Antibiogram and beta-lactamase genes among cefotaxime resistant E. coli from wastewater treatment plant

Background The World Health Organization (WHO) recently classified Enterobacteriaceae resistance to third-generation cephalosporin into the group of pathogens with critical criteria for future research. Methods A study to assess the antibiogram and beta-lactamase genes among the cefotaxime resistant E. coli (CREc) from a South African wastewater treatment plant (WWTP) was conducted using standard phenotypic and molecular biology characterization methods. Results Approximate total E. coli (TEc) concentration (log10 CFU/mL) ranged between 5.7 and 6.8 among which cefotaxime resistant E. coli were between 1.8 and 4.8 (log10 CFU/mL) for cefotaxime antibiotic concentration of 4 and 8 mg/L in the influent samples. Effluent samples, heavily influenced by the chlorination had only 0.3 log10 CFU/mL of TEc. Fifty-one cefotaxime resistant isolates were selected out of an overall of 75 isolates, and subjected to a new round of testing, with a follow up of 36 and 48 isolates for both colistin and gentamicin, respectively as guided by initial results. Selected CREc exhibited resistance to amoxicillin-clavulanic acid (35.3%; n = 51), colistin sulphate (76.5%; n = 36), ciprofloxacin (47.1%; n = 51), gentamicin (87.5%; n = 48) and intermediate-resistance to meropenem (11.8%; n = 51). Extended spectrum-beta-lactamase genes detected, viz.: blaCTX-M (52.6%; n = 38) and blaTEM (84.2%; n = 38) and concurrent blaCTX-M + blaTEM (36.8%; n = 38), but no blaSHV was detected. Carbapenem resistance genes, blaKPC-2 (15.8%; n = 38), blaOXA-1 (57.9%; n = 38), blaNDM-1 (15.8%; n = 38) were also detected. Approximately, 10.5 - 36.8% (n = 38) co-occurrence of two or more beta-lactamase genes was detected in some isolates. Out of the selected number (n = 30), 7(23.3%) were enterotoxigenic E. coli (ETEC), 14 (46.7%) were Enteroaggregative E. coli (EAEC), but no enteropathogenic E. coli (EPEC) was detected. Conclusion Resistance to cefotaxime and the presence of a wide range of beta-lactamase genes exposed the potential risks associated with these pathogens via occupational and domestic exposure during the reuse of treated wastewater.


Introduction
Escherichia coli has been accepted as both a biological indicator of water contamination and a human pathogen implicated in both systemic (e.g. septicemia, urinary tract infection, meningitis, etc) and superficial infections (skin or wound infection) [1][2][3][4][5][6]. E. coli is part of the normal flora of the gastrointestinal tract, shed along with faecal waste also including release by infected and convalescent individuals through poorly managed wastewater [7,8]. The public health impact of the E. coli released depends partly on the antibiotic susceptibility pattern of the organism, as this determines the clinical treatment option(s) available [9]. In specific terms, Park [10] inferred that resistance to third-generation cephalosporin and carbapenem by Gram-negative bacteria in the community is very difficult to manage. In South Africa, cefotaxime is extensively in use [11][12][13], as supported by Section 21 of South African constitution [11]. Koopmans et al. [12] reported that cefotaxime is one of the two most used antibiotics in pediatric ward and pediatric intensive care unit of South African hospitals.
On the global level, the resistance of E. coli (and other members of the Enterobacteriaceae) to cefotaxime has been categorized to be of a critical threat to the public health by the World Health Organization, WHO [14], for which research is seriously required. The threat associated with the resistance to these groups of antibiotics is due to their use as last line of defense against infections. Bacterial species showing resistance to them usually possess both genes and enzymes capable of conferring resistance to other antibiotics [15,16].
Resistance to cefotaxime (cephalosporin) is often due to production of enzymes like extended-spectrum betalactamases, ESBLs encoded by the beta-lactamase genes, such as bla CTX-M [16][17][18][19]. The same bacterial species containing the bla CTX-M may also contain bla TEM and/or bla SHV , especially among clinical isolates [20,21]. If found in municipal wastewater, they might be an indication of clinical origin [21] and also important, because wastewater treatment plants (WWTPs) are hotspots for antibiotic-resistant bacteria or resistance genes and it can be used as early warning signs on the health of the human population. Diwan et al. [22] showed the detection of 88% bla CTX-M gene bearing Enterobacteriaceae isolates while Korzeniewska and Harnisz [18] reported that 55% of the same types of bacterial strain harbored the gene. D'Andrea et al. [19] reported bla CTX-M as the primary basis of resistance to the third-generation cephalosporins in E. coli isolated from clinical and environmental samples. In Nicaragua, bla CTX-M genes were detected among ESBL-producing E. coli from hospital wastewater samples [17].
For cefotaxime resistant E. coli with these ESBLs genes, their threat becomes more critical if they also possess carbapenem resistance genes, bla VIM , bla OXA-1 , bla KPC-2 , bla NDM-1 , etc. [23]. Carbapenem resistance genes code for resistance against imipenem, meropenem, ertapenem, doripenem etc. E. coli with carbapenem resistance and carbapenem resistance genes in the treated hospital effluent possesses potential epidemiological threat globally, as the bacteria may initiate difficult to treat infection among the exposed groups [24][25][26][27]. Transfer of antibiotic resistance genes can also be interspecifically and intraspecifically, given a suitable environment [27]. Most beta-lactamase genes are plasmid based and can easily be transferred in biofilm formed within wastewater systems or in lakes [28,29]. Exchange of plasmids and other mobile genetic elements containing beta-lactamase genes does occur in water, which serves as cushioning matrix in the process [30]. E. coli and other bacteria originating from different sources interact within the wastewater matrix and may modify bacterial ecosystems therein [31], depending on the duration of the interaction. Though WWTPs are to reduce the biological contamination of water, the efficiency of treatment would determine the potential release of bacteria with a critical epidemiological threat like cefotaxime resistant E. coli.
Some reports inferred that the conditions in WWTPs give room for the interaction of antibiotic-resistant bacteria and the exchange of mobile genetic elements [30][31][32]. Surveillance of the release of these WWTPs is imperative, to determine the biological status like the presence of cefotaxime resistant E. coli in the effluent that could constitute a threat. The aim of this study, therefore, was to assess and enumerate the cefotaxime resistant E. coli in influent and effluent samples of a wastewater treatment plant in Durban, South Africa and the corresponding sensitivity pattern to other selected antibiotics, as well as the presence of ESBL genes and other selected beta-lactamase genes in them.

Description of the sampling sites and sample collection
The sampling was done in a WWTP, treating municipal wastewater from Durban, Kwazulu Natal Province, South Africa in February (summer) and August (winter), 2017. The WWTP receives inflow in the range of 12, 000-14,000 m 3 containing industrial, municipal and hospital inflow. The total influent Chemical Oxygen Demand (COD) was 250-1100 mg/L and the total effluent COD was 75 mg/L. Other WWTP information are tabulated in Table 1. The treatment plant contained four primary settling tanks, six trickling filters, six settling tanks and three anaerobic digesters (unheated and unmixed). The plant was designed to provide service for 30,000 people (design capacity = 18.80ML/d; working capacity = 10.98 ML/d mixed). It contained a chlorination step for water disinfection before release to the recipient.
In five sampling rounds representing different seasons both pre-grated influent and final (post-chlorinated) effluent samples were collected. The rationale for collecting pre-grated samples was to have the samples in the exact forms it entered the WWTP. To have the samples in the form in which the effluents released to the recipient water, post-chlorinated final effluent samples were collected. The samples transported in a cold chain analyzed within 12 h of collection.
Quantification and isolation of total E. coli, cefotaxime resistant E. coli, other coliforms and cefotaxime resistant coliforms Quantification of the bacteria was done using standard membrane filtration. Membrane Fecal Coliform (mFC) agar (500 g; Difco ref. 267,720) purchased from Quantum Biotech, South Africa was used as the medium for quantification. The media was prepared with 1% rosolic acid (Difco; ref. 232,281) purchased from Hach International. Ten-fold serial dilutions representative for the influent and effluent ranges of concentration were prepared using sterilized saline solution (0.85% (w/v) NaCl). The prepared samples were filtered through 0.2 μm cellulose acetate filters (Lasec, South Africa) in triplicates. The filters were placed on the mFC agar plates supplemented with 4 mg/L or 8 mg/L cefotaxime antibiotic (100 mg Cefotaxime sodium salt; Sigma; ref. C7039) and on agar plates without antibiotic supplements in line with UNESCO regulations for another ongoing project in our laboratory. Though 4 mg/L was optimized to be adequate for satisfactory results, continuation of both 4 mg/L and 8 mg/L throughout was to see potential variance over a while.
The resulting culture was incubated at 37°C for 24 h. Enumeration of the total number of colonies depended on observation of specific colonial colorations (grey, pink and blue). E. coli forms blue colonies. Grey and/or pink colonies might not be coliforms. The selectivity of the media was determined by estimating the ratio of blue and total colonies.

Species-specific identification of E. coli Extraction of DNA
Isolation of the DNA was done by the boiling method using the protocols of Salehi et al. [33]. The young presumptive E. coli colonies of ≤24 h were suspended in 200 μL aliquots of distilled water. The suspension containing the isolates was mixed by vortexing until a mixture with thoroughly dissipated isolates formed. This was boiled at 95°C for 15 min and subsequently centrifuged at 15000 rpm; at 4°C for 15 min where after, the supernatant was removed and stored at − 20°C for further assays.
PCR based species-specific identification of E. coli PCR based species-specific identification of E. coli was done using the primers (Inqaba Biotechnical Industries, South Africa) specific for a conserved region of E. coli alanine racemase (alr) gene specified in Table 2 [34,35]. The reaction mixture contained 12.5 μL of one taq quick load 2x master mix with standard buffer (Inqaba Biotechnical Industries, South Africa), 20 μM each of the forward and reverse primers, water and 3 μL of DNA template suspension in a final volume of 25 μL.
The cycling conditions for the PCR reaction started with an initial 6 min denaturation step at 95°C, followed by 35 cycles containing denaturation at 95°C for 20s, primer annealing/extension at 72°C for 1 min 30 s, and a final extension for 5 min at 72°C. E. coli WG5 was used as a positive control.
Agarose gel electrophoresis was performed at 80 V for 45 min in 2% agarose gel in Tris Acetate-EDTA (TAE) buffer, stained with Sybr Safe DNA gel stain (Thermo-Fisher Scientific, South Africa).

Multiple antibiotic resistance (MAR) index of cefotaximeresistant E. coli to other antibiotics
Multiple antibiotic resistance index (MAR Index) was determined per isolate as the ratio of the number of antibiotics to which an isolate showed resistance (X) to the total number of antibiotics against which the isolate was tested (Y) [38][39][40].

MARI ¼ X=Y
This was determined on all the isolates concerning all the antibiotics used.
Detection of genes for extended spectrum betalactamases and carbapenemases in cefotaxime-resistant E. coli Multiplex PCR for bla CTX , bla TEM and bla SHV Multiplex PCR assay was carried out to detect bla CTX , bla TEM and bla SHV ( Table 3). The PCR reaction mixture of 25 μL contained 12.5 μL of one taq quick load 2x master mix with standard buffer (Inqaba Biotechnical Industries, South Africa), 20 μM each of the forward and reverse primers, water and 3 μL of DNA template suspension. The cycling conditions for the PCR assay included an initial denaturation at 95°C for 5 min, 30 cycles at 95°C for 30 s and 68°C for 40 s and a final extension at 68°C for 3 min.
Agarose gel electrophoresis also performed in 2% agarose gel in Tris Acetate-EDTA (TAE) buffer, stained with Sybr Safe DNA gel stain (ThermoFisher Scientific, South Africa).
Singleplex PCR for the detection of bla VIM , bla OXA-1 , bla KPC-2 , bla NDM-1 Singleplex PCR was performed for the detection of bla VIM , bla OXA-1 , bla KPC-2 , bla NDM-1 using the primers listed in Table 3. For bla VIM , the cycling conditions included the initial denaturation at 94°C for 3 min, followed by a stage of 36 cycles containing denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and a final extension at 72°C for 5 min. PCR cycling conditions for bla OXA-1 began with initial denaturation at 95°C for 2 min, followed by a stage of 30 cycles containing denaturation at 94°C for 45 s, annealing at 55°C for 30 s and extension at 72°C at 1 min. This was followed by a final extension stage at 72°C for 5 min.
The detection of bla KPC-2 was done with cycling conditions that included an initial denaturation at 95°C for 5 min, followed by a stage of 40 cycles containing denaturation at 95°C for 15 s, annealing at 66.1°C for 30 S, extension at 72°C for the 30 S and final extension at 72°C for 10 min [41]. For bla NDM-1 , the cycling conditions began with denaturation at 95°C for 15 min, followed by a stage of 30 cycles containing 95°C for 1 min denaturation, 61.1°C for 1 min annealing, and extension for the 30 S at 72°C. This was followed by a final extension for 10 min at 72°C [42].

Pathotyping of the E. coli
Isolated and identified E. coli were further pathotyped to determine which strains they were, using the primers

Results
Quantification of total E. coli and cefotaxime-resistant E. coli Large counts of cefotaxime-resistant E. coli were resident in the wastewater. A very high mean total E. coli count of over 6 log 10 CFU/mL was found in the influent sample (represented with Inf SR-3 NA in Fig. 1). Varying concentrations of cefotaxime-resistant E. coli occurred with differences between the two concentrations of the antibiotic supplemented in the laboratory cultivation media. Higher numbers of resistant isolates were enumerated at cefotaxime concentration of 4 mg/L than at 8 mg/L as assumed, though this was inconsistent at one sampling occasion. Higher concentration of cefotaxime (8 mg/L) did not show reduction in the cefotaxime resistant E. coli counts beyond what lower concentration showed in a slight variation. An approximate total E. coli count (log 10 CFU/mL) ranged from 5.7 to 6.8, with cefotaxime resistant E. coli count (log 10 CFU/mL) ranging between 1.8 to 4.9 for cefotaxime concentration of 4 mg/L and 2.3 to 2.8 at 8 mg/L. The influent counts (log 10 CFU/mL) for the total E. coli count (NA) as well as cefotaxime-resistant E. coli (A4 and A8) for all the summer samples (SR-1, SR-2, SR-3) are presented in Fig. 1(a). The results of the other coliforms for summer as depicted in Fig. 1(b) contained a total count (log 10 CFU/mL) that ranged from about 6.4 to 6.6, with one wide outlier of count 0, plotted separately from the range 6.4-6.6. The cefotaxime (4 mg/L) resistant coliforms counts (log 10 CFU/mL) ranged from 0 to 4.7 while those for 8 mg/L ranged from about 2.6 to 2.9, presented in Fig. 1(b).
Seasonal variation was not significant (p < 0.05) with an approximate total E. coli count (log 10 CFU/mL) in winter that ranged from 5.2 to 6.6, cefotaxime-resistant E. coli count (log 10 CFU/mL) ranged from 2.5 to 4.8 at concentration of 4 mg/L and 1.0 to 5.2 at concentration of 8 mg/L. The trend was also similar for other coliforms with a total count (log 10 CFU/mL) that ranged from about 4.7 to 6.3; cefotaxime (4 mg/L) resistant coliforms counts (log 10 CFU/mL) ranged from 2.3 to 3.5 and cefotaxime (8 mg/L) resistant coliforms counts (log 10 CFU/ mL) ranged from 0 to 2.9. The winter counts of E. coli and the other coliforms are represented in Fig. 1(c) and (d), respectively.
Meanwhile, there was consistently no total and cefotaxime-resistant E. coli growth and rarely very low growth (range in log 10 CFU/mL = 0 to 0.3) in the effluent concentration (Fig. 1e). Therefore, the reduction  efficiencies for total E. coli were in the range from 95.6 to 100%, while those estimated for cefotaxime-resistant were between 99 to 100%. Total coliforms in the effluent samples (without cefotaxime) however showed some appreciable counts (log 10 CFU/mL) that ranged from 0 to 3.6 as in Fig. 1(e). Their removal efficiencies inconsistently ranged from 45.5 to 100%. The majority of the coliforms were sensitive to cefotaxime and did not grow on cefotaxime plates. There were more effluent samples without E. coli and coliform growth. Almost all the cefotaxime-supplemented plates for effluent showed no growth. The count of 1 CFU/mL in very few cases was estimated as 0 log 10.

Antibiogram of cefotaxime-resistant E. coli
The results of the antibiotic susceptibility testing from cefotaxime resistant E. coli to additional antibiotics is presented in Table 5  Carbapenem-resistance genes, bla KPC-2 and bla OXA-1 were found in 15.8% of the isolates. bla NDM-1 and bla OXA-1 were also concurrently detected in 10.5%. Both ESBLs genes and carbapenemase genes were concurrently detected in some isolates, with the highest being bla TEM and bla OXA-1 simultaneously detected in 52.6% of the isolates. Fewer isolates showed the presence of the putative genes for carbapenemases than ESBLs. In addition, there was statistically significant difference (p < 0.05) between most carbapenem-resistance genes (bla KPC-2, bla NDM-1, bla KPC-2 + bla OXA-1 and bla NDM-1 + bla OXA-1) and ESBL genes (bla CTX-M and bla TEM ). The details of the beta-lactamase genes are displayed in Table 6.

Discussion
We conducted this study with the aim of quantifying the cefotaxime-resistant E. coli and determining antibiogram to other antibiotics, as well as the presence of betalactamase genes in the Gram-negative bacteria from a WWTP from Durban, South Africa. This aim is in line with the critical criteria placed on Enterobacteriaceae resistant to third-generation cephalosporin and carbapenem antibiotics by WHO [14]. The distribution of resistant E. coli to the environment through wastewater effluents remains an issue of public health concern, depending on downstream human exposure. A large number of total E. coli count (log 10 CFU/mL) that ranged from 5.7 to 6.8 or cefotaxime-resistant E. coli as depicted in Fig. 1(a) -(d) in the influent may not really be of much concern. The former (total E. coli) is expected in influent since E. coli is a normal part of the gut flora [7,8]. The presence of the latter (cefotaxime-resistant E. coli) might also be due to wastewater from hospital inflow. These resistance attributes are expected from the clinical origin [20,21,46] and since the WWTPs are known as early warning system [47], the presence of a large number of cefotaximeresistant E. coli might be a warning sign of probable lack of pretreatment for the hospital outflow. The effluent concentration of both total E. coli and cefotaxime-resistant E. coli (range in log 10 CFU/mL = 0 to 0.3) (Fig. 1e) was low and in most cases zero due to the chlorination practices. Even when the bacterial cells confirmed to be dead after chlorination, studies have shown that large percentage of their genes persists in the effluent [48]. These genes can be picked up by competent environmental strains of E. coli, conferring the antibiotic resistance status of the dead organism on the new [27]. These competent cells might accidentally be among other total coliforms with effluent concentration (log 10 CFU/mL) that ranged from 0 to 3.6. This is possible because transformation only requires a competent cell to pick up resistance genes in the environment, even if the donor is from another genus [27,49]. Though many cefotaxime-resistant Gram-negative bacteria are already recognized as critical threats [11], their further resistance to other antibiotics within the last line of defense poses a greater threat toward total resistance [50,51]. Gentamicin resistance (87.5%) detected in our study was in tandem with the reports by Jakobsen et al. [52]. It was revealed that the isolates showed intermediate resistance to meropenem (11.8%) as well as resistance to ciprofloxacin (47.1%) and colistin sulphate (76.5%) by cefotaxime-resistant E. coli. Fluoroquinolone resistant isolates of E. coli from sewage had also been reported by Colomer-Lluch et al. [53]. The corresponding threat is because fluoroquinolones (e.g. ciprofloxacin) ranked as one of four highest prioritized critically important antimicrobials [54]. A figure of about every 1 in 2 (47.1%) as detected in this study for ciprofloxacin and cefotaxime-resistant E. coli is synonymous with having a 12.5% chance of therapeutic failure or total resistance, in line with the position of WHO [50]. The colistin resistance (76.5%) in Enterobacteriae in this study is in worrying tandem with the report of 100% colistin resistance in lake water and 33.3% in sewage found by Fernandes et al. [55]. Resistance to colistin by environmental or clinical isolates is significantly related to resistance to some other antibiotics in the last line of defense [56]. Colistin administration induces an increase in carbapenemase-producing Enterobacteriaceae around the world and resistance to colistin itself is directly linked with the agricultural use of human antibiotics [56]. The resistance of 35.3% to amoxicillin-clavulanic acid revealed that the basis of resistance in some of the isolates in this study goes beyond beta-lactamase production. This is because clavulanic acid would have inhibited the effect of the enzyme to ensure susceptibility to the antibiotic. This profile highlighted the bacteria as a potential threat for any less kitted workers in WWTPs due to the risks of occupational exposure [57].
The discharge of the antibiotic-resistant bacteria from WWTPs to the environment is reported widely [32,49,51,52,[58][59][60]. Concurrent resistance to multiple antibiotics is  an issue of greater concern. A wide range of multiple antibiotic resistance indices (0.13-0.75) might be a reflection of wide disparity in the static-use and the adaptive-use of these antibiotics [37]. This may signify a consistent administration of these antibiotics without medical prescription or the organisms originating from hospital environment [60]. Large percentage (> 94%) of the cefotaxime-resistant E. coli exhibited multiple antibiotic indices (MAR Index) > 0.2 which means that the isolates might have originated from high risk source (s), where antibiotics are probably abused [38,40,59]. As noted earlier, since the WWTPs are known as early warning systems [47], the MAR Indices in this study revealed that antibiotics are in constant abuse and hence, their high selective pressure [61]. The high resistance to colistin sulphate (76.5%) detected in this study is considered alarming. Over the past few years now, attention around the world has been especially focused on the epidemiology of resistance to colistin in bacteria. Researchers link the emergence of colistin resistance to prior exposure to the sublethal concentration by the bacteria, but studies have reported the isolation of colistin-resistant E. coli strains from individuals without history of colistin usage [62]. In a study in Laos, Olaitan et al. [63] isolated colistin-resistant E. coli from a boy without any record of prior administration of colistin but rather probable zoonotic exposure from the family's pigs. This is why animal or infected human feces like in waste and wastewater are considerable hotspots [64,65]. Colistin is used as the final alternative for resistant isolates against β-lactams, aminoglycosides or quinolones, but colistin-resistant Enterobacteriaceae have been classified with pan-drug resistance [66]. So, infections from the E. coli isolated from the sampled WWTP can be difficult to treat in an individual if contracted via occupational exposure.
Several antibiotic resistance determinants were detected in this study. These include bla TEM , bla CTX-M , bla SHV , bla VIM , bla OXA-1 , bla KPC-2 and bla NDM-1 , which might be responsible for some detected resistance to the cefotaxime and other beta-lactam antibiotics [67]. Though bla VIM was not detected, bla OXA-1 , bla KPC-2 and bla NDM-1 might be responsible for intermediate resistance to meropenem by the cefotaxime-resistant E. coli [67,68]. Several beta-lactamase genes like bla CTX-M , bla SHV and bla TEM have also been reported in both hospital and municipal wastewater effluents [18,69]. These reports, as well as our report, indicate the possibility for the determinants to be disseminated among water systems [70], since the genes may persist after the bacterial cells are dead [48]. Concurrent detection of at least two ESBLs' genes, two carbapenem resistance genes and ESBLs with carbapenem resistance genes (Table 6) depicted the level of threat associated with these bacterial strains.
Reports in other studies [71,72] showed that ESBL producers from WWTP were E. coli and K. pneumoniae. Most rampant putative ESBLs' genes were bla CTX-M genes in which the similar gene types were detected in both clinical and environmental isolates [73,74]. Multidrug resistant E. coli (MDR-Ec), have been isolated from a healthy human [75] and the MDR-Ec is sometimes shed in feces released into WWTP through the user interface. Intraspecific and interspecific transfer of resistance gene was expected to be high in the WWTP due to high bacterial density. This transfer may include rampant bla CTX-M (Coque et al., 2008).
Since 70% (n = 30) of the selected isolates were confirmed as pathogenic due to their pathotypes (Fig. 3), their release into the environment may pose threat to public health and to the environment, as these attributes

Conclusions
The influent samples in the WWTP contained a large number of Cefotaxime-resistant E. coli and other coliforms but the effluent contained only a very scanty concentration of total E. coli and other coliforms. Some of the cefotaxime resistant E. coli also showed resistance to amoxicillin-clavulanic acid, colistin, meropenem, ciprofloxacin, gentamicin, trimethoprim-sulphamethoxazole, nitrofurantoin and tetracycline. The presence of cefotaxime-resistant and indeed multidrug-resistant Enterobacteriaceae in wastewater influent showed that these organisms, classified with critical threats, were possibly harbored in the population. These, cefotaximeresistant E. coli, were pathogenic as revealed by their pathotypes. These resistant isolates bore extendedspectrum beta-lactamase genes and carbapenem resistance genes singly and concurrently, which make them of potential clinical origin. It is imperative for hospitals to pretreat their wastewater effluent to avoid the critical threat associated with these kinds of bacteria to public health, in case of leakage and human exposure.