open community itself (62%), followed by vegetable consumption (29.5%), and contact with farmers (8.5%). A clear link between irrigation water and leafy green vegetable E. coli isolates has been established [54]. As low-income countries have higher AMR abundance in their wastewater [20,22,55,56], the risk of ESBL dissemination is greater. Using contaminated irrigation water may directly link to AMR contamination, mainly if the water is applied to edible parts of fruits and vegetables [57]. The types of nutrients that are frequently present on the leaf surface of leafy green vegetables can be categorized into two: inorganic (ions) and organic molecules (organic acids and carbohydrates) [58]. Carbohydrates (dominant phyllosphere sugars are glucose, fructose and sucrose) are of particular interest due to their capacity to readily support the growth of enteric bacteria such as E. coli. Different factors could again account for these differences, but using untreated wastewater, seasons and manure application as fertilizers might be significant factors [58]. Some authors have previously documented the identification of ESBL E. coli and the possible transmission of antibiotic-resistant bacteria via treated wastewater for irrigation [58,59], while others could not find a clear connection [60].
Concerns about the quality and safety of vegetable salads and fresh produce have increased, despite their essential nutritional benefits to humans [6–8,14,19]. Outbreaks associated with fresh produce portray a dearth of information crucial in understanding the ecology of ESBL E. coli outside animal and human-animal hosts [61]. The microbial incidences reported for fresh produce could be attributed to contaminated cultivated soil, poor storage methods and poor agricultural practices by handlers. The increased microbial occurrence on leafy greens may be linked to the structure of their surface, with many folds and furrows and a corresponding high surface area, which may lend itself to colonization. Fresh produce contamination by pathogenic bacteria is currently emerging as a significant food safety concern, and the occurrence of MDR bacteria can signify an additional hazard. Prevalence of MDR (96.1%) and resistance to aminoglycosides (46.7–66.7) in ESBL-producing Enterobacteriaceae have been reported previously [47,62], which is similar to the findings from this study. Richter et al. [63] reported that 98% of their isolates showed an MDR phenotype, close to the 100% of our research. Vegetables are often eaten without heating processes, leading to a greater risk of acquiring resistant bacteria [6,14,19]. Microbial food quality control maintains a critical factor in continually controlling foodborne illnesses (one of the primary causes of mortality and morbidity) coupled with a cost-intensive loss for food companies.
Contamination by ESBL E. coli of farm environments and vegetable samples from our study hypothetically could primarily be from animal sources. Since the manure used in farm environments is predominantly from animal sources. These animals are usually treated with antibiotics for therapeutic and prophylactic purposes [64,65]. These antibiotic residues could be excreted by the animal in an unmetabolized or poorly metabolized form, resulting in the development and transmission of AMR [66]. But also, harvesting and handling by workers that harbour the pathogens may be a contamination source; thus, cross-contamination is another factor that needs to be considered in spreading antimicrobial-resistant bacteria from foods to humans [67]. In addition, the potential implication of untreated surface water or wastewater for irrigation purposes in Edo State, Nigeria, cannot be overemphasized. An increase in AMR gene carriage in soil has been documented for years [68], pinpointing the role of soil as a reservoir for genes and antibiotic-resistant pathogens. Given that the soil community could harbour these genes and AMR bacteria, contamination during postharvest may also arise from this source in RTE salads and fresh vegetables. This could be an essential route for the spread of antibiotic-resistant pathogens.
Soré et al. [48] documented that resistance to the aminoglycoside, fluoroquinolones and sulfonamides classes was dominant in fresh-cut salads and wastewater samples. An average of ~50% of ESBL-E. coli isolates from our study were resistant to aminoglycoside and fluoroquinolones, with 35.9% resistant to the sulfonamides class of antimicrobials. Zurfluh et al. [47] reported that all isolates from the investigated fresh vegetables were resistant to cephalothin and ampicillin, while 53 (88.3%) were resistant to cefotaxime. All our isolates were resistant to cefotaxime and ceftazidime, with additional resistance to cefpodoxime 96.9% (62/64), ceftriaxone 98.4% (63/64) and ampicillin 48.4% (31/64). Among 12 antibiotics screened by Ramadan et al. [69], only imipenem seems to have remained resilient, which was in line with the carbapenems activity from our study. The high resistance to fluoroquinolones, penicillin, tetracyclines and aminoglycosides is probably a result of easy access, increased use and non-regulation of these antibiotics on the Nigerian market.
Manure from food animals, usually used as a soil fertilizer, may harbour different resistant bacteria and resistance encoding genes from the gut microbiota of food animals [65]. This phenomenon has become a major problem for public health, not only in underdeveloped countries but also in high-performing, socio-economically developed countries. ESBL E. coli was identified in vegetables from vendors of market origin in our study and has also been detected in the Netherlands [70] and Tunisia [27]. It is important to note that vegetables that harbour ESBL E. coli from open markets are usually consumed uncooked or undercooked, where the potential for transfer to humans cannot be ruled out [7]. Outbreaks of foodborne origin (vegetables) consequent of ESBL-E. coli isolates have been documented [71,72]. Above 50% of ESBL E. coli recovered could be disseminated via conjugation through ESBL determinants to other bacteria [27]. The horizontal spread of antibiotic-resistant determinants between soil and human bacteria has been reported previously [73]. The transfer of beta-lactamase elements in E. coli strains could be linked with IncI1 replicon plasmid acquisition [74].
Our results correlate with other studies indicating vegetables as a possible route for the dynamic diffusion of AMR genes in the community [58]. The blaESBL and other resistance genes on vegetables have been described as existing predominantly in opportunistic and saprophytic bacteria, which are thought to represent a background reservoir of antibiotic resistance genes [22]. Iseppi et al. [75] reported that PCR confirmed the occurrence of blaCTX-M-15 in the ESBL E. coli studied. Richter et al. [63] Genotypic characterisation showed the widespread prevalence of CTX-M-1, followed by SHV and TEM. CTX-M14 and CTX-M15 are widespread in the environment, food animals and humans in Europe [76,77]. blaTEM-1 and blaTEM-15 have also been reported from vegetables in Finland [44]. Similar β-lactamase genes to those from our study have been reported in Southern Thailand [45]. ESBL groups identified from our research (CTX-M-15 and CTX-M-1) also correspond to ESBL groups previously reported in Tunisia [27] from vegetables, irrigation water and soil; and in E. coli from healthy humans, food-producing animals, and food [74,78]. Hence, contamination by ESBL E. coli from farm environments and vegetable samples could have occurred from both animal and human origins in Nigeria. The probable consequence of untreated irrigation water can also not be overemphasized as contributing to the spread of MDR pathogens. CTX-M-15 ESBL was detected in our study and has been identified as the most common type of ESBL in Gram-negative bacteria worldwide from different sources, including humans, animals, wildlife and wastewater samples [58].
The finding of resistance genes (blaCTX-M-15, blaCTX-M-1, and blaTEM) commonly linked to human sources [44] highlights the potential transmission route of AMR via food products and emphasizes the importance of hygiene measures. Zurfluh et al. [47] reported that 38.5% (10/26) isolates harboured blaCTX-M-15, and 3.8% (1) were positive for blaCTX-M-1. This is not in line with the findings from our study, as 10.9% (7) strains harboured blaCTX−M−15, while 76.6% (49) harboured blaCTX−M−1. Ramadan et al. [69] reported that 90.4% (179/198) ESBL isolates contained ≥1 blaCTX-M gene, which was similar to the 85.9% (55/64) observed for our isolates. The prevalence of blaCTX-M-15 determinants in isolates has been documented in India following preceding studies from clinical isolates from South India and Delhi [79]. The group CTX-M-15 has progressively been described in community isolates and reported as the utmost Gram-negative ESBL bacteria in Europe [80]. Also, the CTX-M-15 group is especially prevalent in samples from the medical area in Germany [81], while the CTX-M-1 group more often occurs in samples from livestock [82]. The CTX-M-1 group was the most commonly found in this study (49/64; 76.6%, Table 3), which points toward a predominantly livestock origin of the ESBL isolates. Community-onset ESBL-associated infections, including urinary infection and sepsis, are principally caused by E. coli having blaCTX-M ESBLs [54]. Therefore, the relevant β-lactamase genes might contribute to the presence of ESBLs in community-transmitted infections from animal sources to humans via food chains.
The isolates in the present study carried AMR genes conferring resistance against critically essential antimicrobials [83], including aminoglycosides, third- and fourth-generation cephalosporins, tetracyclines and quinolones. All ESBL E. coli isolates from our study were MDR, and some carried integrons, as this characteristic is typical of ESBL-producing bacteria [84]. However, this was different from a survey by Soré et al. [48], where relatively less (93.23%) of the isolates showed MDR (94.40% in wastewater and 91.66% in salads). Co-expression of β-lactamase genes may result in stronger ESBL production and increased antibiotic resistance or MDR [45]. Antibiotic resistance genes encoding 12 major classes of antibiotics, including β-lactam antibiotics, aminoglycoside, macrolide, fluoroquinolone, and others, were found in vegetable samples from the USA, which were similar to the genes detected from samples from our study [46]. Compared to our findings, class 1 integrons were previously identified in MDR beta-lactamase-positive isolates [63,85]. Class II integron was not identified by Richter et al. [63], while it was identified at a low prevalence of 3.1% (2) in our study. Bezanson et al. [86] recounted that class 2 integrons were primarily identified in Canada, accompanied by class 1 integrons from salad vegetables. This is contrary to the findings from our study, where the class I integron was predominant. The diversity of resistance phenotypes observed among the ESBL-E. coli isolates showed that the potential spread of resistant bacteria is not the consequence of disseminating a unique strain. It instead appears that genetic variations of ESBLs aren’t narrowly restricted or localized to a singular ecosystem. Farming practices, such as selecting organic fertilizers, types of irrigation water, and sanitation of vegetable processing environments, may affect the composition of the microbiome and antibiotic resistome in vegetables. Understanding the various potential pathways for antibiotic resistome dissemination through cultivation-irrigation-manure microbiomes is critical for mitigating antibiotic resistance. The limitations of this study are the fact that a single state from Nigeria was covered, and state-of-the-art molecular tools such as Whole Genome Sequencing (WGS), multilocus sequence typing (MLST) were not carried out to determine the sequence types (ST), and clones that are circulating with the farms and the market environments. However, the research group is currently working on attracting funding for such aspects of the research to monitor the microbiome, virulome and resistome using metagenomic tools.
Conclusion
Findings from this study represent the first investigation on the identification and characterization of ESBL E. coli recovered from farm environments, fresh produce and salads in Edo State, Nigeria. This work showed that fresh vegetables and salads could harbour ESBL-E. coli, particularly fresh produce from farms that use untreated water for irrigation. Antimicrobial-resistant pathogens can spread to humans via the food chain, especially when these products are consumed uncooked. Suitable measures, for example, improvement of water quality and agricultural practices, need to be implemented, as well as established global regulatory guidelines to ensure public health and consumer safety. Close antimicrobial resistance surveillance in bacteria from a one-health perspective is crucial to source-track and mitigate the development and spread of such bacteria. Findings from this study could serve as baseline data to evaluate the human health risk and the potential transmission associated with the consumption of food vegetables. The subsequent aspect of this study includes expanding the survey to other zones in Nigeria, use of whole genome sequencing and multilocus sequence typing to characterize the isolates further to understand the exact clones that are circulating in the farm environments across Nigeria,
Supporting information
S1 Table. Primers used in this study.
https://doi.org/10.1371/journal.pone.0282835.s001
(DOCX)
Citation: Igbinosa EO, Beshiru A, Igbinosa IH, Cho G-S, Franz CMAP (2023) Multidrug-resistant extended spectrum β-lactamase (ESBL)-producing Escherichia coli from farm produce and agricultural environments in Edo State, Nigeria. PLoS ONE 18(3): e0282835. https://doi.org/10.1371/journal.pone.0282835
About the Authors:
Etinosa O. Igbinosa
Contributed equally to this work with: Etinosa O. Igbinosa, Abeni Beshiru, Isoken H. Igbinosa, Gyu-Sung Cho, Charles M. A. P. Franz
Roles: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing
E-mail: [email protected] (EOI); [email protected] (CMAPF)
Affiliation: Faculty of Life Sciences, Applied Microbial Processes & Environmental Health Research Group, University of Benin, Benin City, Nigeria
ORICD: https://orcid.org/0000-0001-7441-2145
Abeni Beshiru
Contributed equally to this work with: Etinosa O. Igbinosa, Abeni Beshiru, Isoken H. Igbinosa, Gyu-Sung Cho, Charles M. A. P. Franz
Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing
Affiliations: Faculty of Life Sciences, Applied Microbial Processes & Environmental Health Research Group, University of Benin, Benin City, Nigeria, Department of Microbiology, College of Natural and Applied Sciences, Western Delta University, Oghara, Delta State, Nigeria
Isoken H. Igbinosa
Contributed equally to this work with: Etinosa O. Igbinosa, Abeni Beshiru, Isoken H. Igbinosa, Gyu-Sung Cho, Charles M. A. P. Franz
Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing
Affiliations: Faculty of Life Sciences, Applied Microbial Processes & Environmental Health Research Group, University of Benin, Benin City, Nigeria, Faculty of Life Sciences, Department of Environmental Management & Toxicology, University of Benin, Benin City, Nigeria
Gyu-Sung Cho
Contributed equally to this work with: Etinosa O. Igbinosa, Abeni Beshiru, Isoken H. Igbinosa, Gyu-Sung Cho, Charles M. A. P. Franz
Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing
Affiliation: Department of Microbiology and Biotechnology, Max Rubner–Institut, Federal Research Institute of Nutrition and Food, Kiel, Germany
ORICD: https://orcid.org/0000-0002-3639-6663
Charles M. A. P. Franz
Contributed equally to this work with: Etinosa O. Igbinosa, Abeni Beshiru, Isoken H. Igbinosa, Gyu-Sung Cho, Charles M. A. P. Franz
Roles: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing
E-mail: [email protected] (EOI); [email protected] (CMAPF)
Affiliation: Department of Microbiology and Biotechnology, Max Rubner–Institut, Federal Research Institute of Nutrition and Food, Kiel, Germany
ORICD: https://orcid.org/0000-0001-9726-1845
1. Igbinosa EO, Okeigbemen R, Beshiru A, Akinnibosun O. Detection and characterization of antibiotic resistance of Gram negative bacteria from hospital and non-hospital environments in Benin City Nigeria. UNILAG J Bas Med Sci. 2022; 9(2): 15–24.
2. Igbinosa EO, Beshiru A, Igbinosa IH, Okoh AI. Antimicrobial resistance and genetic characterisation of Salmonella enterica from retail poultry meats in Benin City, Nigeria. LWT 2022; 169: 114049.
3. Beshiru A, Igbinosa IH, Igbinosa EO. An investigation on antibiogram characteristics of Escherichia coli isolated from piggery farms in Benin City, Nigeria. Ann Sci Tech. 2016; 1:8–12.
4. Igere BE, Peter WO, Beshiru A. Distribution/spread of superbug and potential ESKAPE-B pathogens amongst domestic and environmental activities: A public health concern. Discovery 2022; 58(3): 1–20.
5. Igbinosa EO, Beshiru A, Odjadjare EEO. Diversity, antimicrobial characterization and biofilm formation of enterococci isolated from aquaculture and slaughterhouse sources in Benin City, Nigeria. Ife J Sci. 2020; 22(3): 51–63.
6. Igbinosa IH, Beshiru A, Egharevba NE, Igbinosa EO. Distribution of Enterobacteria in ready-to-eat food in cafeterias and retail food outlets in Benin City: Public health implications. J Comm Med Pri Health Care. 2020; 32(2): 80–94.
7. Blaak H, van Hoek AH, Veenman C, van Leeuwen AE, Lynch G, van Overbeek WM, et al. Extended spectrum ß-lactamase and constitutively AmpC-producing Enterobacteriaceae on fresh produce and in the agricultural environment. Int J Food Microbiol. 2014; 169: 8–16.
8. Beshiru A, Okoh AI, Igbinosa EO. Processed ready-to-eat (RTE) foods sold in Yenagoa Nigeria were colonized by diarrheagenic Escherichia coli which constitute a probable hazard to human health. PLoS ONE 2022; 17(4): e0266059.
9. Nüesch-Inderbinen RMT, Stephan S. Fresh fruit and vegetables as vehicles of bacterial foodborne disease: A review and analysis of outbreaks registered by proMED-mail associated with fresh produce. Archiv Für Lebensmittelhygiene. 2016; 67(2):32–39.
10. Afolayan AO, Aboderin AO, Oaikhena AO, Odih EE, Ogunleye VO, et al. An ST131 clade and a phylogroup A clade bearing an O101-like O-antigen cluster predominate among bloodstream Escherichia coli isolates from South-West Nigeria hospitals. Microb Genomics 2022; 8:000863.
11. Akinduti AP, Ayodele O, Motayo BO, Obafemi YD, Isibor PO, Aboderin OW. Cluster analysis and geospatial mapping of antibiotic resistant Escherichia coli O157 in southwest Nigerian communities. One Health 2022; 15: 100447.
12. Odetoyin B, Ogundipe O, Onanuga A. Prevalence, diversity of diarrhoeagenic Escherichia coli and associated risk factors in well water in Ile-Ife, Southwestern Nigeria. One Health Outlook 2022 4(3)
13. Oludare AO, Ogundipe A, Odunjo A, Komolafe J, Olatunji I. Knowledge and food handling practices of Nurses in a Tertiary health care Hospital in Nigeria. J Environ Health. 2016; 78(6): 32–39. pmid:26867289
14. Igbinosa EO, Beshiru A, Igbinosa IH, Ogofure AG, Uwhuba KE. Prevalence and characterization of food-borne Vibrio parahaemolyticus from African salad in Southern Nigeria. Front. Microbiol. 2021; 12:632266.
15. World Health Organization. World health statistics 2009. World Health Organization. 2009; 149p.
16. Franz E, Veenman C, van Hoek AH, de Roda Husman A, Blaak H. Pathogenic Escherichia coli producing Extended-Spectrum β-Lactamases isolated from surface water and wastewater. Sci Rep. 2015; 24(5):14372.
17. Hossain MS, Ali S, Hossain M, Uddin SZ, Moniruzzaman M, Islam MR, et al. ESBL producing Escherichia coli in faecal sludge treatment plants: An invisible threat to public health in Rohingya camps, Cox’s Bazar, Bangladesh. Front Public Health. 2021; 9:783019.
18. Medina-Pizzali ML, Venkatesh A, Riveros M, Cuicapuza D, Salmon-Mulanovich G, Mäusezahl D, et al. Whole-genome characterisation of ESBL-producing E. coli isolated from drinking water and Dog faeces from rural Andean households in Peru. Antibiotics 2022; 11:692.
19. Igbinosa EO, Beshiru A, Isichei-Ukah BO, Oyedoh OP. Isolation and characterization of multiple antibiotic-resistant Salmonella strains from fresh vegetable samples. Nig J Microbiol. 2017; 31(2): 4099–4106.
20. Imanah EO, Beshiru A, Igbinosa EO. Antibiogram profile of Pseudomonas aeruginosa isolated from some selected hospital environmental drains. Asian Pacific J Trop Dis. 2017; 7(10): 604–609.
21. Quarcoo G, Adomako BLA, Abrahamyan A, Armoo S, Sylverken AA, Addo MG, et al. What Is in the Salad? Escherichia coli and antibiotic resistance in lettuce irrigated with various water sources in Ghana. Int J Environ Res Public Health. 2022; 19: 12722.
22. Igbinosa EO, Beshiru A. Characterization and identification of antibiotic-resistant Acinetobacter baumannii from hospital wastewaters. Nig J Microbiol. 2017; 31(2): 3926–3932.
23. Coque TM, Baquero F, Canton R. Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro Surveill. 2008; 13(47):19044. pmid:19021958
24. Gekenidis MT, Rigotti S, Hummerjohann J, Walsh F, Drissner D. Long-term persistence of blaCTX-M-15 in soil and lettuce after introducing extended-spectrum β-lactamase (ESBL)-producing Escherichia coli via manure or water. Microorganisms 2020; 8(11):1646.
25. Lopes R, Fuentes-Castillo D, Fontana H, Rodrigues L, Dantas K, Cerdeira L, et al. Endophytic lifestyle of global clones of extended-spectrum β-lactamase producing priority pathogens in fresh vegetables: A Trojan horse strategy favoring human colonization? mSystems 2021; 6:e01125–20.
26. Hamilton-Miller JM, Shah S. Identity and antibiotic susceptibility of enterobacterial flora of salad vegetables. Int J Antimicrob Agents. 2001; 18(1):81–3. pmid:11463532
27. Ben-Said L, Jouini A, Klibi N, Dziri R, Alonso CA, Boudabous A, et al. Detection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in vegetables, soil and water of the farm environment in Tunisia. Int J Food Microbiol. 2015; 203:86–92. pmid:25791254
28. Montero L, Irazabal J, Cardenas P, Graham JP, Trueba G. Extended-spectrum beta-lactamase producing-Escherichia coli isolated from irrigation waters and produce in Ecuador. Front. Microbiol. 2021; 12:709418.
29. Said SL, Jouini A, Klibi N, Dziri R, Alonso CA, Boudabous A, et al. Detection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in vegetables, soil and water of the farm environment in Tunisia. Int J Food Microbiol. 2015; 203: 86–92. pmid:25791254
30. Chotinantakul K, Woottisin S, Okada S. The emergence of CTX-M-55 in ESBL-producing Escherichia coli from vegetables sold in local markets of northern Thailand. Japanese J Infect Dis 2021; 139: Article ID JJID.
31. Pintor-Cora A, Álvaro-Llorente L, Otero A, Rodríguez-Calleja JM, Santos JA. Extended-spectrum beta-lactamase producing Enterobacteriaceae in fresh produce. Foods. 2021; 10: 2609. pmid:34828891
32. Song J, Kim J, Oh S, Shin J. Multidrug-resistant extended-spectrum β-Lactamase-producing Escherichia coli isolated from vegetable farm soil in South Korea. Microb Drug Res. 2021; 27(11)
33. Djuikoue CI, Wega F, Dayomo AL, Nana CDS, Ekeu DN, Guegang CG, et al. Freshwater linked resistance profile and prevalence of Escherichia coli producing ESBL Type CTX-M strains in Cameroon urban cities. Front Environ Microbiol. 2022; 8(3): 55–61.
34. Ramaite K, Ekwanzala MD, Momba MNB. Prevalence and molecular characterisation of extended-spectrum beta-lactamase-producing shiga toxin-producing Escherichia coli, from Cattle farm to aquatic environments. Pathog. 2022; 11: 674.
35. Burgess SA, Moinet M, Brightwell G, Cookson AL. Whole genome sequence analysis of ESBL-producing Escherichia coli recovered from New Zealand freshwater sites. Microb Genomics. 2022; 8:000893.
36. Clinical and Laboratory Standards Institute antimicrobial (CLSI). Performance Standards for Antimicrobial Susceptibility Testing This document includes updated tables for the Clinical and Laboratory Standards Institute antimicrobial susceptibility testing standards M02, M07, and M11. A CLSI supplement for global application. 30th Edition. Wayne, USA. 2020; 332pp.
37. Bej AK, DiCesare JL, Haff L, Atlas RM. Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Appl Environ Microbiol. 1991; 57:1013–1017.
38. Igbinosa IH, Amolo CN, Beshiru A, Akinnibosun O, Ogofure AG, El-Ashker M, et al. identification and characterization of MDR virulent Salmonella spp isolated from smallholder poultry production environment in Edo and Delta States, Nigeria. PLoS ONE. 2023; 18(2): e0281329.
39. Talukdar PK, Rahman M, Nabi A, Islam Z, Hoque MM, Endtz HP, et al. Antimicrobial resistance, virulence factors and genetic diversity of Escherichia coli isolates from household water supply in Dhaka, Bangladesh. PLoS One 2013; 8(4):e61090.
40. Sáenz Y, Briñas L, Domínguez E, Ruiz J, Zarazaga M, Vila J. et al. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins. Antimicrob. Agents Chemother. 2004; 48:3996–4001.
41. Lee E, Radu S, Jambari NN, Abdul-Mutalib NA. Prevalence and antibiogram profiling of extended-ppectrum beta-lactamase (ESBL) producing Escherichia coli in raw vegetables, in Malaysia. Biol Life Sci Forum. 2021; 6: 44.
42. Mesa RJ, Blanc V, Blanch AR, Cortes P, Gonzalez JJ, Lavilla S, et al. Extended-spectrum β-lactamase-producing Enterobacteriaceae in different environments (humans, food, animal farms and sewage). J Antimicrob Chemoth. 2006; 58: 211–215.
43. Kaesbohrer A, Bakran-Lebl K, Irrgang A, Fischer J, Kämpf P, Schiffmann A, et al. Diversity in prevalence and characteristics of ESBL/pAmpC producing E. coli in food in Germany. Vet Microbiol. 2019; 233: 52–60.
44. Kurittu P, Khakipoor B, Aarnio M, Nykäsenoja S, Brouwer M, Myllyniemi AL, et al. Plasmid-borne and chromosomal ESBL/AmpC genes in Escherichia coli and Klebsiella pneumoniae in Global Food Products. Front Microbiol. 2021; 12:592291.
45. Romyasamit C, Sornsenee P, Chimplee S, Yuwalaksanakun S, Wongprot D, Saengsuwan P. Prevalence and characterization of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated from raw vegetables retailed in Southern Thailand. PeerJ 2021; 9:e11787.
46. Moon SH, Udaondo Z, Abram KZ, Li X, Yang X, DiCaprio EL, et al. Isolation of AmpC- and extended spectrum β-lactamase-producing Enterobacterales from fresh vegetables in the United States. Food Cont. 2022; 132: 108559.
47. Zurfluh K, Nüesch-Inderbinen M, Morach M, Zihler-Berner A, Hächler H, Stephan R. Extended-spectrum β-lactamase-producing Enterobacteriaceae isolated from vegetables imported from the Dominican Republic, India, Thailand, and Vietnam. Appl Environ Microbiol. 2015; 81(9): 3115–3120.
48. Soré S, Sawadogo Y, Bonkoungou JI, Kaboré SP, Béogo S, Sawadogo C, et al. Detection, identification and characterization of extended-spectrum beta-lactamases producing Enterobacteriaceae in wastewater and salads marketed in Ouagadougou, Burkina Faso. Int J Biol Chem Sci. 2020; 14(8): 2746–2757.
49. Roer L, Overballe-Petersen S, Hansen F, Johannesen TB, Stegger M, Bortolaia V, et al. ST131 fimH 22 Escherichia coli isolate with a bla CMY-2/IncI1/ST12 plasmid obtained from a patient with bloodstream infection: highly similar to E. coli isolates of broiler origin. J Antimicrob Chemother. 2019; 74:557–560.
50. Valcek A, Roer L, Overballe-Petersen S, Hansen F, Bortolaia V, Leekitcharoenphon P, et al. IncI1 ST3 and IncI1 ST7 plasmids from CTX-M-1-producing Escherichia coli obtained from patients with bloodstream infections are closely related to plasmids from E. coli of animal origin. J Antimicrob Chemother. 2019; 74: 2171–2175.
51. Mughini-Gras L, Dorado-García A, van Duijkeren E, van den Bunt G, Dierikx CM, Bonten MJM, et al. Attributable sources of community-acquired carriage of Escherichia coli containing β-lactam antibiotic resistance genes: a population-based modelling study. Lancet Planet Health. 2019; 3: e357–e369.
52. Njage PMK, Buys EM. Pathogenic and commensal Escherichia coli from irrigation water show potential in transmission of extended-spectrum and AmpC β-lactamases determinants to isolates from lettuce. Microb Biotechnol. 2014; 8: 462–473.
53. Costa EF, Hagenaars TJ, Dame-Korevaar A, Brouwer MSM, de Vos CJ. Multidirectional dynamic model for the spread of extended-spectrum-β-lactamase-producing Escherichia coli in the Netherlands. Microb Risk Anal. 2022; 22: 100230.
54. Ratshilingano MT, du Plessis EM, Duvenage S, Korsten L. Characterization of multidrug-resistant Escherichia coli isolated from two commercial lettuce and spinach supply chains. J Food Prot. 2022; 85(1): 122–132.
55. Igbinosa EO, Beshiru A, Onwunma E, Akinnibosun O. Evaluation and characterization of Salmonella and Shigella species from abattoir effluents and receiving watersheds in Ikpoba River, Benin City, Nigeria. NIPES J Sci Technol Res. 2021; 3(3): 28–39.
56. Beshiru A, Igbinosa IH. Biomonitoring of the physicochemical variables, Vibrio and Salmonella pathogens from raw shrimp in selected rivers in Southern region Nigeria. NIPES J Sci Technol Res. 2020; 3(1):28–42.
57. FAO and WHO. Joint FAO/WHO Expert Meeting in Collaboration with OIE on Foodborne Antimicrobial resistance: Role of the environment, crops and biocides–meeting report. Microbiol Risk Assess Series. 2019; 34. Rome: FAO.
58. Chelaghma W, Loucif L, Bendjama E, Cherak Z, Bendahou M, Rolain JM. Occurrence of extended spectrum cephalosporin-, carbapenem- and colistin-resistant Gram-negative bacteria in fresh vegetables, an increasing human health concern in Algeria. Antibiotics. 2022; 11: 988. pmid:35892378
59. Pignato S, Coniglio MA, Faro G, Weill FX, Giammanco G. Plasmid-mediated multiple antibiotic resistance of Escherichia coli in crude and treated wastewater used in agriculture. J Water Health. 2009; 7: 251–258.
60. Negreanu Y, Pasternak Z, Jurkevitch E, Cytryn E. Impact of treated wastewater irrigation on antibiotic resistance in agricultural soils. Environ Sci Technol. 2012; 46: 4800–4808. pmid:22494147
61. Jackson CR, Randolph KC, Osborn LO, Tyler HL. Culture-dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables. BMC Microbiol. 2013; 13:1–12.
62. Richter L, Du Plessis EM, Duvenage S, Korsten L. Occurrence, identification, and antimicrobial resistance profiles of extended-spectrum and AmpC β-lactamase-producing Enterobacteriaceae from fresh vegetables retailed in Gauteng Province, South Africa. Foodborne Pathog Dis. 2019; 16(6): 421–27.
63. Richter L, du Plessis EM, Duvenage S, Korsten L. Occurrence, phenotypic and molecular characterization of extended-spectrum- and AmpC-β-lactamase producing Enterobacteriaceae isolated from selected commercial spinach supply chains in South Africa. Front Microbiol. 2020; 11:638.
64. Igbinosa EO, Beshiru A. Characterization of antibiotic-resistant and species diversity of staphylococci isolated from apparently healthy farm animals. Afr J Clin Exp Microbiol. 2019; 20(4): 289–298.
65. Igbinosa IH, Beshiru A, Ikediashi SC, Igbinosa EO. Identification and characterization of Salmonella serovars isolated from Pig farms in Benin City, Edo State, Nigeria: One health perspective. Microb Drug Res. 2021; pmid:32589500
66. Igbinosa IH, Beshiru A. Detection and antibiotic-resistant of Salmonella species and Escherichia coli from selected captive animals in the Ogba Zoological garden Benin City. Nig J Pure Appl Sci. 2018; 31(1): 3171–3179.
67. Beshiru A, Igbinosa IH, Igbinosa EO. Characterization of enterotoxigenic Staphylococcus aureus from ready-to-eat seafood (RTES). LWT. 2021; 135: 11004.
68. Knapp CW, McCluskey SM, Singh BK, Campbell CD, Hudson G, Graham DW. Antibiotic resistance gene abundances correlate with metal and geochemical conditions in archived Scottish soils. PLoS One. 2011; 6: e27300. pmid:22096547
69. Ramadan AA, Abdelaziz NA, Amin MA. Novel blaCTX-M variants and genotype-phenotype correlations among clinical isolates of extended-spectrum beta-lactamase-producing Escherichia coli. Sci Rep. 2019; 9: 4224.
70. Reuland EA, Al Naiemi N, Raadsen SA, Savelkoul PHM, Kluytmans JAJW, Vandenbroucke-Grauls CMJE. Prevalence of ESBL-producing Enterobacteriaceae in raw vegetables. Eur J Clin Microbiol Infect Dis. 2014; 33: 1843–1846. pmid:24848131
71. Buchholz U, Bernard H, Werber D, Böhmer MM, Remschmidt C, Wilking H, et al. German outbreak of Escherichia coli O104:H4 associated with sprouts. N Engl J Med 2011; 365: 1763–1770.
72. King LA, Nogareda F, Weill FX, Mariani-Kurkdjian P, Loukiadis E, Gault G, et al. Outbreak of Shiga toxin-producing Escherichia coli O104:H4 associated with organic fenugreek sprouts, France, June 2011. Clin Infect Dis. 2012; 54: 1588–1594.
73. Forsberg K, Reyes A, Wang B, Selleck E, Sommer M, Dantes G. The shared antibiotic-resistome of soil bacteria and human pathogens. Sci. 2012; 337: 1107–1111. pmid:22936781
74. Ben-Sallem R, Ben-Slama K, Rojo-Bezares B, Porres-Osante N, Jouini A, Klibi N, et al. IncI1 plasmids carrying blaCTX-M-1 or blaCMY-2 genes in Escherichia coli from healthy humans and animals in Tunisia. Microb Drug Resist. 2014; 20: 495–500.
75. Iseppi R, de Niederhausern S, Bondi M, Messi P, Sabia C. Extended-Spectrum β-lactamase, AmpC, and MBL-producing Gram-negative bacteria on fresh vegetables and ready-to-eat salads sold in local markets. Microb Drug Res. 2018; pmid:29451428
76. Bollache L, Bardet E, Depret G, Motreuil S, Neuwirth C, Moreau J, et al. Dissemination of CTX-M-producing Escherichia coli in freshwater fishes from a French watershed (Burgundy). Front. Microbiol. 2019; 9:3239.
77. Galler H, Luxner J, Petternel C, Reinthaler FF, Habib J, Haas D, et al. Multiresistant bacteria isolated from intestinal faeces of farm animals in Austria. Antibiotics 2021; 10:466. pmid:33923903
78. Ben-Sallem R, Ben-Slama K, Sáenz Y, Rojo-Bezares B, Estepa V, Jouini A, et al. Prevalence and characterization of extended-spectrum beta-lactamase (ESBL)- and CMY-2-producing Escherichia coli isolates from healthy food-producing animals in Tunisia. Foodborne Pathog Dis. 2012; 9:1137–1142.
79. Hawkey P. Prevalenceand clonality of extended-spectrum beta-lactamases in Asia. Clin Microbiol Infect. 2008; 14:159–165. pmid:18154540
80. Rossi F, Rizzotti L, Felis GE, Torriani S. Horizontal gene transfer among microorganisms in food: Current knowledge and future perspectives. Food Microbiol. 2014; 42:232–243. pmid:24929742
81. Pietsch M, Eller C, Wendt C, Holfelder M, Falgenhauer L, Fruth A. Molecular characterisation of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from hospital and ambulatory patients in Germany. Vet Microbiol. 2017; 200:130–137.
82. Wu G, Day MJ, Mafura MT, Nunez-Garcia J, Fenner JJ, Sharma M. Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany. PloS ONE. 2013; 8(9):e75392.
83. WHO. Critically Important Antimicrobials for Human Medicine, 6th Revision. Geneva: World Health Organization. 2019.
84. Schmiedel J, Falgenhauer L, Domann E, Bauerfeind R, Prenger-Berninghoff E, Imirzalioglu C, et al. Multi-resistant extended-spectrum β- lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany. BMC Microbiol. 2014; 14: 187.
85. Ye Q, Wu Q, Zhang S, Zhang J, Yang G, Wang H. Antibiotic-resistant extended spectrum ß-Lactamase- and plasmid-mediated AmpC-producing Enterobacteriaceae isolated from retail food products and the Pearl River in Guangzhou China. Front Microbiol. 2017; 8:96.
86. Bezanson GS, MacInnis R, Potter G, Hughes T. Presence and potential for horizontal transfer of antibiotic resistance in oxidase-positive bacteria populating raw salad vegetables. Int J Food Microbiol. 2008; 127(1–2):37–42. pmid:18632174
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Abstract
Antimicrobial resistance (AMR) is a major public health concern, especially the extended-spectrum β-lactamase-producing (ESBL) Escherichia coli bacteria are emerging as a global human health hazard. This study characterized extended-spectrum β-lactamase Escherichia coli (ESBL-E. coli) isolates from farm sources and open markets in Edo State, Nigeria. A total of 254 samples were obtained in Edo State and included representatives from agricultural farms (soil, manure, irrigation water) and vegetables from open markets, which included ready-to-eat (RTE) salads and vegetables which could potentially be consumed uncooked. Samples were culturally tested for the ESBL phenotype using ESBL selective media, and isolates were further identified and characterized via polymerase chain reaction (PCR) for β-lactamase and other antibiotic resistance determinants. ESBL E. coli strains isolated from agricultural farms included 68% (17/25) from the soil, 84% (21/25) from manure and 28% (7/25) from irrigation water and 24.4% (19/78) from vegetables. ESBL E. coli were also isolated from RTE salads at 20% (12/60) and vegetables obtained from vendors and open markets at 36.6% (15/41). A total of 64 E. coli isolates were identified using PCR. Upon further characterization, 85.9% (55/64) of the isolates were resistant to ≥ 3 and ≤ 7 antimicrobial classes, which allows for characterizing these as being multidrug-resistant. The MDR isolates from this study harboured ≥1 and ≤5 AMR determinants. The MDR isolates also harboured ≥1 and ≤3 beta-lactamase genes. Findings from this study showed that fresh vegetables and salads could be contaminated with ESBL-E. coli, particularly fresh produce from farms that use untreated water for irrigation. Appropriate measures, including improving irrigation water quality and agricultural practices, need to be implemented, and global regulatory guiding principles are crucial to ensure public health and consumer safety.
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; Beshiru, Abeni; Igbinosa, Isoken H; Gyu-Sung Cho
; Charles M. A. P. Franz