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Article

Resistome Profile of Treated Wastewater Using Metagenomic Approach

by
Amarachukwu Obayiuwana
1,
Abasiofiok M. Ibekwe
2,* and
Chinelo Eze
3
1
Department of Biological Sciences, Augustine University Ilara-Epe, Lagos 106101, Lagos State, Nigeria
2
George E. Brown Salinity Laboratory, USDA-ARS, 450 West Big Springs Road, Riverside, CA 92507, USA
3
Department of Pharmaceutical Microbiology and Biotechnology, University of Nsukka, Nsukka 410001, Enugu State, Nigeria
*
Author to whom correspondence should be addressed.
Water 2025, 17(6), 867; https://doi.org/10.3390/w17060867
Submission received: 11 February 2025 / Revised: 10 March 2025 / Accepted: 13 March 2025 / Published: 18 March 2025
(This article belongs to the Section Wastewater Treatment and Reuse)
Browse Figure
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Abstract

:
The occurrence and dissemination of resistomes within environmental compartments are worrisome, considering the huge public health challenge they may pose. Treated wastewater from wastewater treatment plants (WWTPs) has been shown to contain enormous and diversified bacterial communities with antibiotic resistance gene (ARG) profiles, and it provides tolerable environments for their prefoliation and dissemination. This study considered the ARG profiles of a municipal WWTP that also collects wastewater from diverse production factories with the aim to determine the efficacy of the wastewater treatment plant and the prevalence of ARGs in the various compartments. The WWTP employs the conventional activated sludge system in its treatment. Our study employed metagenomic screening of ARGs and mobile genetic elements (MGEs), using different PCR assays of untreated wastewater (UTW) and treated wastewater (TWW) from the WWTP. Downstream- (RWD) and upstream- (RWU) receiving river water was also screened. Twenty-nine ARGs and two mobile genetic elements (MGEs) were screened for. Antibiotic resistance genes to all the classes of antibiotics studied were detected in all the samples. Of the twenty-nine ARGs screened for, twenty-four (82.8%) were detected, and one MGE was detected from the two screened for in the samples with the highest ARG prevalence detected in the UTW. This study shows that ARGs proliferate in every compartment of the WWTP, even in the treated water and the receiving rivers, portraying that the WWTP system was not efficient in getting rid of the resistomes, creating an important channel for human acquisition of resistant determinants to antibiotics.

1. Introduction

Recently, antibiotic resistance in human and animal therapy is receiving extensive attention worldwide [1,2]. It has become a major setback in the application of antibiotics in clinical infections and animal husbandry, posing a threat to public health. This threat has also been acknowledged by the World Health Organization (WHO) [3]. This also invariably has an impact on food availability and safety, with a resultant impact on the world’s economy. Globally, there have been consequential reports on the high levels of resistance in the treatment of infectious diseases [4]. A high level of mortality has been recorded globally and is estimated to increase exponentially in the coming years [5]. Studies have shown that antibiotic resistance is a result of the effect of the many natures of the reservoirs of antibiotic resistance genes (ARGs) in the proliferation and spread of antibiotic resistance bacteria (ARB) [6,7]. ARB are widespread in different environmental compartments, and there are extensive reports on the proliferation of ARGs to many classes of antibiotics in these compartments as well [7,8,9,10].
The prevalence of the resistomes in many environmental compartments has been linked to many complex interwoven factors such as the disproportionate use of antibiotics in human and veterinary medicine [11] and in crops [12]. These factors and many more have compromised the effective application of antibiotics [13] in the management of bacterial infections, contributing to an increased presence of ARGs in the environment and the major driving force for new resistance development [14] in pathogenic bacteria. With the complexity in understanding the evolution of antibiotic resistance and ARG-associated gene transfer events, there have been many reports on the positive correlation linking residual antibiotics, ARGs, and ARB in hospital wastewater [15,16,17]. Aside from hospital wastewater, there have been reports of ARGs associated with other environmental compartments such as aquatic environments, which may serve as genetic reactors that lead to the exchange of antimicrobial resistance and virulence genes among the bacteria community [18,19] by introducing sub-lethal levels of antibiotics to the native bacterial community within the compartment, resulting in selective pressures and emergence of resistant strains [20,21]. These can also include agricultural runoff [22,23], reclaimed water [24], and even treated wastewater from WWTPs [7,8,9,10].
Wastewater treatment plants have been reported in many studies as reservoirs for ARB and ARGs in many instances [7,9]. These reports recognize them as important systems for the development and dissemination of ARGs. WWTP compartments have provided platforms for the possible exchange of ARGs among bacteria species of the same origin or different species through the HGT pathway [6], such that are likely to occur between native environmental bacterial communities and pathogenic bacteria [25]. Also, the loci of resistomes on mobile genetic elements (MGEs) make their transfer achievable among bacteria of the same or different origins [26]. Chemicals of emerging concerns (CECs), such as antibiotics in the WWTPs, have promoted their dissemination. In recent studies, high concentrations of antibiotic residues have been found in WWTPs, even in treated wastewater [27]. This assertion depends greatly on the design and the applied technology operational in the plants [28]. The majority of the low-income nations are not able to pass their sewage through conventional treatment in the WWTPs. The majority of the sewages are channeled directly to public drainages and, ultimately, neighboring water bodies.
This study aims at evaluating the efficacy of the wastewater treatment plant under study, particularly in its ability to remove the resistomes. To carry out this study effectively, our study will determine the presence of ARGs in the sewage before treatment and the ARG integrity at the end of the treatment of the sewage up to the river points receiving the effluents. Hence, this research screened a municipal wastewater treatment plant for the presence of ARGs and MGEs, which are the resistomes that can be transferred to human pathogens through the effluent receiving points in the river. In this study, we investigated the incidence of ARG and MGE profiles in DNA samples obtained from untreated sewage and effluent at the end of treatment in a municipal WWTP in Ogun State, Nigeria. This plant employs conventional approaches such as sedimentation, activated sludge, aeration of the pond, etc., in the treatment of wastewater. This plant receives wastewater from industrial facilities, especially pharmaceutical wastewater and household sewage from residential quarters within the estate. Our previous studies [7,9] carried out in the same WWTP evaluated the resistomes in genomic DNA obtained from bacterial isolates from the WWTP. This study will evaluate the metagenomic DNA samples obtained from the WWTP without prior bacterial isolation.

2. Materials and Methods

Study site and sample collection. The study site and location (Table S1) was a municipal wastewater treatment plant located in an industrial estate in Agbara, Ogun State, Nigeria (Figures S1 and S2). The WWTP receives its influents from production wastewater coming from manufacturing industries located within the estate and sewages from households in the residential areas of the estate. Samples from untreated wastewater (UTW) were collected directly from the influent unit of the plant. Samples of the treated wastewater (TWW) were taken from the effluent unit. Also, samples of river water (RW) that receive the effluents were taken from both the upstream (RWU) and downstream (RWD) sections. Table S1 in the Supplementary Materials shows the description of sampled points and the sample types collected. Wastewater was collected in duplicates using 2 L dark-brown glass bottles and transported aseptically to the laboratory immediately for processing. In the laboratory, composite samples were taken by pooling together duplicate samples. All samples were stored at 4 °C until DNA extraction.
Extraction of Metagenomic DNA. Total DNA was extracted from the untreated (influent samples) and treated (effluent samples) wastewater of the WWTP and river water samples using the FastDNA™ SPIN kit for soil (MP Biomedicals, Solon, OH, USA) following the manufacturer’s instructions. The DNA isolation was carried out following the filtration of the wastewater and river water samples through a 0.2 µL membrane, as previously reported [7,9], and DNA was quantified using NanoDrop ND-1000 (Nanodrop, Foster City, CA, USA) and stored at −80 °C before further use.
Primers and PCR screening of ARGs and Integrons. Primer sequences and their annealing temperatures used in this study are shown in Tables S3–S7 in the Supplementary Materials and were synthesized at Sangon Industries Limited, Beijing, China. The primer solutions were stored at −20 °C for the PCR amplification. The DNA samples isolated from the two categories of wastewater (treated and untreated) were taken from the WWTP, and two of the RW samples (downstream and upstream) were screened for possible resistomes. A total of 29 specific ARGs and 2 Intl genes were screened, and these included ARGs encoding resistance to tet, aminoglycoside, beta-lactamase and penicillins, chloramphenicol, and sulphonamides. All the tet genes, gene classifications, and their resistance mechanisms tested in this study are listed in Tables S3–S7. These include tet A, B, C, D, E, G, 30, and J (efflux protein), tet M, O, Q, and Bp (ribosomal protection protein), and inactivating enzyme (tetX). Eight ARGs conferring resistance to the aminoglycoside class of antibiotics were screened. The 3 groups screened for included aminoglycoside acetyl-transferases (3) [aac(3)-II, aac(3)-IV, and aac(6′)-Ib (aacA4)], aminoglycoside nucleotidyl-transferases (3) [(ant(3″)-Ia (aadA), ant(2″)-Ia (aadB), and ant(6)-I (aadE)], and aminoglycoside phosphor-transferases (2) [aph(3″)-I (strA) and aph(6)-Id(strB)]. Four bla genes were screened, including TEM, OXA, CTX-M, and IMP, as well as two chloramphenicol acetyl-transferases genes (catA1) and specific exporters (cmlA). PCR reactions were carried out in 50 µL PCR tubes (Takara, Dalian, China) as previously described [7,9].

3. Results

3.1. Screening of ARGs: tet Genes

The result of the PCR screening shows that at least a tet gene was detected in all the samples collected from the WWTP (UTW and TWW) and the river water upstream (RWU) and downstream (RWD) (Table 1 and Table 2). Table 1 and Table 2 show the summary of the profiles of all the resistance genes screened in both WWTP and RW samples, while Figure 1 shows their prevalence. The prevalences of the tet genes in UTW, TWW, RWU, and RWD are 77.9%, 53.8%, 38.5%, and 30.8%, respectively (Table S2) (Figure 1). The tetracycline resistance genes that were common to all the study sites were tet(A) and tet(G), which are tet genes encoding efflux proteins. The tet(30), tet(J), tet(M), and tet(Q) genes were detected only in the untreated wastewater in the WWTP but were not present in the other sites tested (Table 1). The tet(X) gene that encodes for enzymatic modification proteins was detected only in the WWTP samples (UTW and TWW). The river water sites, RWU, showed an expression of five tet genes, while the RWD site expressed four of the tet genes (Table 2). The screened tet genes under study encoding for ribosomal protection and enzymatic modification were not detected in the river water samples (RWU and RWD) except for tet(BP) detected in RWD. The results show that the untreated wastewater samples of the WWTP had the most abundant tet genes in the study.

3.2. Aminoglycoside Resistance Genes

Figure 1 and Table 1 show the result of aminoglycoside resistance genes tested in the WWTP and RW samples. Overall, a total of six (75%) out of eight genes (Table 1) were detected (Table 1). aadA, strA, and strB were the most frequently detected aminoglycoside resistance genes in the samples (Table 1). The UTW samples expressed the highest number of aminoglycoside resistance genes in this study, with a total of five (83.3%) genes of the six detected in all the samples (Table 2). Only three (50%) of the genes were detected in TWW, whereas there was no gene expression for samples obtained from RWU samples, except for aadB, an aminoglycoside resistance gene encoding nucleotidyl-transferase enzymes detected in RWD. None of the genes encoding phosphor-transferase enzymes were found in the RW samples.

3.3. Detection of bla Genes

bla genes were detected only in UTW and TWW samples of the WWTP (Table 2). Only one type of bla gene out of five screened was detected in the sampled sites, with only blaOXA identified in only two of the sampled sites (Table 2). The gene blaOXA was detected only in the UTW and TWW samples (Table 1). No screened bla gene was detected in the RWU and RWD samples (Table 1). blaTEM, blaCTX-M, and blaIMP were not detected in any of the samples screened.

3.4. Phenicol, Sulfa Resistance Genes, and Mobile Genetic Elements

The encoding genes for chloramphenicol acetyl-transferase genes (catA1 and cmlA) were detected in the sites (Table 1). The results revealed that catA1 was expressed in all the study sites, whereas cmlA was detected in samples of the WWTP only. Invariably, the untreated and treated wastewater of the WWTP expressed catA1 and cmlA. The catA1 gene was detected in both river water samples of RWU and RWD, whereas cmlA was not detected in any of the two sites. The prevalence of catA1 and cmlA in the WWTP compartment is 100%, whereas in the RW samples, catA1 is 100% prevalent (Figure 1). sul genes were detected in both the WWTP and RW compartments of the study sites (Table 1). sulI and sulII were detected in all the studied sites (Table 2). The prevalence of both dihydropteroate synthase encoding genes (sulI and sulII) was 100% in all the study sites (Figure 1). The wastewater treatment plant for both the untreated and treated wastewater samples showed 100% prevalence of Intl1, a mobile genetic element (MGE) tested in the study (Table 1). The Intl1 genes were identified in WWTP compartments, in both the untreated and treated wastewater samples.

4. Discussion

Wastewater has been widely reported to harbor antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) [7,9] and plays a significant role in their dissemination. Primers of known antibiotic resistance genes obtained from the published literature (Tables S3–S5) were used for this study. Also, river water samples from upstream and downstream points were studied. This river receives effluents from the WWTP after treatment. Our results showed that ARGs and MGEs were widely distributed within the wastewater treatment plant and the receiving river. The untreated wastewater (UTW) samples, as expected, had the most prevalence of ARGs as presented (Figure 1). This compartment (UTW) was positive for 21 ARGs and MGEs of the 29 ARGs and 2 MGEs (Table 1), with the highest prevalence for tetracycline and aminoglycoside resistance genes.
Similarly, the treated wastewater (TWW) samples showed a significantly high prevalence compared to UTW. TWW was positive for 16 ARGs and MGEs. The question remains how efficient is the WWTP in the treatment of the wastewater and removal of ARGs. A significant number of ARGs were also recorded for the receiving water bodies, both for the downstream and upstream points (Table 1). Overall, a total of eight ARGs were reported for both the RWU and RWD, respectively. These findings suggest that surface water points receiving effluent from these WWTPs are hotspots for possible horizontal gene transfer (HGT) within the environmental bacterial community [6].
Our result revealed that the ARGs that encode resistance to the antibiotic tetracycline were the most prevalent in this study. The UTW samples obtained from the wastewater treatment plant showed more prevalence for tetracycline resistance genes (tet) (Figure 1). The presence of the tet genes has been widely reported in activated sludges and untreated and treated wastewater in their different compartments from wastewater treatment plants [9,10,29]. This may be a result of the wide applications of tetracycline antibiotics in both human and animal husbandry [30]. Our data showed that efflux protein-encoding genes were the most prevalent, as shown by tet genes encoding for transport proteins obtained from the environment [31]. The tetracycline resistance genes that were common to the study sites were tet(A) and tet(G), which encode efflux proteins. The final effluent (TWW) had a significant amount of tet genes compared to the UTW from the WWTP. It shows that the conventional treatment procedure applied in the WWTP may not have been effective in removing the tet genes. As expected, the receiving river samples (RWU and RWD) had less prevalence of tet genes (Figure 1). This may be a result of the dilution effect of the flowing current of the river. The tet(X) is an ARG that confers resistance to a third-generation tetracycline, tigecycline [32]. This tet gene was only detected in WWTP samples but was not detected in the river water samples. Even at that, and with the strict regulation in the use of tigecycline, the dissemination of tet(X) within the environmental compartments must be minimized. Moreover, the literature has reported the presence of these genes in human pathogens [33], as we also had reported during our last study from these sites [9].
The results from this study show that ARGs encoding resistance to aminoglycoside [34] were detected only in WWTP samples, except for aadB, a nucleotidyl-transferase encoding gene detected in RWD. The resistance genes for aminoglycoside resistance were readily present in UTW samples (Table 1). In our samples, three aminoglycoside resistance genes screened for were positive, and these were aadA, strA, and strB (Table 1). These three ARGs were all present in both the untreated and treated wastewater. The aadB genes were only present in RWD samples. Although several aminoglycoside resistance genes were detected in this study from the samples, reports of previous works on aminoglycoside ARGs showed their presence in bacterial communities. The genes aacC1, aacC2, aacC3, and aacC4 were previously detected in bacteria from sewage treatment plants [35,36,37].
The attack of the beta-lactam ring is largely responsible for the resistance encountered in the use of the beta-lactams class of antibiotics. Strains of bacteria that show resistance to this class of antibiotics harbor genes that can encode for a large range of enzymes known as beta-lactamases [38] that can hydrolyze bla drugs. Of the four ARGs encoding for the beta-lactamases tested in this study, only blaOXA was detected in the WWTP samples, both in the untreated and treated samples (Table 1). This result agrees with the reports of research works on resistance to the beta-lactams class of antibiotics, especially for penicillins, that despite their wide use as one of the most common antibiotic agents, these agents are not often found in TWW and activated sludge samples [10,39]. Also, the result agrees with similar studies within WWTPs, where blaOXA-58 genes that confer resistance to carbapenems were frequently reported [39,40]. In our study, bla genes were not detected in the RW samples. Contrary to the findings reported above, in a similar study of ARG prevalence in four different pharmaceutical wastewater [41], each of the study sites expressed at least one type of the bla gene. The study site that had the most abundance of the ARGs analyzed in that study was positive for all the bla genes tested.
catA1 and cmlA were tested and detected in this study, and these two genes encode chloramphenicol acetyl-transferases and specific exporters, respectively, and these two mechanisms are responsible for chloramphenicol antibiotics detected. This is in agreement with our previous study with bacterial samples where catA1 and cmlA were both detected in a wide range of bacterial isolates obtained from a similar environment, with catA1 genes having a higher prevalence bacterial species than the cmlA genes [9]. It appears that both catA1 and cmlA were not eliminated or eliminated during the treatment of the wastewater. In the receiving river water samples, only catA1 was detected in both the RWU and RWD water samples. Also, the catA1 and cmlA genes in different environmental compartments have been reported in other studies [42].
The common mechanism responsible for resistance to sulfonamides is the synthesis of the dihydropteroate synthase (DHPS) enzyme, which has a lower affinity to sulfonamides encoded by sulI, sulII, and sulIII genes, amidst other diverse mechanisms [43]. These genes are carried on mobile genetic elements such as transposons and plasmids and reported in diverse species of bacteria [44]. Our results revealed a high distribution of sulI and sulII genes in our samples (UTW and TWW) and the receiving river water samples (RWU and RWD). The two sul genes were present in all the study sites during this study but not in all the isolates during our previous study and from similar environments from previous studies and similar samples [45]. The genomic DNA obtained from the bacterial isolates had a prevalence of 31.7% and 21.7% for sulI and sulII, respectively [7]. Our findings agree with other reports that have demonstrated the occurrence of sul genes in WWTPs where sul and tet resistance genes and bacteria have been identified and may potentially be released into the environment [45,46]. Sul genes were more dominant in the effluent samples in comparison to influent [47].
Lateral gene transfer occurs among bacteria of the same or different origin through their possession of mobile genetic elements and are known as agents for the transfer of genetic materials among bacteria in the environment [48]. In this study, class I integron (Intl1) genes were detected only in the wastewater treatment plant samples (Figure 1). They were detected in the UTW and TWW samples alike (Table 1). Our study is in agreement with other studies where wastewater effluents have been shown to contain high concentrations of MGEs [49]. The integrase-encoding genes, Intl2 and Tn15/545, were not detected in any of the WWTP and RW samples. Also, Intl1 was not detected in the RW samples (RWU and RWD). The results contradict our results in a previous work where similar environments had at least one type of MGE in their different compartments [9,41]. The presence of the MGEs in the WWTP may be very worrisome, particularly with the discharge of the final effluents into rivers that serve other purposes to the human populace and animals. Even though in this report MGEs were not detected in the river water, possibly as a result of the dilution effect by the water currents, there is a very high possibility of the proliferation of MGEs over some time. Unfortunately, in many developing countries, there may be no secondary or tertiary treatment levels in most WWTPs before this water is discharged to the environment or receiving water bodies. This creates a potential hazard to the people living downstream of these WWTPs. When you compare this to data from WWTPs from developed countries, where secondary and tertiary treatments are included, most of the contaminants are reduced, though not eliminated. In some cases, reverse osmosis may be included, and the concentrations of emerging contaminants are significantly reduced to the extent that they are not transferable to crops [50,51,52].
This study demonstrated that treated wastewater contains a substantial amount of resistome profile, which indicates the inefficiency of the WWTP to eliminate ARGs from the municipal wastewater, which is a task that is not readily achievable without the implementation of secondary and tertiary approaches of treatment of wastewater. The WWTP may be effective in the reduction in biological oxygen demand, but it is not able to significantly remove the ARGs. This condition is very worrisome, considering the number of pharmaceutical factories that empty their wastewater into the WWTP. As presented in our earlier works [9], pharmaceutical wastewater is a potential reservoir of resistomes, which may harbor resistance genes with possible risk to public health. Also, in a recent study, treated sewage effluent on a waterway served as a concentrated source of ARGs and MGEs, which appear to collect in the sediments [53]. This situation is complicated with the discharge of untreated wastewater from both municipal and industrial sources without prior treatment, which appears to be the common practice in most developing countries, including Nigeria. These practices have made treatment of infectious diseases very challenging with the proliferation of ARGs in water bodies and other environmental compartments. These ARGs in question are targeted mostly against the most commonly used ARGs in treatment of clinical infections.

5. Conclusions

This study shows that WWTPs are most likely to play a major part in the dissemination of ARGs and MGEs. We have also shown that WWTPs are major reservoirs of diverse ARGs and MGEs in the various compartments of WWTPs and RW. The WWTP under study was not able to significantly remove the resistomes present in the wastewater, and that is why some ended up in the receiving river in the downstream and upstream of the river. This applies to most of the resistomes screened for different antibiotics. This raises a question about the functionality of the WWTP. This inefficiency in function of the WWTP can be associated to inefficiency in energy generation to run the impellers for aeration of the pond. This is a very limiting factor in developing countries. The PCR assays of the metagenomic DNA in this study were very sensitive and provided rapid results in comparison to genomic DNA from bacterial isolates previously used [9] but not without its limitations. Other similar studies from many laboratories have used different PCR techniques, and our future work will include the use of quantitative PCR, HT-qPCR, and ddPCR for proper qualitative and quantitative ARG analyses. In order to have improved removal of resistomes from the WWTP, the treatment of wastewater should include both secondary and tertiary treatments to reduce the environmental contamination of ARGs and MGEs from municipal wastewater. The government should make the monitoring of activities of industrialists and municipal wastewater a priority.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/w17060867/s1: Figure S1: Map of Nigeria highlighting the study areas: Ogun and Lagos States; Figure S2: A section of the map of Nigeria showing Agbara Industrial Area in Ogun State; Table S1: Description of sites and sources of metagenomic DNA of wastewater treatment plant; Table S2: Raw Data for the prevalence of ARGs and MGEs in the wastewater compartment and receiving river; Table S3: Primers and conditions used to amplify tetracycline resistance genes by the PCR techniques; Table S4: Primers and conditions used to amplify aminoglycosides resistance genes by PCR techniques; Table S5: Primers and conditions used to amplify β-Lactam resistance genes by PCR techniques. Table S6: Primers and conditions used to amplify sulphonamide and chloramphenicol resistance genes by PCR technique. Table S7: Primers and conditions used to amplify some genetic elements by PCR technique. References [54,55,56,57,58,59,60,61,62,63,64] are citied in the Supplementary Materials.

Author Contributions

Conceptualization, A.M.I.; Methodology, A.O. and A.M.I.; Formal analysis, A.O. and C.E.; Resources, A.O. and A.M.I.; Data curation, C.E.; Writing–original draft, A.O.; Writing–review & editing, A.O. and A.M.I.; Visualization, C.E.; Supervision, A.M.I. All authors have read and agreed to the published version of the manuscript.

Funding

This work was sponsored by the Organization for Women in Science for the Developing World (OWSD) and The State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Science.

Data Availability Statement

The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 17 00867 g001
Figure 1. Percent prevalence of antibiotics resistance genes and Intl1 detected from a wastewater treatment plant and receiving river water.
Figure 1. Percent prevalence of antibiotics resistance genes and Intl1 detected from a wastewater treatment plant and receiving river water.
Water 17 00867 g001
Table 1. Distribution of selected antibiotics resistance genes from WWTP and receiving river water.
Table 1. Distribution of selected antibiotics resistance genes from WWTP and receiving river water.
Gene Function/Mechanism of Drug ResistanceResistance GenesWWTP and Receiving River WaterTotal No of Points with Positive Genes
UTWTWWRWURWD
Effluxtet(A)++++4
tet(B)−−+−1
tet(C)++−−2
tet(D)−+++3
tet(E)−−+−1
tet(G)++++4
tet(30)+−−−1
tet(J)+−−−1
Ribosomal protectiontet(M)+−−−1
tet(O)++−−2
tet(Q)+−−−1
tet(BP)++−+3
Enzymatic modificationtet(X)++−−2
Acetyl-transferasesaac(3)-II−−−−0
aac(3)-IV+−−−1
aacA4−−−−
Nucleotidyl-transferasesaadA++−−
aadB−−−+
aadE+−−−1
Phosphor-transferasesstrA++−−2
strB++−−2
BlablaTEM−−−−0
blaOXA++−−2
blaCTX-M−−−−0
blaIMP−−−−0
Chloramphenicol acetyl-transferasecatA1++++4
cmlA++−−2
Dihydropteroate synthaseSulI++++4
SulII++++4
IntegraseIntl1++−−2
Intl2−−−−0
Notes: UTW—Untreated wastewater; TWW—treated wastewater; RWU—river water upstream; and RWD—river water downstream. + means a positive result; − means a negative result.
Table 2. ARGs and MGEs from TWW and receiving river water.
Table 2. ARGs and MGEs from TWW and receiving river water.
Pharmaceutical FacilitiesUTWTWWRWURWD
Tetracycline Resistance Genestet(A), tet(BP), tet(C), tet(G), tet(J), tet(M), tet(O), tet(Q), tet(X), tet(30)tet(A), tet(BP), tet(C), tet(D), tet(G), tet(O), tet(X)tet(A), tet(B), tet(D), tet(E), tet(G)tet(A), tet(BP), tet(D), tet(G)
Aminoglycoside Resistance GenesaadA, aadE, aac(3)-IV, strA, strBaadA, strA, strB-aadB
β-Lactams Resistance GenesblaOXAblaOXA--
Phenicol Resistance GenescatA1, cmlAcatA1, cmlAcatA1catA1
Sulphonamide Resistance GenessulI, sulIIsulI, sulIIsulI, sulIIsulI, sulII
Mobile Genetic ElementsIntl1Intl1--
Notes: UTW—Untreated wastewater; TWW—treated wastewater; RWU—river water upstream; and RWD—river water downstream.
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Obayiuwana, A.; Ibekwe, A.M.; Eze, C. Resistome Profile of Treated Wastewater Using Metagenomic Approach. Water 2025, 17, 867. https://doi.org/10.3390/w17060867

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Obayiuwana A, Ibekwe AM, Eze C. Resistome Profile of Treated Wastewater Using Metagenomic Approach. Water. 2025; 17(6):867. https://doi.org/10.3390/w17060867

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Obayiuwana, Amarachukwu, Abasiofiok M. Ibekwe, and Chinelo Eze. 2025. "Resistome Profile of Treated Wastewater Using Metagenomic Approach" Water 17, no. 6: 867. https://doi.org/10.3390/w17060867

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Obayiuwana, A., Ibekwe, A. M., & Eze, C. (2025). Resistome Profile of Treated Wastewater Using Metagenomic Approach. Water, 17(6), 867. https://doi.org/10.3390/w17060867

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