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Article

Analysis of Wastewater Reveals the Spread of Diverse Extended-Spectrum β-Lactamase-Producing E. coli Strains in uMgungundlovu District, South Africa

by
Siyabonga N. Gumede
1,
Akebe L. K. Abia
1,*,
Daniel G. Amoako
1,2,* and
Sabiha Y. Essack
1
1
Antimicrobial Research Unit, College of Health Sciences, University of KwaZulu-Natal, Durban 4000, South Africa
2
Centre for Respiratory Diseases and Meningitis, National Institute for Communicable Diseases, Johannesburg 2131, South Africa
*
Authors to whom correspondence should be addressed.
Antibiotics 2021, 10(7), 860; https://doi.org/10.3390/antibiotics10070860
Submission received: 16 June 2021 / Revised: 8 July 2021 / Accepted: 9 July 2021 / Published: 15 July 2021
(This article belongs to the Special Issue Antibiotic Resistance in Wastewater and Its Treatment)
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Abstract

:
Wastewater treatment plants (WWTPs) are major reservoirs of antibiotic-resistant bacteria (ARB), favouring antibiotic resistance genes (ARGs) interchange among bacteria and they can provide valuable information on ARB circulating in a community. This study characterised extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli from the influent and effluent of four WWTPs in uMgungundlovu District, KwaZulu-Natal, South Africa. E. coli was enumerated using the membrane filtration method and confirmed using the API 20E test and real-time polymerase chain reaction. ESBL-producers were phenotypically identified by their susceptibility to the third-generation cephalosporins using the disc diffusion and the double-disc synergy methods against cefotaxime (30 µg) with and without 10 µg clavulanic acid. Genotypic verification was by PCR of the TEM, SHV, and CTX-M genes. The clonality of isolates was assessed by ERIC-PCR. The highest E. coli count ranged between 1.1 × 105 (influent) and 4.3 × 103 CFU/mL (effluent). Eighty pure isolates were randomly selected, ten from the influent and effluent of each of the four WWTP. ESBLs were phenotypically confirmed in 49% (n = 39) of the isolates, of which 77% (n = 30) were genotypically confirmed. Seventy-three percent of the total isolates were multidrug-resistant (MDR). Only two isolates were susceptible to all antibiotics. Overall, resistance to first and second-generation cephalosporins was higher than to third and fourth generation cephalosporins. Also, 15% of the isolates were resistant to carbapenems. The CTX-M-type ESBL (67%; n = 20) was the most common ESBL antibiotic resistance gene (ARG) followed by TEM (57%; n = 17) and SHV-types (27%; n = 8). Also, a substantial number of isolates simultaneously carried all three ESBL genes. ERIC-PCR revealed a high diversity of isolates. The diversity of the isolates observed in the influent samples suggest the potential circulation of different ESBL-producing strains within the studied district, requiring a more comprehensive epidemiological study to prevent the spread of ESBL-producing bacteria within impoverished communities.

1. Introduction

Antibiotic resistance is one of the major public health concerns globally. This is commonly demonstrated in bacteria such as E. coli, found on the WHO global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics [1,2]. Antibiotic-resistant E. coli and antibiotic resistance genes (ARGs) have been detected in various environmental samples, including soils, surface water, and wastewater [3,4,5]. Extended-spectrum β-lactamase (ESBL)-producing bacteria, including E. coli, are among the most commonly encountered multidrug-resistant (MDR) bacteria today [6,7,8] and are frequently associated with a high mortality rate and prolonged hospitalisation [9,10]. This may be because clinical E. coli isolates frequently co-carry multiple ESBL genes with varying hydrolysis spectra to different antibiotics, leading to treatment failure [10,11,12,13].
ESBLs are a cluster of enzymes that efficiently hydrolyse third-generation oxyimino-cephalosporins such as cefotaxime, ceftazidime, and ceftriaxone and monobactams (aztreonam), but spare the cephamycins (cefotetan, cefoxitin) and carbapenems (imipenem, meropenem, ertapenem, biapenem, and doripenem) [8,14,15]. However, carbapenems, considered last-resort antibiotics used to treat severe infections caused by ESBL-producing E. coli, are also gradually losing their efficacy due to carbapenemases [16,17,18,19]. Most ESBL variants evolved from the parent chromosomal or plasmid-mediated Temoniera (TEM), Sulfhydryl Variable (SHV), and Cefotaximase-München (CTX-M) β-lactamases [6,7,20]. These ESBLs are among the primary causes of clinical resistance to β-lactam antibiotics in E. coli associated with common infections [21]. CTX-M-type ESBLs, mainly CTX-M-15, are currently the most prevalent in communities and clinical settings [7,22,23]. This family of ESBLs has superseded TEM and SHV in prevalence [6].
Wastewater treatment plants (WWTPs) have been reported as reservoirs of ESBL-producing E. coli and other ARB [24]. This is because of the high bacterial biomass, antibiotic metabolites, and ARGS, usually from animal and human-associated wastewater [25,26]. This cocktail of microbial species, antibiotic residues and antibiotic resistance genes (ARGs) create a favourable environment for bacterial proliferation, DNA exchange and selection of resistance among bacteria [25,27,28,29]. Given that WWTPs receive wastewater from entire communities, they can be considered as microcosms, and the analysis of wastewater from these systems could indicate the antibiotic-resistant bacteria (ARB) circulating within a given population. This has engendered the novel paradigm known as wastewater-based epidemiology as a rapid alternative public health assessment approach at the community level [30]. Data obtained from such analyses could serve as an early warning system for potential ARB-associated disease outbreaks and inform public health practitioners on the need for rapid intervention measures to present such, especially within resource-limited settings.
Thus, the current study investigated the phenotypic and genotypic characteristics of ESBL-producing E. coli in the influent and effluent of four WWTPs in the uMgungundlovu District, KwaZulu-Natal, South Africa. The study results would provide information regarding the diversity of ESBL-producing E. coli strains circulating within the community and the potential contribution of the WWTPs to these bacteria’s presence in the environment.

2. Results

2.1. Enumeration of Isolates

The total E. coli count in the influent water samples ranged between 6.5 × 104 and 10.8 × 104 CFU/mL, while in the effluent water samples, it ranged between 1.8 × 103 and 4.3 × 103 CFU/mL (Figure 1). Thus, the influent samples had relatively higher counts than effluent samples. In addition, all the WWTPs showed relatively high efficiency to eliminate bacterial contaminants (between 96.0–98.1%).

2.2. Antimicrobial Susceptibility

Resistance to the penicillins, i.e., ampicillin (94%; 75/80) and amoxicillin (88%; 70/80) was the highest except for piperacillin (57%; 46/80) and the penicillin-inhibitor combination amoxicillin-clavulanate (30%; 24/80) (Table 1). Resistance to first- and second-generation cephalosporins (cephalexin—68% [54/80]; cephalothin—66% [53/80]; cefuroxime—63% [50/80]) was higher than to third- and fourth-generation cephalosporins (ceftazidime—21% [17/80] and cefepime—20% [16]) except for cefixime (55%; 44/80) and cefotaxime (56%; 45/80). Fifteen percent of the isolates were resistant to carbapenems, while low percentage resistance was noted for nitrofurantoin (9%; 7/80), amikacin (11%; 9/80), chloramphenicol, tigecycline (16%; 13/80), and gentamicin (18%; 14/80). Variable percentage resistances were observed across the WWTPs with a general trend of higher percentages in effluent samples (Table 1).

2.3. Prevalence of Multidrug Resistance in E. coli Isolates

Individual isolates displayed diverse antibiograms, except for six influent isolates and thirteen effluent isolates that shared similar patterns (Table S1; Supplementary Materials). A total of 32 MDR phenotypes were evident in 73% (n = 58) of the isolates, with some isolates resistant to up to 20 antibiotics (Table S1; Supplementary Materials). However, effluent isolates (93%; n = 37) had a relatively higher number of MDR isolates than influent isolates (53%; n = 21). Most MDR isolates (76%) demonstrated resistance to at least 50% (11 or more) of the tested antibiotics. Of these, nine isolates were extremely drug-resistant, with resistance to more than twenty antibiotics. The remainder (24%) showed resistance profiles ranging from three up to ten antibiotics simultaneously. Twenty-five percent of the isolates were resistant to at least two antibiotics or more but were not assigned as MDR, and only two isolates were susceptible to all antibiotics.

2.4. ESBL Resistance Genes

Of the 39 (49%) ESBL-positive E. coli isolates, ESBL ARGs were detected in 77% (n = 30), with more being isolated from effluent samples (n = 19) compared to influent samples (n = 11). The CTX-M-type ESBL (67%; n = 20) was the most prevalent ARG followed by TEM (57%; n = 17) and SHV-type (27%; n = 8). Ten percent (n = 3) of isolates co-carried CTX-M and TEM-type ARGs, 13% (n = 4) co-carried CTX-M and SHV ARGs, and 13% (n = 4) carried all the three ESBL types. Nine isolates (30%) carried CTX-M alone, 33% (n = 10) carried TEM alone, and no isolates carried SHV alone (Table 2).

2.5. Strain Typing by ERIC-PCR

The Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR analysis of the E. coli isolated from influent (n = 40) and effluent (n = 40) water samples showed high diversity (Figure 2a and Figure 2b, respectively). A similarity index of ≥50% was used to group the isolates into different ERIC-types. Influent E. coli isolates were classified into 17 ERIC-types (Ⅰ-XVII), while effluent E. coli strains were classified into 13 ERIC-types (Ⅰ-XIII). From the influent E. coli isolates three major ERIC-types were identified, viz VIII (n = 7), XIII (n = 5) and XVI (n = 5) while in the effluent E. coli isolates four major ERIC-types were identified, viz I (n = 5), II (n = 12), VII (n = 5), IX (n = 5). Major ERIC-types from influent E. coli consisted of isolates from different WWTPs, with A, B, and D isolates falling under the same ERIC-types. Major ERIC-types from effluent E. coli isolates consisted of a mixture of isolates from all sampling sites.

3. Discussion

The analysis of wastewater can indicate the resistant bacterial species circulating within a community. Also, the release of ARGs and ARB into aquatic environments may create environmental reservoirs of ARB and ARGs, posing significant challenges to human health [31] and the evolution of the environmental microbial population [25,32,33]. This study demonstrated diverse ESBL-producing E. coli circulating within South African communities and the subsequent discharge of these bacteria by WWTPs into receiving surface waters.

3.1. Enumeration of E. coli

The average total E. coli count in the influent water samples was about 105 CFU/mL in all WWTPs, and the effluent counts were smaller by up to two orders of magnitude than raw influent. The low E. coli count in the effluent indicates the WWTPs’ process efficiency, with bacterial reduction of more than 96% across all WWTPs (Table 1). However, significant bacterial biomass, including some ARB, was released with the effluent into surrounding surface water bodies. Nevertheless, the highest microbial load in all the four WWTPs’ effluents were below the limits of 105 CFU/100 mL of effluent wastewater stipulated in the South African National Water Act [34].

3.2. Antibiotic Susceptibility

Antibiotic sensitivity testing of E. coli isolates showed variable percentages of resistance across the WWTPs with a general trend of higher percentages in effluent samples (Table 1). Ampicillin is highly used in human and veterinary medicine for treatments and prophylaxis [35]. Its ready availability and extensive use could have favoured the development of resistance to this antibiotic. Interestingly, previous studies within the same locality have reported high resistance to ampicillin in humans [36], animals (poultry and pigs) [37,38], and the environment (river water) [39]. Together with the current study’s findings, these reports are substantial evidence that resistance to penicillins is of public health significance within the uMgungundlovu District. Disinfection by chlorination used in WWTPs alters the microbial community composition [40,41] but may also contribute to selecting antibiotic-resistant E. coli [42]. Chlorine tolerance and chlorine-induced upregulation of ARGs in bacteria have been associated with resistance to β-lactams and other antibiotics [42]. Resistance to ampicillin (94%) and amoxicillin (88%) was the highest in this study, with increased percentages evident in chlorine-treated effluent samples in all WWTPs. This finding is further supported by the fact that a high percentage of resistance to these antibiotics had previously been reported in one of the main rivers that serve as a receiving water body for some of the WWTPs studied [39].

3.3. Multidrug Resistance and ESBL Production

ESBL-production is among the commonly encountered mechanisms for MDR in bacteria [6,7]. The phenotypically determined presence of ESBL in 49% of isolates was genotypically corroborated in 31% of the isolates, and five isolates carried TEM-1 ARG (Table 2). The variation could be attributed to unexplored ESBLs or other resistance mechanisms, such as the possible presence of unexamined AmpC β-lactamase in some of the isolates. Six isolates that were genotypically negative for ESBL-production showed resistance to cefoxitin associated with AmpC cephalosporinase activity. Overexpressed AmpC β-lactamase can either mask the phenotypic detection of an ESBL or present as an ESBL-phenotype in the absence of an ESBL [43]. Moreover, other studies have also indicated the discrepancy of phenotypic methods of ESBL detection [44,45,46]. For example, Ojer-Usoz et al. [45] examined WWTP water samples in Navarra, Spain. They identified ESBL-producing Enterobacterales (n = 185) using Chrom ID ESBL agar plates (100%), with varying levels of detection compared to the combination disk test (86.5%), double-disk synergy test (75.7%), and PCR (71.3%) as a confirmatory method. As a result, molecular techniques remain the gold standard in the identification of ESBL-producing bacteria.
The prevalence of ESBL-producing E. coli in this study was similar to the findings of Nzima et al. [24], who reported an ESBL prevalence of 28.6% in E. coli isolated from WWTP and effluent-receiving river in Durban, South Africa. The β-lactamase-producers were relatively higher in the effluent isolates (48%; n = 19) than influent isolates (28%; n = 11). A recent multi-country study (Denmark, Spain, and the United Kingdom) revealed that although wastewater arising from urban areas may contain large amounts of ESBL and carbapenamase-associated ARGs, their concentrations were drastically reduced within the sewer systems, resulting in lower values during sewer convergence [47]. This observation could explain the lower ESBL prevalence in the influent samples in our study. However, unlike [47] that further reported that the ESBL ARG concentration dropped even further after the treatment process, the current study results revealed that the WWTP favoured the proliferation of ESBL-producing bacteria, resulting in higher counts in the effluent samples. These discrepancies could be due to the sample size and the sampling regime used, as the current study was a point-prevalence study within a single locality, compared to the multi-country approach used in the previous study. Our findings were nevertheless similar to a previous study [48], that reported a higher prevalence of ESBL-producing E. coli in the effluent samples (0.6%) compared to the influent samples (0.3%) recovered in a WWTP in Besançon, France, indicating a higher tolerance of ESBL-producers to the water treatment protocol of the WWTP for the elimination of bacteria. The increased frequency of ESBL-producers in the effluent could also be attributed to high bacterial biomass and nutrient levels in the WWTPs, favouring proliferation and interchange of ARGs among bacteria [49,50].
ESBL-producing E. coli isolates demonstrated varying resistance percentages to cephalosporins irrespective of the ESBL-type or variant they carried. ESBL-positive isolates were resistant to at least one or more cephalosporins tested, with higher resistance observed in the first and second-generation cephalosporins (>76%). The highest resistance to the third- and fourth-generation cephalosporins was observed to cefixime (72%) and cefotaxime (80%) compared to ceftazidime and cefepime (52%). Isolates carrying a gene that encodes for CTX-M-28 and CTX-M-3 variants demonstrated a similar hydrolytic efficiency for cephalosporins (i.e., LEX-CEF-CFM-CXM-CTX) except for one CTX-M-3-producing isolate, which was resistant to all cephalosporins. Ten isolates that carried one ARG or co-carried multiple ARGs viz blaCTX-M-15, blaSHV-28, blaTEM-181 (n = 3), blaCTX-M-15, blaSHV-28, blaTEM-213 (n = 1), blaCTX-M-15, blaSHV-28 (n = 4), blaTEM-116 (n = 1) and blaCTX-M-15 (n = 1) also demonstrated resistance to all the cephalosporins tested. The World Health Organization listed late-generation cephalosporins among priority antibiotics for human medicine [51], and thus the occurrence and spread of bacterial strains resistant to these and other antibiotics present a serious clinical threat. ESBL-producing E. coli were isolated in influent samples leading from WWTPs A and D, which process hospital wastewater. Thus, the isolates may be of hospital origin, further highlighting the role of WWTPs as a proxy for the types of ESBL-producing strains circulating within the community. This necessitates the pre-treatment of hospital effluent using methods such as coagulation-flocculation and flotation [52] before discharge into the municipal sewage system [26].
WWTPs receive animal and human-associated wastewater frequently containing contaminants such as metals, biocides, disinfectants, antibiotics, ARGs, and high bacterial biomass [25,29,33,53]. The pressure by these contaminants on microbial populations in the WWTPs may exert selection pressure that can promote upregulation or enrichment of ESBL and other ARGs carried by bacteria through selection or co-selection. Studies have indicated that ESBL-producers demonstrate co-resistance to other antibiotic classes, including fluoroquinolones, aminoglycosides, tetracyclines, and trimethoprim/sulfamethoxazole [32,45]. Resistance to ciprofloxacin (60%), nalidixic (72%), tetracycline (88%), and trimethoprim/sulfamethoxazole (92%) was observed among ESBL-producing isolates in the current study. Similarly, Dolejska et al. [54] reported high resistance to ciprofloxacin (100%), nalidixic (100%), tetracyclines (73%), and trimethoprim/sulfamethoxazole (73%) in ESBL-positive E. coli isolated from a WWTP in the Czech Republic. Nzima et al. [24] also reported high resistance to tetracyclines (80%) in ESBL-producing E. coli isolates recovered in a WWTP in Durban, South Africa. Isolates that produced CTX-M-3 and CTX-M-28 were resistant to fluoroquinolones, and CTX-M-15-producing isolates demonstrated varying percentages of resistance to this antibiotic class. Non-ESBL TEM-1-producing isolates also demonstrated some resistance to these antibiotics. Co-resistance to aminoglycosides (36%) was also observed in some ESBL-producing E. coli, but resistance to this antibiotic class was generally low. Lower resistance percentages were noted for nitrofurantoin, chloramphenicol, tigecycline, and carbapenems (Table S1; Supplementary Materials).
The CTX-M-type ESBLs were the most prevalent ARGs detected in this study (Table 2). CTX-Ms are currently the most prevalent ESBLs, classified into six subgroups [55,56]. The predominance of the CTX-M ESBL observed in E. coli isolates in the current study was similar to a previous study [32] that found a higher prevalence of CTX-M producing E. coli isolates in the effluent samples (100%) of a WWTP in Curitiba, Brazil. This indicates that WWTPs are a microcosm of ARGs circulating within a community and a hotspot for proliferation and dissemination of CTX-M-producing E. coli in the environment via effluent discharge.
CTX-M-type ESBLs belonging to the CTX-M-1 sub-lineage were detected in this study. The sub-lineages CTX-M-1 and CTX-M-9 constitute the most common variants within the CTX-M family [55,57]. CTX-M-15, the most globally dispersed genotype within the CTX-M family [8,58], was the most frequently detected ESBL variant mainly detected in effluent water samples in all WWTPs. CTX-M-15-producing bacteria are clinically relevant [59], and the presence of CTX-M-15 producing E. coli in WWTP effluent implicates isolates of clinical origin, hence a picture of their presence in the study community. Furthermore, some CTX-M-15 producing E. coli isolates co-carried other ESBLs genes, a common phenomenon among clinical isolates that may increase drug resistance. Studies have indicated that the global prevalence of CTX-M-15 could be attributed to specific clones [60]. However, the CTX-M-15 producing E. coli detected in this study were evident in multiple isolates.

3.4. Clonality of Isolates

ERIC-PCR fingerprinting of E. coli isolates using a similarity index of ≥50% showed a high diversity of E. coli strains. A higher number of clonal clusters was observed in the influent sample than the effluent sample (Figure 1). This could be attributed to the fact that the sewage network leading to WWTPs is vast, consisting of diverse bacterial strains of different origins, suggesting that different ESBL-producing E. coli strains could be circulating within the different communities they serve. This was further supported by the decline in the number of clonal clusters in the effluent isolates. The WWTPs used the same treatment processes resulting in the observed decrease in clonal clusters. The decreased clonal clusters in the effluent samples could also be attributed to some clones being more susceptible to the wastewater treatment processes of the WWTP for the elimination of bacteria. Despite the high diversity, some E. coli isolates recovered from influent samples of WWTP A, B and D showed some similarity in major ERIC-types XVI and XIII. Similar ERIC-types in geographically distinct collection points indicate the endemicity of certain clones in the uMgungundlovu District communities. More than 60% of the influent E. coli isolates from the same sampling point of the three WWTPs (A, B, and C) fell under different ERIC-types, except for the clonal cluster designated as ERIC-type VIII, which constituted of several isolates from WWTP D influent sample, indicating the predominance of this clone in the region around this WWTP.
While the current study reveals that diverse ESBL-producing E. coli clones circulate within the studied community, the presence of these bacteria was not determined in the surrounding aquatic environment. Further studies that would also sample the receiving water bodies into which these WWTPs discharge their effluent could provide more information on whether these resistant bacteria are present in rivers within the community. Ascertaining this would help prevent the risk of infection with resistant bacteria in parts of the community using the water directly for different needs.

4. Materials and Methods

4.1. Study Sites

The study site consisted of four wastewater treatment plants (A–D) in the uMgungundlovu District. WWTPs A and D treat industrial wastewater, hospital wastewater, and domestic wastewater from 117,303 of the 679,039 persons with flush toilets connected to the sewage system within Msunduzi local municipality. These WWTPs have an average inflow capacity of 76 (A) and 0.5 (D) megalitres/day. WWTP B has an inflow capacity of 5.6 megalitres/day, including industrial wastewater, hospital wastewater, and domestic wastewater from 31,322 of the 109,867 persons with flush toilets connected to the sewage system within uMngeni local municipality. WWTP C has an inflow capacity of 0.54 megalitres/day, mainly domestic wastewater from 11596 persons residing in the surrounding suburbs. These WWTPs use aeration basins for biological nutrient removal, clarifiers for the separation process, and chlorine disinfection.

4.2. Sampling

Water samples were collected once-off from the WWTPs’ influent in the primary sedimentation tank, while the effluent samples were collected at the treated wastewater discharge point in the chlorine contact tank. Samples were collected using sodium thiosulfate (20 mg/L) coated sterile 500 mL bottles (JVL Laboratories, Durban, South Africa), following standard procedures prescribed for examining wastewater [61]. Water samples were transported to the Antimicrobial Research Unit (ARU), University of KwaZulu-Natal, in a portable 4 °C cooler-box for processing within six hours from collection.

4.3. Quantitative Analysis and Isolate Identification

Ten millilitres (10 mL) of each water sample were serially diluted (101 to 106) in 90 mL of sterile distilled water. A thoroughly homogenised mixture was vacuum filtered through a 0.45 µm pore membrane filter (Pall Corp, Port Washington, NY, USA) using the Sterifil Aseptic System apparatus (Pall Corp). The membrane filter was aseptically inoculated onto eosin methylene blue agar plates (Oxoid™, Waltham, MA, USA) and incubated at 37 °C for 24 h. Colonies with morphology and colour characteristics of E. coli (i.e., green metallic sheen with dark centres) were counted in each plate and recorded as colony forming units per millilitre (CFU/mL). Randomly selected presumptive E. coli colonies were sub-cultured on nutrient agar (Oxoid™) and incubated at 37 °C for 18–24 h, after which isolates were phenotypically identified using the API 20E test kits (BioMerieux, Marcy-l’Étoile, France). Ten putative E. coli isolates per sampling point were randomly selected and individually stored in a Muller Hinton broth: glycerol (70:30) mixture in cryovials at −80 °C for further processing, giving 40 influent and 40 effluent isolates from the four WWTPs.

4.4. Molecular Confirmation of E. coli by Real-Time Polymerase Chain Reaction (PCR)

Genomic DNA of overnight Muller Hinton broth (Oxoid™) cultures (20 h at 37 °C) was extracted using the GeneJet Genomic DNA purification kit (ThermoFisher Scientific™, Waltham, MA, USA) to detect the presence of the E. coli housekeeping gene, malate dehydrogenase (mdh), using real-time PCR. DNA purity and concentration were determined using a NanoDropTM 8000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 260/280 nm wavelength. DNA samples were subsequently stored at −20 °C for further examination.
PCR assays were performed in a 10-µL final volume comprising of 5 µL of Luna® Universal qPCR Master Mix (New England BioLabs® Inc., Ipswich, MA, USA), 1 µL of nuclease-free water (ThermoFisher Scientific™), 0.5 µL of each forward and reverse primer at a final concentration of 0.5 µM (Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa) and 3 µL of template DNA. The primers used were mdh-F (5′GGTATGGATCGTTCCGACCT-3′) and mdh-R-(5′-GGCAGAATGGTAACACCAGAGT-3′) [62], under conditions previously described [63]. All reactions were run in a QuantStudioTM 5 Applied Biosystems real-time thermocycler (ThermoFisher Scientific™). E. coli ATCC 25922 (American Type Culture Collection, ATCC, Manassas, VA, USA) was used as a positive control. The no template control contained all the reagents except template DNA. Results were interpreted by melt curve analysis using the QuantStudio Design and Analysis software version 1.4.3 (ThermoFisher Scientific™) under default mode.

4.5. Antibiotic Susceptibility Testing and Determination of ESBL Production

Antibiotic sensitivity profiles of E. coli isolates were examined against 23 antibiotics using the standard disc diffusion method [64]. The following antibiotic panel (Oxoid™) was used: amoxicillin/clavulanic acid (AMC 20/10 μg), amoxicillin (AMX 10 μg), amikacin (AMK 30 μg), ampicillin (AMP 10 μg), cefepime (FEP 30 μg), cephalothin (CEF 30 μg), cefotaxime (CTX 30 μg), cefoxitin (FOX 30 μg), cefixime (CFM 5 μg), ceftazidime (CAZ 30 μg), cephalexin (LEX 30 μg), cefuroxime (CXM 30 μg), chloramphenicol (CHL 30 μg), ciprofloxacin (CIP 5 μg), gentamicin (GEN 10 μg), imipenem (IPM 10 μg), meropenem (MEM 10 μg), nitrofurantoin (NIT 100 μg), piperacillin (PIP 100 μg), tetracycline (TET 30 μg), tigecycline (TGC 15 μg), nalidixic (NAL 30 μg) and trimethoprim/sulfamethoxazole (SXT 1.25/23.75 μg). Susceptibility profiles were determined according to the Clinical Laboratory Standard Institution (CLSI) guidelines [65], and E. coli ATCC 25922 was used for quality control. The double-disc synergy test [66] using 30 µg cefotaxime in the presence and absence of 10 µg clavulanic acid (MAST Group Ltd., Bootle, UK) was undertaken to identify ESBL-producing isolates. E. coli isolates displaying resistance to one or more antibiotics from three different antibiotic classes were assigned as multidrug-resistant (MDR) isolates.

4.6. Genotypic Characterisation of ESBLs

The previously extracted template DNA was used to detect ESBLs-encoding genes (blaCTX-M and blaSHV) by real-time PCR. These reactions were carried out in a 25 µL final volume consisting of 12.5 µL of Luna® Universal qPCR Master Mix (New England BioLabs® Inc., Ipswich, MA, USA), 5.5 µL of nuclease-free water (ThermoFisher Scientific™), 1 µL of each forward and reverse primer with a final concentration of 1 µM and 5 µL of template DNA. Amplification reactions were conducted in a QuantStudioTM 5 Applied Biosystems (ThermoFisher Scientific™) using specific primer sets (Inqaba Biotechnology Industries (Pty) Ltd.) (Table S2; Supplementary Materials). The cycling conditions included a hot-start initial activation at 98 °C for 50 s, followed by 35 cycles of denaturation at 95 °C for 10 s, final extension at 72 °C for 20 s and annealing temperature at 58 °C for 1 min. Results were interpreted by melt curve analysis using the QuantStudio Design and Analysis software version 1.4.3 (ThermoFisher Scientific™).
The TEM gene was amplified in a Thermal Cycler T100TM (Bio-Rad, Hercules, CA, USA) in 25 µL final volume using 12.5 µL of DreamTaq Green PCR Master Mix (ThermoFisher Scientific™), 5.5 µL of nuclease-free water (ThermoFisher, Scientific™), 1 µL of each forward and reverse primer (Inqaba Biotechnology Industries (Pty) Ltd.) at a final concentration of 1 µM and 5 µL of template DNA. PCR conditions were an initial activation at 94 °C for 3 min, and 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 90 s and a final extension at 72 °C for 7 min. Five microliters of each PCR product was subsequently electrophoresed on a 1.5% agarose gel for 3 h at 70 V using 1 × Tris-acetate EDTA (TAE) buffer and stained for 30 min by immersing in 0.1 mg/mL ethidium bromide solution. Amplicons were visualised using a Bio-Rad molecular imager Gel DocTM XR+ imaging system (Bio-Rad). Previously confirmed blaCTX-M, blaTEM, and blaSHV positive isolates from the ARU laboratory collections were used as internal positive controls. The no template control, consisting of all reagents except DNA, was concurrently run during amplification reactions. PCR products (20 µL) of CTX-M-positive isolates (n = 20), TEM-positive isolates (n = 17), and SHV-positive isolates (n = 8) were sequenced using the Sanger sequencing method at Inqaba Biotechnology Industries (Pty) Ltd. to identify ESBL genes and ascertain point mutations conferring novelty (if any). Sequence results were interpreted using Chromas (http://technelysium.com.au/wp/chromas/, accessed on 26 April 2020) and BioEdit (https://bioedit.software.informer.com, accessed on 26 April 2020) software.

4.7. Strain Typing by ERIC-PCR

The Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR assay was executed in a 20 µL final volume comprising of 16 µL of DreamTaq PCR Master Mix (2X) (ThermoFisher Scientific™), 1 µL of nuclease-free water, 0.5 µL of Eric 1 primer (ATGTAAG CTCCTGGGGATTCAC) and 0.5 µL of Eric 2 primer (AAGTAAGTGACTGGGGT GAGCG) (Inqaba Biotechnology Industries, (Pty) Ltd.) [67] and 3 µL of template DNA. Amplification reactions were conducted in a Bio-Rad T100TM thermal cycler (Bio-Rad) under the following cycling conditions: initial activation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 1 min, extension at 65 °C for 8 min and a final extension at 65 °C for 16 min. Five microliters of each PCR product was subsequently electrophoresed on a 1% agarose gel for 3 h at 70 V using 1 × Tris-acetate EDTA (TAE) buffer and stained for 30 min by immersing in 0.1 mg/mL ethidium bromide solution. Amplicons were visualised using the Bio-Rad molecular imager Gel DocTM XR+ imaging system (Bio-Rad), and E. coli ATCC 25922 was used as a positive control. Captured gel images were analysed using the BioNumerics software version 6.6 (Applied Maths NV, Sint-Martens-Latem, Belgium) as previously described by McIver et al. [37]. Clusters were determined at a 50% similarity cut-off value [68].

5. Conclusions

The current study demonstrated the ubiquity of antibiotic-resistant ESBLs-producing E. coli in the uMgungundlovu District by examining four major WWTPs. Despite the high removal efficiency observed in the WWTPs, substantial ARB numbers were discharged into the receiving water bodies through the effluents, which may present a route for wide dispersion of ESBLs-producing E coli. This poses a public health threat to communities who use waters contaminated with WWTPs effluent discharge for their domestic water demands. The high prevalence of clinically relevant CTX-M-15 producing E. coli in WWTPs indicates they might be of clinical origin. Therefore, while measures are developed and implemented to curb the problem of AMR, specifically those linked to ESBL, within communities in South Africa, the need for pre-treatment of hospital wastewater before it is channelled to municipal WWTPs should be highlighted to minimise transmission.

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/antibiotics10070860/s1, Table S1: Multidrug-resistant E. coli phenotypes identified in the study, Table S2: Primer sets for the amplification of TEM, SHV, and CTX-M-type ESBLs.

Author Contributions

Conceptualization, A.L.K.A., D.G.A. and S.Y.E.; methodology, S.N.G., A.L.K.A..; validation, A.L.K.A., D.G.A. and S.Y.E.; investigation, S.N.G.; resources, S.Y.E.; data curation, A.L.K.A. and D.G.A.; writing—original draft preparation, S.N.G.; writing—review and editing, A.L.K.A., D.G.A. and S.Y.E.; supervision, A.L.K.A., D.G.A. and S.Y.E.; project administration, S.Y.E.; funding acquisition, S.Y.E. All authors have read and agreed to the published version of the manuscript.

Funding

This work supported by the Antimicrobial Research Unit (ARU) and College of Health Sciences (CHS) of University of KwaZulu-Natal, NRF Competitive Grant for Rated Researchers (Grant no.: 106063), and the DST/NRF South African Research Chair in Antibiotic Resistance and One Health (Grant No. 98342).

Institutional Review Board Statement

Ethical approval was obtained from the University of KwaZulu-Natal Biomedical Research Ethics Committee (Reference BCA444/16). The management of Umgeni Water authorised sample collection in relevant wastewater treatment plants (WWTPs).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data related to this work have been included in the manuscript and its accompanying supplementary materials.

Conflicts of Interest

S.Y.E. is the chairperson of the Global Respiratory Infection Partnership and a member of the Global Hygiene Council, both sponsored by unrestricted educational grants from Reckitt and Benckiser (Pty), UK. All the other authors had no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. World Health Organization. Global Priority List of Antibiotic-Resistant Batceria to Guide Research, Discovery, and Development of New Antibiotics; WHO: Geneva, Switzerland, 2017. [Google Scholar]
  2. Ramadan, A.A.; Abdelaziz, N.A.; Amin, M.A.; Aziz, R.K. Novel bla CTX-M variants and genotype-phenotype correlations among clinical isolates of extended spectrum beta lactamase-producing Escherichia coli. Sci. Rep. 2019, 9, 4224. [Google Scholar] [CrossRef] [PubMed]
  3. Franz, E.; Veenman, C.; Van Hoek, A.H.A.M.; Husman, A.D.R.; Blaak, H. Pathogenic Escherichia coli producing Extended-Spectrum β-Lactamases isolated from surface water and wastewater. Sci. Rep. 2015, 5, 14372. [Google Scholar] [CrossRef] [Green Version]
  4. Furlan, J.P.R.; Stehling, E.G. Presence of Β-Lactamases Encoding Genes in Soil Samples from Different Origins. Water Air Soil Pollut. 2017, 228, 11270. [Google Scholar] [CrossRef]
  5. Adegoke, A.A.; Madu, C.E.; Aiyegoro, O.A.; Stenström, T.A.; Okoh, A.I. Antibiogram and beta-lactamase genes among cefotaxime resistant E. coli from wastewater treatment plant. Antimicrob. Resist. Infect. Control 2020, 9, 46. [Google Scholar] [CrossRef]
  6. Bush, K. Past and present perspectives on β-lactamases. Antimicrob. Agents Chemother. 2018, 62, e01076-18. [Google Scholar] [CrossRef] [Green Version]
  7. Doi, Y.; Iovleva, A.; Bonomo, R.A. The ecology of extended-spectrum β-lactamases (ESBLs) in the developed world. J. Travel Med. 2017, 24, S44–S51. [Google Scholar] [CrossRef]
  8. Ur Rahman, S.; Ali, T.; Ali, I.; Khan, N.A.; Han, B.; Gao, J. The Growing Genetic and Functional Diversity of Extended Spectrum Beta-Lactamases. Biomed Res. Int. 2018, 2018, 9519718. [Google Scholar] [CrossRef] [PubMed]
  9. Mita, Y.; Shigemura, K.; Osawa, K.; Kitagawa, K.; Kotaki, T.; Shirakawa, T.; Miyara, T.; Fujisawa, M. Clinical Risk Factors for Death Caused by Extended-Spectrum Beta-Lactamase: Producing Bacteria. Urol. Int. 2019, 102, 181–186. [Google Scholar] [CrossRef]
  10. Pandit, R.; Awal, B.; Shrestha, S.S.; Joshi, G.; Rijal, B.P.; Parajuli, N.P. Extended-Spectrum β-Lactamase (ESBL) Genotypes among Multidrug-Resistant Uropathogenic Escherichia coli Clinical Isolates from a Teaching Hospital of Nepal. Interdiscip. Perspect. Infect. Dis. 2020, 2020, 6525826. [Google Scholar] [CrossRef] [Green Version]
  11. Jena, J.; Sahoo, R.K.; Debata, N.K.; Subudhi, E. Prevalence of TEM, SHV, and CTX-M genes of extended-spectrum β-lactamase-producing Escherichia coli strains isolated from urinary tract infections in adults. Biotech 2017, 7, 244. [Google Scholar] [CrossRef] [PubMed]
  12. Mirkalantari, S.; Masjedian, F.; Irajian, G.; Siddig, E.E.; Fattahi, A. Determination of the frequency of β-lactamase genes (bla SHV, bla TEM, bla CTX-M) and phylogenetic groups among ESBL-producing uropathogenic Escherichia coli isolated from outpatients. J. Lab. Med. 2020, 44, 27–33. [Google Scholar] [CrossRef] [Green Version]
  13. Alqasim, A.; Abu Jaffal, A.; Alyousef, A.A. Prevalence of multidrug resistance and extended-spectrum β -Lactamase carriage of clinical uropathogenic Escherichia coli isolates in Riyadh, Saudi Arabia. Int. J. Microbiol. 2018, 2018, 3026851. [Google Scholar] [CrossRef] [Green Version]
  14. Ghafourian, S.; Sadeghifard, N.; Soheili, S.; Sekawi, Z. Extended spectrum beta-lactamases: Definition, classification and epidemiology. Curr. Issues Mol. Biol. 2014, 17, 11–22. [Google Scholar]
  15. Ashok, A.K.; Jaryal, S.C.; Thakur, K.; Sood, A.; Gupta, P.K.; Thakur, S. Detection of Inducible and Non-inducible (constitutive) AmpC β-lactamase-producing Gram-Negative Bacteria among Family Enterobacteriaceae by Two Phenotypic Methods-Disk Antagonism Test (DAT) and AmpC disk Test at a Tertiary Care Hospital, Himachal Pradesh. Int. J. Curr. Microbiol. Appl. Sci. 2016, 5, 133–139. [Google Scholar] [CrossRef] [Green Version]
  16. Antunes, N.T.; Lamoureaux, T.L.; Toth, M.; Stewart, N.K.; Frase, H.; Vakulenko, S.B. Class D β-lactamases: Are they all carbapenemases? Antimicrob. Agents Chemother. 2014, 58, 2119–2125. [Google Scholar] [CrossRef] [Green Version]
  17. Jeon, J.H.; Lee, J.H.; Lee, J.J.; Park, K.S.; Karim, A.M.; Lee, C.R.; Jeong, B.C.; Lee, S.H. Structural basis for carbapenem-hydrolyzing mechanisms of carbapenemases conferring antibiotic resistance. Int. J. Mol. Sci. 2015, 16, 9654–9692. [Google Scholar] [CrossRef]
  18. Kotsakis, S.D.; Flach, C.F.; Razavi, M.; Joakim Larsson, D.G. Characterization of the first OXA-10 natural variant with increased carbapenemase activity. Antimicrob. Agents Chemother. 2019, 63, e01817-18. [Google Scholar] [CrossRef] [Green Version]
  19. Shaikh, S.; Fatima, J.; Shakil, S.; Rizvi, S.M.D.; Kamal, M.A. Antibiotic resistance and extended spectrum beta-lactamases: Types, epidemiology and treatment. Saudi J. Biol. Sci. 2015, 22, 90–101. [Google Scholar] [CrossRef] [Green Version]
  20. Bogner, C.; Miethke, T.; Wantia, N.; Gebhard, F.; Busch, D.; Hoffmann, R. Differences in ESBL Genes between E. coli, Klebsiella spp. and Enterobacter Cloacae Strains. Int. J. Clin. Med. Microbiol. 2016, 1, 106. [Google Scholar] [CrossRef]
  21. Dangelo, R.G.; Johnson, J.K.; Bork, J.T.; Heil, E.L. Treatment options for extended-spectrum beta-lactamase (ESBL) and AmpC-producing bacteria. Expert Opin. Pharmacother. 2016, 17, 953–967. [Google Scholar] [CrossRef]
  22. Bajaj, P.; Singh, N.S.; Virdi, J.S. Escherichia coli β-lactamases: What really matters. Front. Microbiol. 2016, 7, 417. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Bevan, E.R.; Jones, A.M.; Hawkey, P.M. Global epidemiology of CTX-M β-lactamases: Temporal and geographical shifts in genotype. J. Antimicrob. Chemother. 2017, 72, 2145–2155. [Google Scholar] [CrossRef] [Green Version]
  24. Nzima, B.; Adegoke, A.A.; Ofon, U.A.; Al-Dahmoshi, H.O.M.; Saki, M.; Ndubuisi-Nnaji, U.U.; Inyang, C.U. Resistotyping and extended-spectrum beta-lactamase genes among Escherichia coli from wastewater treatment plants and recipient surface water for reuse in South Africa. New Microbes New Infect. 2020, 38, 100803. [Google Scholar] [CrossRef]
  25. Hocquet, D.; Muller, A.; Bertrand, X. What happens in hospitals does not stay in hospitals: Antibiotic-resistant bacteria in hospital wastewater systems. J. Hosp. Infect. 2016, 93, 395–402. [Google Scholar] [CrossRef]
  26. King, T.L.B.; Schmidt, S.; Essack, S.Y. Antibiotic resistant Klebsiella spp. from a hospital, hospital effluents and wastewater treatment plants in the uMgungundlovu District, KwaZulu-Natal, South Africa. Sci. Total Environ. 2020, 712, 135550. [Google Scholar] [CrossRef]
  27. Rodriguez-Mozaz, S.; Chamorro, S.; Marti, E.; Huerta, B.; Gros, M.; Sànchez-Melsió, A.; Borrego, C.M.; Barceló, D.; Balcázar, J.L. Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river. Water Res. 2015, 69, 234–242. [Google Scholar] [CrossRef] [PubMed]
  28. Yi, T.; Kim, T.G.; Cho, K.S. Fate and behavior of extended-spectrum β-lactamase-producing genes in municipal sewage treatment plants. J. Environ. Sci. Health Part A Toxic/Hazard. Subst. Environ. Eng. 2015, 50, 1160–1168. [Google Scholar] [CrossRef]
  29. Aubertheau, E.; Stalder, T.; Mondamert, L.; Ploy, M.C.; Dagot, C.; Labanowski, J. Impact of wastewater treatment plant discharge on the contamination of river biofilms by pharmaceuticals and antibiotic resistance. Sci. Total Environ. 2017, 579, 1387–1398. [Google Scholar] [CrossRef]
  30. Sims, N.; Kasprzyk-Hordern, B. Future perspectives of wastewater-based epidemiology: Monitoring infectious disease spread and resistance to the community level. Environ. Int. 2020, 139, 105689. [Google Scholar] [CrossRef]
  31. Fernando, D.M.; Tun, H.M.; Poole, J.; Patidar, R.; Li, R.; Mi, R.; Amarawansha, G.E.A.; Dilantha Fernando, W.G.; Khafipour, E.; Farenhorst, A.; et al. Detection of antibiotic resistance genes in source and drinking water samples from a first nations community in Canada. Appl. Environ. Microbiol. 2016, 82, 4767–4775. [Google Scholar] [CrossRef] [Green Version]
  32. Conte, D.; Palmeiro, J.K.; da Silva Nogueira, K.; de Lima, T.M.R.; Cardoso, M.A.; Pontarolo, R.; Degaut Pontes, F.L.; Dalla-Costa, L.M. Characterization of CTX-M enzymes, quinolone resistance determinants, and antimicrobial residues from hospital sewage, wastewater treatment plant, and river water. Ecotoxicol. Environ. Saf. 2017, 136, 62–69. [Google Scholar] [CrossRef]
  33. Osińska, A.; Korzeniewska, E.; Harnisz, M.; Niestępski, S. The prevalence and characterization of antibiotic-resistant and virulent Escherichia coli strains in the municipal wastewater system and their environmental fate. Sci. Total Environ. 2017, 577, 367–375. [Google Scholar] [CrossRef]
  34. South African Department of Water and Environmental Affairs National Water Act 36 of 1998. In Wastewater Limit Values Applicable to the Irrigation of Any Land or Property up to 50 Cubic Metres; Government Gazette No. 19182; Department of Water Affairs: Pretoria, South Africa, 2013; pp. 17–18.
  35. South African National Department of Health. Standard Treatment Guidelines and Essential Medicines List for South Africa: Hospital Level (Adults), 4th ed.; National Deaprtment of Health: Pretoria, South Africa, 2015; ISBN 9781920031718.
  36. Kubone, P.Z.; Mlisana, K.P.; Govinden, U.; Luther, A.; Abia, K.; Essack, S.Y. Antibiotic Susceptibility and Molecular Characterization of Uropathogenic Escherichia coli Associated with Community-Acquired Urinary Tract Infections in Urban and Rural Settings in South Africa. Trop. Med. Infect. Dis. 2020, 5, 1–13. [Google Scholar]
  37. McIver, K.S.; Amoako, D.G.; Abia, A.L.K.; Bester, L.A.; Chenia, H.Y.; Essack, S.Y. Molecular epidemiology of antibiotic-resistant escherichia coli from farm-to-fork in intensive poultry production in KwaZulu-Natal, South Africa. Antibiotics 2020, 9, 850. [Google Scholar] [CrossRef]
  38. Abdalla, S.E.; Abia, A.L.K.; Amoako, D.G.; Perrett, K.; Bester, L.A.; Essack, S.Y. From Farm-to-Fork: E. Coli from an Intensive Pig Production System in South Africa Shows High Resistance to Critically Important Antibiotics for Human and Animal Use. Antibiotics 2021, 10, 178. [Google Scholar]
  39. Mbanga, J.; Amoako, D.G.; Abia, A.L.K.; Allam, M.; Ismail, A.; Essack, S.Y. Genomic Insights of Multidrug-Resistant Escherichia coli From Wastewater Sources and Their Association With Clinical Pathogens in South Africa. Front. Vet. Sci. 2021, 8, 137. [Google Scholar] [CrossRef]
  40. Shi, P.; Jia, S.; Zhang, X.X.; Zhang, T.; Cheng, S.; Li, A. Metagenomic insights into chlorination effects on microbial antibiotic resistance in drinking water. Water Res. 2013, 47, 111–120. [Google Scholar] [CrossRef]
  41. Kutilova, I.; Medvecky, M.; Leekitcharoenphon, P.; Munk, P.; Masarikova, M.; Davidova-Gerzova, L.; Jamborova, I.; Bortolaia, V.; Pamp, S.J.; Dolejska, M. Extended-spectrum beta-lactamase-producing Escherichia coli and antimicrobial resistance in municipal and hospital wastewaters in Czech Republic: Culture-based and metagenomic approaches. Environ. Res. 2021, 193, 110487. [Google Scholar] [CrossRef]
  42. Liu, S.S.; Qu, H.M.; Yang, D.; Hu, H.; Liu, W.L.; Qiu, Z.G.; Hou, A.M.; Guo, J.; Li, J.W.; Shen, Z.Q.; et al. Chlorine disinfection increases both intracellular and extracellular antibiotic resistance genes in a full-scale wastewater treatment plant. Water Res. 2018, 136, 131–136. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Connelly, C.M.; Ph, D.; Torres, M.P.; Ph, D.; Lechner, J.; Porter, L.R.; Morrow, S.; Ascp, M.T.; Hanson, N.D. Plasmid-mediated β-lactamases: Nomadic microbial assassins lurking in multidrug resistant Gram-negative strains. Biotechniques 2013, 55, 212. [Google Scholar] [CrossRef] [Green Version]
  44. Korzeniewska, E.; Harnisz, M. Extended-spectrum beta-lactamase (ESBL)-positive Enterobacteriaceae in municipal sewage and their emission to the environment. J. Environ. Manag. 2013, 128, 904–911. [Google Scholar] [CrossRef]
  45. Ojer-Usoz, E.; González, D.; García-Jalón, I.; Vitas, A.I. High dissemination of extended-spectrum β-lactamase-producing Enterobacteriaceae ineffluents from wastewater treatment plants. Water Res. 2014, 56, 37–47. [Google Scholar] [CrossRef]
  46. Njage, P.M.K.K.; Buys, E.M. Pathogenic and commensal Escherichiacoli from irrigation water show potential in transmission of extended spectrum and AmpC ? ²-lactamases determinants to isolates from lettuce. Microb. Biotechnol. 2015, 8, 462–473. [Google Scholar]
  47. Li, L.; Nesme, J.; Quintela-Baluja, M.; Balboa, S.; Hashsham, S.; Williams, M.R.; Yu, Z.; Sørensen, S.J.; Graham, D.W.; Romalde, J.L.; et al. Extended-Spectrum β-Lactamase and Carbapenemase Genes are Substantially and Sequentially Reduced during Conveyance and Treatment of Urban Sewage. Environ. Sci. Technol. 2021, 55, 5939–5949. [Google Scholar] [CrossRef]
  48. Bréchet, C.; Plantin, J.; Sauget, M.; Thouverez, M.; Talon, D.; Cholley, P.; Guyeux, C.; Hocquet, D.; Bertrand, X. Wastewater treatment plants release large amounts of extended-spectrum β-lactamase-producing escherichia coli into the environment. Clin. Infect. Dis. 2014, 58, 1658–1665. [Google Scholar] [CrossRef]
  49. Li, Q.; Chang, W.; Zhang, H.; Hu, D.; Wang, X. The role of plasmids in the multiple antibiotic resistance transfer in ESBLs-producing Escherichia coli isolated from wastewater treatment plants. Front. Microbiol. 2019, 10, 633. [Google Scholar] [CrossRef]
  50. Pärnänen, K.M.M.; Narciso-Da-Rocha, C.; Kneis, D.; Berendonk, T.U.; Cacace, D.; Do, T.T.; Elpers, C.; Fatta-Kassinos, D.; Henriques, I.; Jaeger, T.; et al. Antibiotic resistance in European wastewater treatment plants mirrors the pattern of clinical antibiotic resistance prevalence. Sci. Adv. 2019, 5, eaau9124. [Google Scholar] [CrossRef] [Green Version]
  51. World Health Organization. WHO List of Critically Important Antimicrobials for Human Medicine (WHO CIA List); World Health Organization: Geneva, Switzerland, 2019. [Google Scholar]
  52. Suarez, S.; Lema, J.M.; Omil, F. Pre-treatment of hospital wastewater by coagulation-flocculation and flotation. Bioresour. Technol. 2009, 100, 2138–2146. [Google Scholar] [CrossRef]
  53. Amos, G.C.A.; Hawkey, P.M.; Gaze, W.H.; Wellington, E.M. Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment. J. Antimicrob. Chemother. 2014, 69, 1785–1791. [Google Scholar] [CrossRef] [PubMed]
  54. Dolejska, M.; Frolkova, P.; Florek, M.; Jamborova, I.; Purgertova, M.; Kutilova, I.; Cizek, A.; Guenther, S.; Literak, I. CTX-M-15-producing Escherichia coli clone B2-O25b-ST131 and Klebsiella spp. isolates in municipal wastewater treatment plant effluents. J. Antimicrob. Chemother. 2011, 66, 2784–2790. [Google Scholar]
  55. D’Andrea, M.M.; Arena, F.; Pallecchi, L.; Rossolini, G.M. CTX-M-type β-lactamases: A successful story of antibiotic resistance. Int. J. Med. Microbiol. 2013, 303, 305–317. [Google Scholar] [CrossRef]
  56. Palzkill, T. Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC β-lactamases. Front. Mol. Biosci. 2018, 5, 1–19. [Google Scholar] [CrossRef] [Green Version]
  57. Alfaresi, M.; Kim Sing, G.; Senok, A. First Report of bla CTX-M-28 in Enterobacteriaceae Isolates in the United Arab Emirates. J. Pathog. 2018, 2018, 1304793. [Google Scholar] [CrossRef] [Green Version]
  58. Zhao, W.H.; Hu, Z.Q. Epidemiology and genetics of CTX-M extended-spectrum β-lactamases in Gram-negative bacteria. Crit. Rev. Microbiol. 2013, 39, 79–101. [Google Scholar] [CrossRef]
  59. Raven, K.E.; Ludden, C.; Gouliouris, T.; Blane, B.; Naydenova, P.; Brown, N.M.; Parkhill, J.; Peacock, S.J. Genomic surveillance of Escherichia coli in municipal wastewater treatment plants as an indicator of clinically relevant pathogens and their resistance genes. Microb. Genomics 2019, 5, e000267. [Google Scholar] [CrossRef]
  60. Paulshus, E.; Thorell, K.; Guzman-Otazo, J.; Joffre, E.; Colque, P.; Kühn, I.; Möllby, R.; Sørum, H.; Sjöling, Å. Repeated isolation of extended-spectrum-β-lactamase-positive Escherichia coli sequence types 648 and 131 from Community wastewater indicates that sewage systems are important sources of emerging clones of antibiotic-resistant bacteria. Antimicrob. Agents Chemother. 2019, 63, e00823-19. [Google Scholar] [CrossRef] [Green Version]
  61. Bridgewater, L.; APHA/AWWA/WEF. Standard Methods for the Examination of Water and Wastewater, 22nd ed.; Rice, E.W., Bridgewater, L., Eds.; American Public Health Association: Washingdon, DC, USA, 2012; ISBN 0738133175. [Google Scholar]
  62. Tarr, C.L.; Large, T.M.; Moeller, C.L.; Lacher, D.W.; Tarr, P.I.; Acheson, D.W.; Whittam, T.S. Molecular characterization of a serotype O121:H19 clone, a distinct Shiga toxin-producing clone of pathogenic Escherichia coli. Infect. Immun. 2002, 70, 6853–6859. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Abia, A.L.K.; Ubomba-Jaswa, E.; Momba, M.N.B. Impact of seasonal variation on Escherichia coli concentrations in the riverbed sediments in the Apies River, South Africa. Sci. Total Environ. 2015, 537, 462–469. [Google Scholar] [CrossRef] [PubMed]
  64. Bauer, A.; Kirby, W.; Sherris, J.; Turck, M. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 1966, 45, 493. [Google Scholar] [CrossRef]
  65. CLSI Performance Standards for Antimicrobial Susceptibility Testing, 27th ed.; Informational Supplement M100-S27; CLSI: Wayne, PA, USA, 2017.
  66. Ezeanya, C.; Agbakoba, N.; Ejike, C.; Okwelogu, S. Evaluation of a Chromogenic Medium for the Detection of ESBL with Comparison to Double Disk Synergy Test. Br. J. Med. Med. Res. 2017, 21, 1–11. [Google Scholar] [CrossRef] [Green Version]
  67. Versalovic, J.; Koeuth, T.; Lupski, R. Distribution of repetitive DNA sequences in eubacteria and application to finerpriting of bacterial enomes. Nucleic Acids Res. 1991, 19, 6823–6831. [Google Scholar] [CrossRef] [PubMed]
  68. Pusparini, N.; Waturangi, D.E.; Usia, T.; Nikastri, E. Genetic diversity of Escherichia coli isolated from ice cube production sites. BMC Res. Notes 2018, 11, 4–11. [Google Scholar] [CrossRef] [PubMed]
Antibiotics 10 00860 g001 550
Figure 1. Total E. coli counts and percentage reduction in the studied wastewater treatment plants (WWTPs). Error bars represent the standard deviation. A–D = designation of the different WWTPs.
Figure 1. Total E. coli counts and percentage reduction in the studied wastewater treatment plants (WWTPs). Error bars represent the standard deviation. A–D = designation of the different WWTPs.
Antibiotics 10 00860 g001
Antibiotics 10 00860 g002a 550Antibiotics 10 00860 g002b 550
Figure 2. Dendrogram of ESBL-producing E. coli from the (a) influent and (b) effluent of the wastewater treatment plants (WWTPs). I–XVII = ERIC Types. A–D = Designation of the different WWTPs.
Figure 2. Dendrogram of ESBL-producing E. coli from the (a) influent and (b) effluent of the wastewater treatment plants (WWTPs). I–XVII = ERIC Types. A–D = Designation of the different WWTPs.
Antibiotics 10 00860 g002aAntibiotics 10 00860 g002b
Table
Table 1. Overall percentage resistance of E. coli to different antibiotics.
Table 1. Overall percentage resistance of E. coli to different antibiotics.
AntibioticsABCD
InfluentEffluentInfluentEffluentInfluentEffluentInfluentEffluent
FEP30 30 30 20 10 10 10 20
CAZ30 30 30 30 10 10 10 20
CTX30 50 40 90 20 70 60 90
CFM40 50 40 90 20 60 50 90
FOX50 20 30 20 10 10 10 20
LEX50 60 80 80 30 60 80 100
CXM30 60 50 100 20 9060 90
CEF30 60 60 100 30 80 70 100
MEM30 10 20 20 10 10 0 20
IPM30 10 20 20 10 10 0 20
AMK20 10 20 20 10 10 0 20
GEN20 20 20 20 10 20 10 20
TGC30 20 20 20 10 10 0 20
TET30 80 60 100 20 100 80 90
NAL4080 50 90 30 70 70 90
CIP20 50 30 90 20 60 70 80
CHL50 20 20 0 10 20 0 10
NIT20 10 0 10 10 10 0 10
SXT70 90 60 100 40 90 90 90
AMC50 20 30 20 20 20 10 30
AMX70 90 80 100 80 100 90 90
PIP30 70 30 80 40 60 60 80
AMP90 90 90 10080 100 100 100
A–D = wastewater treatment plants; AMC = amoxicillin/clavulanic acid, AMX = amoxicillin, AMK = amikacin, AMP = ampicillin, FEP = cefepime, CEF = cephalothin, CTX = cefotaxime, FOX = cefoxitin, CFM = cefixime, CAZ = ceftazidime, LEX = cephalexin, CXM = cefuroxime, CHL = chloramphenicol, CIP = ciprofloxacin, GEN = gentamicin, IPM = imipenem, MEM = meropenem, NIT = nitrofurantoin, PIP = piperacillin, TET = tetracycline, TGC = tigecycline, NAL = nalidixic acid, SXT = trimethoprim/sulfamethoxazole.
Table
Table 2. Distribution of the β-lactamase variants among the isolates from the different WWTPs.
Table 2. Distribution of the β-lactamase variants among the isolates from the different WWTPs.
β-Lactamase Enzymes Detected
WWTPSampling PointCTX-M-Type Variants (n)SHV-Type Variants (n)TEM-Type Variants (n)
A InfluentCTX-M-15 (2)SHV-28 (2)TEM-181 (2)
TEM-213 (1)
EffluentCTX-M-15 (3)
CTX-M-3 (1)		SHV-28 (1)TEM-181 (2)
B InfluentCTX-M-15 (1)Not detectedTEM-116 (1)
TEM-1 (1)
EffluentCTX-M-15 (3)SHV-28 (2)TEM-181 (1)
TEM-215 (1)
C InfluentCTX-M-15(1)
CTX-M-3 (1)		Not detectedTEM-1 (1)
TEM-181 (1)
EffluentCTX-M-28 (1)
CTX-M-15 (3)		SHV-28 (1)TEM-1 (3)
D InfluentNot detectedNot detectedTEM-1 (2)
EffluentCTX-M-3 (1)
CTX-M-28 (1)
CTX-M-15 (2)		SHV-28 (2)TEM-181 (1)
TOTAL 20817
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Gumede, S.N.; Abia, A.L.K.; Amoako, D.G.; Essack, S.Y. Analysis of Wastewater Reveals the Spread of Diverse Extended-Spectrum β-Lactamase-Producing E. coli Strains in uMgungundlovu District, South Africa. Antibiotics 2021, 10, 860. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10070860

AMA Style

Gumede SN, Abia ALK, Amoako DG, Essack SY. Analysis of Wastewater Reveals the Spread of Diverse Extended-Spectrum β-Lactamase-Producing E. coli Strains in uMgungundlovu District, South Africa. Antibiotics. 2021; 10(7):860. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10070860

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Gumede, Siyabonga N., Akebe L. K. Abia, Daniel G. Amoako, and Sabiha Y. Essack. 2021. "Analysis of Wastewater Reveals the Spread of Diverse Extended-Spectrum β-Lactamase-Producing E. coli Strains in uMgungundlovu District, South Africa" Antibiotics 10, no. 7: 860. https://0-doi-org.brum.beds.ac.uk/10.3390/antibiotics10070860

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