Reviewing the Phenomenon of Antimicrobial Resistance in Hospital and Municipal Wastewaters: The Crisis, the Challenges and Mitigation Methods

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The silent threat from the release of antimicrobial resistant genes into the environment from hospital and municipal sources, which is going unnoticed, and the existing techniques for remediation of ARG polluted waters have been highlighted. Additionally, prospective nano-based applications have been suggested.

Abstract

Antibiotic resistance is a major crisis that the modern world is confronting. This review highlights the abundance of different types of antibiotic resistance genes (ARGs) in two major reservoirs in the environment, namely hospital and municipal wastewater, which is an unforeseen threat to human lives across the globe. The review helps understand the current state of affairs and the whereabouts on the dissemination of ARGs in both these environments. The various traditional wastewater treatment methods, such as chlorination and UV treatment, and modern methods, such as electrochemical oxidation, are discussed, and the gaps in these technologies are highlighted. The need for the development of newer techniques for wastewater treatment with enhanced efficiency is urgently underscored. Nanomaterial applications for ARG removal were observed to be less explored. This has been discussed, and prospective nanomaterials and nanocomposites for these applications are proposed.

1. Introduction

Antimicrobial resistance (AMR) is burgeoning as one of the critical medical emergencies that is estimated to claim 10 million people per year by 2050 [1] and thus it is feared that the AMR death toll will surpass the death toll caused by other dreadful diseases such as tuberculosis and cancer. This serious human health threat needs to be addressed with a coordinated global action plan. AMR in human pathogens is principally caused by the usage of irrelevant antisbiotics with suboptimal dosages and inadequate/prolonged antibiotic usage. Pervasive practices such as self-medication and off-label prescription of antibiotics are also considered as additional causes for antibiotic resistance. The uncontrolled usage of antibiotics as feed additives in animal husbandry in developing and under-developed countries is yet another cause of AMR [2]. Therefore, maximum surveillance and the generation of adequate AMR data in high-probability sources such as hospital and municipal wastewater becomes necessary.

Across the globe, there are various environments from which AMR pathogens/genes arise and become promulgated into the ecosystem. Of these, the most important sources include municipal waste, hospitals wastes, animal farming and pharmaceutical wastes [3,4,5,6] (Figure 1). Municipal wastewaters (MWW) are one of the largest ecological niches with a very complex microbial composition due to their rich nutritional status. The constant release of antimicrobial compounds, heavy metals and other organic molecules into municipal waste facilitates the evolution of microbes with resistance against various antibiotics. This has led to municipal wastewaters becoming reservoirs of microbes with AMR through their corresponding AMR genes (ARGs) [7]. Across the globe, many municipal wastewater systems have been studied for AMR microbes and their ARGs [8,9,10,11,12,13,14,15,16,17].

Apart from municipal waste, hospital wastewaters (HWW) are also a rich reservoir of AMR microbes and ARGs. It is presumed that the highly frequent and irrational usage of a wide variety of antibiotics in the hospital environments led to a release of microbial AMR and ARGs in hospital waste. Various studies were conducted to explore the patterns of antibiotic resistance in microbial populations and their corresponding genes in hospital wastewaters across the globe [18,19,20,21,22]. Considering the high probability of dissemination of AMR microbes and ARGs from hospital and municipal wastewater systems to various environmental niches, it is vitally important to understand the complex antibiotic resistance profiles of municipal and hospital waste that pose a serious threat from the one health perspective. Figure 2 gives an overview of the various ARGs reported from MWW and HWW sources.

The present review focuses on the status quo of antibiotic resistance of the highly probable contaminant sources like hospital and municipal wastewaters. The disinfecting methods to check the spread of AMR pathogens and their genes have also been compiled and presented.

2. Methods for AMR Detection

Recently, analytical techniques related to AMR detection have been elaborately reviewed [23]. In brief, traditionally, AMR in bacteria and fungi has been detected using culture-based techniques such as broth dilution assay [24,25], agar dilution assay [25,26], disk diffusion tests and E-strip tests [27,28,29]. The minimum inhibitory concentration (MIC) of antibiotics is generally used to measure the drug susceptibility of microbial isolates, which also provides information on AMR. Despite their accuracy, these methods are limited only to cultivable bacteria, which represent less than 3% of the environmental microbiome. In addition, these methods are highly time-consuming and require skilled workers [30]. To compensate for the shortcomings of the culture-based AMR detection techniques, molecular methods such as polymerase chain reaction (PCR) and microarrays came into play for AMR assessment in microbes. In PCR-based AMR detection, the presence of AMR genes has been detected by the amplification of AMR genes using specific primers designed to amplify AMR genes [31,32]. On the other hand, microarray technology detects and quantifies AMR genes and its expression patterns by hybridizing probe DNA/RNA sequences to the complementary target sequences on a microarray plate [33,34]. This method is particularly useful for the multiplexed detection of various ARGs simultaneously [35]. While these technologies have improved the efficiency of AMR detection, these methods still depend on the designed probes and primers, which restrain their detection to only known gene sequences. Conversely, metagenomic sequencing techniques offer more extensive details by directly sequencing the entire genomic content of environmental samples using next-generation sequencing techniques (NGS). Such methods allow for the identification of the entire composition (microbial and ARG) of samples including novel genes and organisms providing a comprehensive understanding of the microbial diversity and AMR [36,37,38,39]. Apart from these techniques, mass spectrometry (MS) techniques such as liquid chromatography–mass spectrometry (LC-MS) [40,41] and gas chromatography-based mass spectrometry (GC-MS) [42,43], Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) [44] has also emerged as vital tools for the detection of AMR. The mass-to-charge ratio of molecules is detected by MS enabling the precise and rapid identification of molecules that take part in the microbial AMR process. These methods have proven to be useful in clinical studies and continue to play an important role in advancing our understanding of AMR [23].

3. Antibiotic Resistance in Hospital and Municipal Wastewater

3.1. Hospital Wastewater

Hospitals are regarded as one of the most important AMR reserves, due to the high and frequent usage of a wide range of antibiotics for the treatment of various diseases that create AMR through selection pressure and horizontal gene transfer between pathogens in hospital wastewater [45]. Similarly, the improperly treated hospital waste and other municipal waste disposed into the sewage also cause multiple drug resistance in microorganisms in municipal wastewater systems.

Hospital wastewater is notorious for their unique ARG profile, due to the usage of a wide variety of antibiotics including Colistin, a last-resort antibiotic [46]. Hence, studies deciphering various patterns of the ARG profile have become a global requirement. Several studies on the antibiotic resistance of hospital and municipal wastewaters have been conducted [20,47,48,49,50,51,52,53,54,55,56]. The employment of molecular methods to detect AMR began as early as 2004 while real-time PCR (RT PCR) was used to detect AMR genes, namely VanA of Enterococci, ampC of Enterobacter and mecA of Staphylococci in various municipal and clinical wastewater samples by Volkmann et al. [57]. This study also revealed that mecA was found to be prevalent in clinical wastewater samples; however, in municipal water, ampC (~80%) was the most dominant. Moges et al. [47] reported the abundance of multidrug-resistant (MDR) bacterial isolates such as Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli in hospital wastewater samples (>80%) when compared with non-hospital wastewater samples (~45%).

A similar study was conducted to compare the AMR strains in hospital wastewater and water from the urban Clinton River for the presence of three groups of extended spectrum beta-lactamases (ESBLs) including bla_SHV, bla_TEM and bla_CTX-M using the RTPCR technique [51]. The authors assessed the AMR of 156 isolates, out of which 34 isolates belonged to the ESBL category. It was found that the concentration of the bla_TEM gene was 100-fold higher in HWW than river water. In addition, the microbial isolates were reported to exhibit resistance against other antibiotics such as cefotaxime (40–65%), chloramphenicol (25–40%), tetracycline (30–40%), ciprofloaxin (25–65%) and gentamicin (30–50%). HWW analysis by quantitative PCR (qPCR) in a Romanian hospital revealed 14 different ARGs conferring resistance to aminoglycosides, chloramphenicol, tetracycline, beta-lactam and sulfonamides; among those ARGs, sulI (sulfonamide resistance) was the most abundant ARG [21]. Yet another study using qPCR technique was employed for the analysis of ARG composition in a primary, secondary and tertiary hospital in East China [58] which showed quinolone-resistant ARGs (qnrA), and beta-lactam-resistant ARGs (bla_GES-1, bla_GES-10 and bla_TEM) to be the prevalent ARGs. Lien et al. [59] assessed the antibiotic susceptibility of HWW samples from both rural and urban hospital settings through disk diffusion and E-strip tests. Genes resistant against ceftriaxone, ceftazidime, gentamicin, amoxicillin/clavulanic acid, ciprofloxacin, fosfomycin and MDR were observed in both rural and urban hospital wastewater in different concentrations. For example, 49% of bacterial isolates were found to have ceftriaxone resistance and 40% were resistant towards antibiotics such as ceftazidime, gentamicin and amoxicillin/clavulanic acid in rural HWW. However, the Co-trimoxazole and ceftriaxone resistant genes were carried by 71% and 39% of isolates in urban HWWs, respectively. The study also revealed that the most common ARGs found were ESBL genes such as blaCTX-M and blaTEM and the most common quinolone resistance gene found was qepA.

Shotgun metagenomics is the advanced high-throughput DNA sequencing tool that has been scarcely used to study antibiotic resistomes from various environmental samples. Petrovich et al. [60] characterized the antibiotic resistome of a hospital wastewater using the shotgun metagenomic sequencing method. The HWW sewage effluent was mainly composed of resistance genes against aminoglycoside (AAC(6′) lb9, aadA11), macrolide (mefA, mel), cephalosporin and penem (GES-5). Shotgun metagenomics was extended by Manoharan et al. [61] to study the AMR genes in the HWW treatment plants at a medical center in Daegu, South Korea, and the samples were reported to contain AMR genes corresponding to MDR (>53%), MLS (9%), bacitracin (4.4%), tetracycline (3.4%) and beta-lactam resistance (3.3%) genes in hospital wastewater. Guo et al. [62] analyzed the AMR genes present in untreated wastewater samples of three different hospitals affiliated with Southwest Medical University in China, namely a general hospital, a TCM hospital and a stomatology hospital using shotgun metagenomic sequencing methods. The resistome was observed to differ with respect to the type of hospital. Tetracycline-, penicillin-, tobramycin- and sisomycin-resistant genes were highly abundant in wastewaters from general hospitals, whereas tetracycline, marcolide and bacitracin resistance genes dominated the TCM hospital wastewater. The most prevalent ARGs found in the stomatology hospital wastewater were bacitracin, tetracycline, sulfonamide and cephalosporin resistance genes. A similar investigation by Ma et al. [63] in wastewater samples of a general and an eye hospital in Zhejiang, China also revealed that there was a certain difference in the composition of AMR genes between general and eye hospital samples. While tet39 (tetracycline resistance gene) was the most abundant ARG in general hospital wastewater, sulI (sulfonamide) was found to be more abundant in eye hospital wastewater. Wang et al. [64] also used shotgun metagenomics to assess the AMR profile in HWW in Jedda, Saudi Arabia, after the COVID-19 pandemic. It revealed the prevalence of carbapenems, macrolides, tetracycline and sulfonamides resistance genes in those samples. The most abundant ARGs were found to be msrE, sul2 and tet 36. Shotgun metagenomic analysis of wastewater samples from six Indian hospitals (general) exhibited ARGs against carbapenem, trimethoprim, and sulfonamide antibiotics in all the samples. In addition, for the first time, a mobile colistin resistance gene, mcr-5.1, was also detected in those wastewater samples [48]. More recently, dental hospital wastewater in China was also explored for the ARG profile by shotgun metagenomics [65]. The study claimed that a total number of 1208 types of ARGs, belonging to 29 antibiotic types/subtypes, were present in the samples.

3.2. Regional Variation of ARGs in HWWs

The diversity of ARGs in HWW varies significantly across the globe. The variation in ARGs depends on the antibiotic usage patterns and the effectiveness of WWT in that region. Based on the research conducted on the number of AMR gens from the wastewater samples in Asia (China, India and Iran) [11,48,62,63,65], Europe (Spain and Sweeden) [22,66,67] and South America (Brazil) [22,68], the developing countries such as China, India and Brazil showed the highest AMR gene copy numbers (10⁵–10⁷ ARG copies/mL) in HWW samples [22,48,63,65]. While the HWW samples from Spain and Iran exhibited moderate AMR genes (10³–10⁵copies/mL) [11,22]. The ARGs survey conducted in Sweden HWW showed the least (10¹–10³ copies) among the above countries [67]. Of these countries, studies carried out in the Chinese cities of Zheijang, Shantou and Linan have reported the predominance of bla_OXA, aac(6′)-lb-cr, sul1, sul2, tetW and tetA carried by pathogens such as E. coli, P. aeruginosa, Aeromonas sp. and Acinetobacter sp. across HWW samples [62,63,65]. On the other hand, ARGs such as blaNDM-1, sul1, sul2 and mcr-5.1 were found to be the most prevalent ARGs in Indian hospitals. K. pneumoniae and E. coli were two of the prominent hosts of ARGs in Indian HWWs [48]. Similarly, HWWs of Brazil are primarily composed of ARGs such as bla_KPC and bla_TEM carried by K. pneumoniae, P. aeruginosa and Enterobacter sp. Apart from resistance towards a single antibiotic, these regions comprise the highest relative number of MDR genes and MDR pathogens. It was found that the prominent ARGs in Iranian HWW were bla_KPC, qnrS2, tetW and bla_TEM; most of these ARGs were associated with E. coli and P. aeruginosa. In addition, MDR genes found in Proteus mirabilis and Klebsiella pneumoniae raise concerns about their level of resistance [11]. Whereas in Spain, the HWW resistome is dominated by fluoroquinolone resistance genes such as qnrS2 carried by Aeromonas hydrophila and Klebsiella pneumoniae. Even though these regions are not as critical as in China or India, there is a need for continuous monitoring of ARGs, to prevent ARG spikes in the environment. Lastly, the study by Hutinel et al. [67] revealed ARGs (the most predominant such as mcr-1 and optrA) with concentration ranges between 10¹ and 10²copies/mL in HWW. It was identified that pathogens such as Enterococcus species and Staphylococcus species were the major carriers of such ARGs.

3.3. Municipal Wastewater

One important challenge concerning hospital wastewater is that in most of the developing and developed countries, HWW are discharged into MWW, making it complex (i.e., AMR and its resistome) and difficult to treat [69,70]. In addition, the waste effluents from various sources, such as pharmaceutical industries, animal farms, dairy and poultry farms, are being discharged in municipal sewage and generate resistomes with a unique composition in MWW. The inefficient removal of ARGs by the wastewater treatment plants (WWTPs) results in the presence of significant amounts ARGs and their respective host bacteria in the reclaimed water, which might spread to fresh water bodies, food crops and plants along with the other effluents of WWTPs that might enter into the human and animal food chain and ultimately affect human life. Hence, WWTPs are regarded as vital resources of ARGs.

Table 1 and Table 2 depict the resistome of various municipal and hospital wastewater samples from different regions, respectively. The data shows that tetracycline resistance was the most frequently detected AMR in HWW and was followed by the aminoglycoside, sulfonamide and macrolide resistance. The resistome of MWW also comprised higher concentrations of tetracycline resistance genes followed by ciprofloxacin resistance genes. The ARG profile of the HWW was found to be more diverse comprising resistance genes against aminoglycosides, beta-lactam and even last-resort antibiotics such as colistin. The AMR in HWW and MWW is presumed to have resulted owing to extensive usage of a variety of antibiotics [71]. However, in contrast to the study of Zhang et al. [15] that showed the abundance of tetracycline and macrolides resistance in MWW, the resistance against disinfectants wex-cide-128 was found to be higher in HWW.

In 2010, it was reported that China was the second largest consumer of antibiotics. According to Wang et al. [72], 65% of prescriptions from primary healthcare units across China was included with antibiotics for any kind of disease treatment. Such exorbitant usage of antibiotics might have resulted in the development of the complex resistome in MWW, making it essential for the surveillance of ARGs in MWW [73]. In this context, Chen and Zhang. [74] detected the presence of ARGs in four MWW systems along with eight rural domestic wastewater systems in eastern China using qPCR. It was revealed that tetracycline resistance genes such as tetM, tetO, tetQ and tetW and sulfonamide resistance genes such as sulI and sulII were predominant in both MWW samples and rural domestic wastewater samples. A similar study carried out by Mao et al. [13] in two activated sludge WWTPs in northern China and used qPCR/RT PCR to reveal the presence of ARGs that confer resistance towards tetracycline (tetA, tetB, tetE, tetG, tetH, tetS, tetT, tetX), sulfonamides (sulI, sulII), quinolones (qnrB) and macrolides (ermC). Fluorescent RT-PCR-based surveillance was further extended to detect the presence of ARGs in sewage wastewater treatment plants in Shanzhan, Southern China [75]. The ARGs such as tetA, tetB, tetC, tetX, tetD, tetQ, and tetW (tetracycline resistance genes) and sul I, sul II and sul III (sulfonamide resistance genes) were found to be predominant in the influents of WWTP in which the most abundant ARGs were sulfonamide resistance genes (sulI (10−5.44–10−4.38)) and tetracycline resistant genes (tet C (10−5.68)).

Further, RT PCR was employed by Munir et al. [56] to quantify the ARGs from five wastewater treatment plants in Michigan, USA. The ARG profile of wastewater influents across all WWTPs were found to be predominantly composed of ARGs such as sulI (sulfonamide resistance), tetO and tetW (tetracycline resistance). Similarly, Parnanen et al. [17] conducted an analysis for the detection of ARGs in urban wastewater treatment plants (UWWTP) across various European countries including, Portugal, Spain, Ireland, Cyprus, Germany, Finland, and Norway using a highly parallel quantitative polymerase chain reaction. The authors reported the presence of ARGs such as aadA (Aminoglycoside), cmxA (chloramphenicol), blaoxa (beta-lactam), qacE∆11 (MDR), ermF (Marcolide) and tetQ (tetracycline) genes, which were commonly found in higher abundances across the samples. Likewise, Makowska et al. [76] detected the presence of methicillin resistance genes in 29 S. aureus strains (out of 79) and vancomycin resistance VanA gene in 11 (out of 101) strains of Enterococcus species isolated from an urban MWW treatment plant in Kozeigowy, Poland.

Metagenomic sequencing has been employed for the characterization of the ARG profile of various MWWTPs across China, Singapore and South Korea [52,53,54,55,77]. Using the metagenomic sequencing method, ARGs were analyzed from 116 wastewater samples collected from 32 WWTPs in 17 cities of China including Beijing, Hong Kong, Hangzhou and Lasa. The results showed that out of the 128 ARGs detected across 80% of samples, the ARGs against aminoglycosides, MLS, tetracycline and beta-lactam were the most predominant ones [77]. Ng et al. [52] investigated the presence of ARGs in a full-scale MWW treatment plant in Singapore using metagenomic sequencing methods. The researchers found that the MWW effluent was composed of ARGs resistant towards aminoglycoside [aadA, aph(6)-I, aph(3′)-I, aac(6′)-I, aac(6′)-II, ant(2″)-I], beta-lactams [class A, class C, class D beta-lactamases (bla_OXA)], chloramphenicol (acetyltransferase, exporters, floR, cmIA), fosmidomycin (rosAB), macrolide-lincosamide-streptogramin (macAB, ereA, ermFB), multidrug resistance (subunits of transporters), polymyxin (arnA), quinolone (qnrS), rifamycin (arr), sulfonamide (sul1, sul2) and tetracycline (tetM, tetG, tetE, tet36, tet39, tetR, tet43, tetQ, tetX).

Table 1. Consolidated list of ARGs reported in MWW.

S. No	Samples	Concentrations of ARGs	Resistance against Antibiotics	ARGs	References
1	MWW	3.35 × 107 CFU/100 mL of ARB	Gentamicin, chloramphenicol and ceftidazime	AAC(3)-1, cmlA1, ctx-m-32	[11]
2	MWW	Sulfonamide: 9.73 × 1010 copies/kizumL Tetracycline 1.81 × 1011 copies/mL	Sulfonamide and tetracycline	SulI, sulII, tet A. tetB, tetC, tetG, tetL, tetM, tetO, tetW and tetX	[78]
3	MWW		Beta-lactam and quinolone	qnrA, qnrB, qnrVC, qnrC, qnrS, qnrD, incU, aac(6′)-IB, blaoxa	[66]
3	MWW	-	Sulfonamide, aminoglycoside and marcolide	blaGES, qacE, sul1, mph (E), blaOXA, blaGES and msrE	[79]
4	MWW	Upto 1 × 10−3 copies of ARG /16SrRNA gene	Linezolid, Colisitin, aminoglycoside and sulfonamide	gar, sul4, mcr(1–5) genes	[67]
5	MWW		Tetracycline and marcolide	Tet40, tet37, tetQ, tet36, tetW, tetO, emrB, emrF, ereB, ereA and ermX	[15]
6	MWW	-	Beta-lactam, marcolides, Fluroquinolones, tetracycline and sulfonamides	qnrS, teteW, sul1, blaTEM and ermB	[22]
7	MWW	Tet genes 0.82 × 10−2 to 1.57 × 10−2 level of abundance	Sulfonamides and tetracycline	Sul1, sul2, tetM, tetO, tetQ and tetW	[74]
		Sul genes: 2.82 × 10−3 to 4.76 × 10−2 level of abudance
8	MWW	−8 log copies/16SrRNA gene	Sulfonamides, beta-lactam methicillin, tetracycline and marcolide	mecA	[14]
		−7.35 log copies/16SrRNA gene		Sul2
		−7 log copies/16SrRNA gene		Sul1
		−7 log copies/16SrRNA gene		ermB
		−7.75 log copies/16SrRNA gene		blaTEM
		−7.4 log copies/16SrRNA gene		tetO
		−7.5 log copies/16SrRNA gene		blaCTX-M
9	MWW	-	MDR, tetracycline, aminoglycoside, beta-lactam, MLS and amphenicol	aadA, cmxA, blaoxa, quacEdelta1, ermF and tetQ	[17]
10	MWW	1.106 copies per 16S rRNA gene	Beta-lactam, sufonamide, tetracycline, chloramphenicol and aminoglycoside	blaOXA, sul1, tetM, ermFB, aadA, sul2 and tetX	[52]
11	MWW	Approximately 105 to 109	Tetracycline and sulfonamide	tetO, tetW and sulI	[56]
12	MWW	5.2 × 108 genes/ml	Beta-lactam and carbapenems	BlaCTX-M, blaNDM, blaOXA-48 and blaVIM	[76]
13	MWW	1.59–145.57 ppm	Aminoglycosides, bacitracin, beta-lactams, MDR, tetracycline, sulfonamide and MLS	TetQ, TetW, TetM, aadA, blaOXA and ermF	[55]
14	MWW		MDR, MLS, beta-lactam, tetracycline and aminoglycosides	mexF, mexL and Rm3	[53]

Table 1. Consolidated list of ARGs reported in MWW.

S. No	Samples	Concentrations of ARGs	Resistance against Antibiotics	ARGs	References
1	MWW	3.35 × 107 CFU/100 mL of ARB	Gentamicin, chloramphenicol and ceftidazime	AAC(3)-1, cmlA1, ctx-m-32	[11]
2	MWW	Sulfonamide: 9.73 × 1010 copies/kizumL Tetracycline 1.81 × 1011 copies/mL	Sulfonamide and tetracycline	SulI, sulII, tet A. tetB, tetC, tetG, tetL, tetM, tetO, tetW and tetX	[78]
3	MWW		Beta-lactam and quinolone	qnrA, qnrB, qnrVC, qnrC, qnrS, qnrD, incU, aac(6′)-IB, blaoxa	[66]
3	MWW	-	Sulfonamide, aminoglycoside and marcolide	blaGES, qacE, sul1, mph (E), blaOXA, blaGES and msrE	[79]
4	MWW	Upto 1 × 10−3 copies of ARG /16SrRNA gene	Linezolid, Colisitin, aminoglycoside and sulfonamide	gar, sul4, mcr(1–5) genes	[67]
5	MWW		Tetracycline and marcolide	Tet40, tet37, tetQ, tet36, tetW, tetO, emrB, emrF, ereB, ereA and ermX	[15]
6	MWW	-	Beta-lactam, marcolides, Fluroquinolones, tetracycline and sulfonamides	qnrS, teteW, sul1, blaTEM and ermB	[22]
7	MWW	Tet genes 0.82 × 10−2 to 1.57 × 10−2 level of abundance	Sulfonamides and tetracycline	Sul1, sul2, tetM, tetO, tetQ and tetW	[74]
		Sul genes: 2.82 × 10−3 to 4.76 × 10−2 level of abudance
8	MWW	−8 log copies/16SrRNA gene	Sulfonamides, beta-lactam methicillin, tetracycline and marcolide	mecA	[14]
		−7.35 log copies/16SrRNA gene		Sul2
		−7 log copies/16SrRNA gene		Sul1
		−7 log copies/16SrRNA gene		ermB
		−7.75 log copies/16SrRNA gene		blaTEM
		−7.4 log copies/16SrRNA gene		tetO
		−7.5 log copies/16SrRNA gene		blaCTX-M
9	MWW	-	MDR, tetracycline, aminoglycoside, beta-lactam, MLS and amphenicol	aadA, cmxA, blaoxa, quacEdelta1, ermF and tetQ	[17]
10	MWW	1.106 copies per 16S rRNA gene	Beta-lactam, sufonamide, tetracycline, chloramphenicol and aminoglycoside	blaOXA, sul1, tetM, ermFB, aadA, sul2 and tetX	[52]
11	MWW	Approximately 105 to 109	Tetracycline and sulfonamide	tetO, tetW and sulI	[56]
12	MWW	5.2 × 108 genes/ml	Beta-lactam and carbapenems	BlaCTX-M, blaNDM, blaOXA-48 and blaVIM	[76]
13	MWW	1.59–145.57 ppm	Aminoglycosides, bacitracin, beta-lactams, MDR, tetracycline, sulfonamide and MLS	TetQ, TetW, TetM, aadA, blaOXA and ermF	[55]
14	MWW		MDR, MLS, beta-lactam, tetracycline and aminoglycosides	mexF, mexL and Rm3	[53]

Table 2. Consolidated list of ARGs reported in HWW.

S. No	Samples	Abundances of ARGs	Resistance against Antibiotics	Most Abundant ARGs	References
1	HWW	(2.793 ± 0.55) × 104	Tetracycline, marcolide, suflonamide, quinolones and multidrug resistant	qnrA	[20]
		(4.845 ± 0.71) × 104		tetM
		(3.057 ± 1.075) × 107		tetO
		(3.07 ± 0.94) × 104		ereA
		(1.93 ± 0.33)		oqxB
		(2.73 ± 0.56) × 106		ermB
		(3.81 ± 0.80) × 107.		sulI
		(4.42 ± 0.29) × 106		sulII
		(4.49 ± 0.24) × 104		sulIII
		(3.073 ± 0.34) × 106		tetX
		(2.34 ± 0.97) × 103		qnrD
		1.88		ermA
2	HWW	Mean relative abundances of all ARGs = 1501	Multidrug, aminoglycoside, cephalosporin, macrolide, penam, tetracycline, and fluoroquinolone	blaGES-5, mef A and aac, mel, AAC-A (6)lb9′, aadA11, sulI, tet36, msrE, LCR1 and mexW	[60]
3	HWW		MDR, MLS, bacitacin, tetacycline and beta-lactam		[61]
4	HWW	-	Extended spectrum beta-lactam and ciprofloxacin	qepA blaCTX-M and blaTEM	[59]
5	GHWW		Tetracycline, penicillin, tobramycin and sisomycin	N.Ds	[62]
	THWW		Tetracycline, marcolide and nacitracin
	SHWW		Bacitracin, tetracycline, sulfonamide and cephalosporin
6	HWW	101–103 order	Extended spectrum beta-lactam	blaTEM, blaSHV, and blaCTX-M	[51]
7	HWW	Beta-lactam = 4.87 × 106 copies/mL Quinolone genes 5.38 × 104 copies/mL	Quinolone and beta-lactam resistance	blaOXA-1, blaOXA-10, blaTEM-1, blaDHA-1, blaSHV-1, blaGES-1, qnrA, qnrS, qnrD, and qepA	[58]
8	HWW	100% of E. coli	Beta-lactam, sulfonamide and quinolone	blaSHV	[80]
		50% of E. coli		blaCTX
		100% of E. coli		blaTEM
		60% of E. coli		blaVIM
		100% of E. coli		SulI
		50% of E. coli		QnrS
9	HWW	1.883 ± 0.451 ARG/16S rRNA,	Tetracycline, aminoglycoside, beta-lactam and multidrug	tet 39, aph(3″)-I, sulI, aadA	[63]
	HWW (Eye Hospital)	1.614 ± 0.177 ARG/16S rRNA	Sulfanomide, aminoglycoside, multidrug and beta-lactam	Sul1, aadA, qacEΔ1 and bacA	[63]
10	HWW	-	Aminoglycoside, beta-lactam, sulfonamide, marcolide	aads, ErmF, msrE, Bla-VEB	[48]
11	HWW (Dental)	-	MDR, aminoglycoside, tetracycline and flouroquinolone	tetE, MexE, TEM (84 subtypes), tet 39, tet 41, AAC-3 (2 subtypes), AAC-2, VanB, and dfrC	[65]
12	HWW	-	Beta-lactam, marcolides, tetracycline, aminoglycoside and methicillin	blaTEM, blaKPC, mph(A), mel tetD, tetM, tetA, strB and mecA	[68]
13	HWW	Tetracycline 1.81 × 1011 copies/mL Sulfonamide: 9.73 × 1010 copies/mL	Tetracycline and sulfonamide	SulI, sulII, tet A. tetB, tetC, tetG, tetL, tetM, tetO, tetW and tetX	[78]
14	HWW MWW	-	Beta-lactam, marcolides, Fluroquinolones, tetracycline and sulfonamides	qnrS, teteW, sul1, blaTEM and ermB	[22]
15	HWW	HWW 4.19 × 107 CFU/100 mL of ARB	Gentamicin, chloramphenicol and ceftidazime	AAC(3)-1, cmlA1, ctx-m-32	[11]
16	HWW		Quinolone resistance and beta-lactamases	qnrA, qnrB, qnrVC, qnrC, qnrS, qnrD, incU, aac(6′)-IB, blaoxa	[66]
17	HWW	Upto 1 × 10−3 copies of ARG/16SrRNA gene	Linezolid, Colisitin, and sulfonamide	gar, sul4 and mcr(1–5) genes	[67]

Table 2. Consolidated list of ARGs reported in HWW.

S. No	Samples	Abundances of ARGs	Resistance against Antibiotics	Most Abundant ARGs	References
1	HWW	(2.793 ± 0.55) × 104	Tetracycline, marcolide, suflonamide, quinolones and multidrug resistant	qnrA	[20]
		(4.845 ± 0.71) × 104		tetM
		(3.057 ± 1.075) × 107		tetO
		(3.07 ± 0.94) × 104		ereA
		(1.93 ± 0.33)		oqxB
		(2.73 ± 0.56) × 106		ermB
		(3.81 ± 0.80) × 107.		sulI
		(4.42 ± 0.29) × 106		sulII
		(4.49 ± 0.24) × 104		sulIII
		(3.073 ± 0.34) × 106		tetX
		(2.34 ± 0.97) × 103		qnrD
		1.88		ermA
2	HWW	Mean relative abundances of all ARGs = 1501	Multidrug, aminoglycoside, cephalosporin, macrolide, penam, tetracycline, and fluoroquinolone	blaGES-5, mef A and aac, mel, AAC-A (6)lb9′, aadA11, sulI, tet36, msrE, LCR1 and mexW	[60]
3	HWW		MDR, MLS, bacitacin, tetacycline and beta-lactam		[61]
4	HWW	-	Extended spectrum beta-lactam and ciprofloxacin	qepA blaCTX-M and blaTEM	[59]
5	GHWW		Tetracycline, penicillin, tobramycin and sisomycin	N.Ds	[62]
	THWW		Tetracycline, marcolide and nacitracin
	SHWW		Bacitracin, tetracycline, sulfonamide and cephalosporin
6	HWW	101–103 order	Extended spectrum beta-lactam	blaTEM, blaSHV, and blaCTX-M	[51]
7	HWW	Beta-lactam = 4.87 × 106 copies/mL Quinolone genes 5.38 × 104 copies/mL	Quinolone and beta-lactam resistance	blaOXA-1, blaOXA-10, blaTEM-1, blaDHA-1, blaSHV-1, blaGES-1, qnrA, qnrS, qnrD, and qepA	[58]
8	HWW	100% of E. coli	Beta-lactam, sulfonamide and quinolone	blaSHV	[80]
		50% of E. coli		blaCTX
		100% of E. coli		blaTEM
		60% of E. coli		blaVIM
		100% of E. coli		SulI
		50% of E. coli		QnrS
9	HWW	1.883 ± 0.451 ARG/16S rRNA,	Tetracycline, aminoglycoside, beta-lactam and multidrug	tet 39, aph(3″)-I, sulI, aadA	[63]
	HWW (Eye Hospital)	1.614 ± 0.177 ARG/16S rRNA	Sulfanomide, aminoglycoside, multidrug and beta-lactam	Sul1, aadA, qacEΔ1 and bacA	[63]
10	HWW	-	Aminoglycoside, beta-lactam, sulfonamide, marcolide	aads, ErmF, msrE, Bla-VEB	[48]
11	HWW (Dental)	-	MDR, aminoglycoside, tetracycline and flouroquinolone	tetE, MexE, TEM (84 subtypes), tet 39, tet 41, AAC-3 (2 subtypes), AAC-2, VanB, and dfrC	[65]
12	HWW	-	Beta-lactam, marcolides, tetracycline, aminoglycoside and methicillin	blaTEM, blaKPC, mph(A), mel tetD, tetM, tetA, strB and mecA	[68]
13	HWW	Tetracycline 1.81 × 1011 copies/mL Sulfonamide: 9.73 × 1010 copies/mL	Tetracycline and sulfonamide	SulI, sulII, tet A. tetB, tetC, tetG, tetL, tetM, tetO, tetW and tetX	[78]
14	HWW MWW	-	Beta-lactam, marcolides, Fluroquinolones, tetracycline and sulfonamides	qnrS, teteW, sul1, blaTEM and ermB	[22]
15	HWW	HWW 4.19 × 107 CFU/100 mL of ARB	Gentamicin, chloramphenicol and ceftidazime	AAC(3)-1, cmlA1, ctx-m-32	[11]
16	HWW		Quinolone resistance and beta-lactamases	qnrA, qnrB, qnrVC, qnrC, qnrS, qnrD, incU, aac(6′)-IB, blaoxa	[66]
17	HWW	Upto 1 × 10−3 copies of ARG/16SrRNA gene	Linezolid, Colisitin, and sulfonamide	gar, sul4 and mcr(1–5) genes	[67]

Further, Gupta et al. [53] explored the resistome of WWTP situated in Gwangju city, South Korea. It was revealed that the effluent was primarily composed of MDR genes and ARGs towards MLS, beta-lactam, tetracycline and aminoglycosides. The MDR genes mexF and mexI and beta-lactam resistance gene Rm3 were found to be the most abundant ARGs in the MWW influent. However, Yoo and Lee. [55] detected the presence of ARGs in MWW samples collected from a WWTP in another South Korean city, Busan. The results unveiled the incidence of genes resistant against aminoglycosides, bacitracin, beta-lactam, sulfonamide, tetracycline and MLS in which the most abundant ARGs detected were TetQ, TetW, TetM (tetracycline resistant), aadA (aminoglycoside resistant), blaOXA (beta-lactam resistant) and ermF (macrolide resistant). More recently, a similar study was carried out to elucidate the ARG profile of a MWW treatment plant in Seoul, South Korea, using nanopore sequencing based metagenomic approach that exhibited that the ARGs sul1 (sulfonimine resistance), aadA1, aadA2 and aadA13 (aminoglycoside resistance genes) were the most predominant ARGs [54]. Thus, the AMR profile of either HWW or MWW has been extensively investigated for the presence of either assessing the antibiotic resistance of pathogenic cultures or their ARGs. In addition to that, there are few other studies that exhibited the difference in the AMR pattern, (ARG compositions) in both MWW and HWW.

Verela et al. [66] conducted a study isolating 112 (quinolone-resistant) nalidixic acid-resistant (NA-r) Aeromonas strains from hospital effluent, raw inflow and treated effluent urban WWTP in Portugal. The NA-r strains were also checked for their resistance against other antibiotic such as ciprofloxacin, amoxicillin, ticarcillin, cephalothin, ceftazidime, streptomycin, sulfamethoxazole/trimethoprim, tetracycline, gentamicin colistin sulfate and meropenem using the agar diffusion method. Further, the antibiotic resistance-based grouping through screening for the presence of AMR genes such as quinolone resistance genes (qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC, qepA, oqxAB, aac(6′)-Ib-cr, bla_OXA, incU) and beta-lactam resistance genes (blaOXA-1-4, bla_OXA-7, bla_OXA-10-11, bla_OXA-13-17, bla_OXA-19, bla_OXA-21, bla_OXA-28, bla_OXA-30-32 and bla_OXA-34-35) in those isolates was performed using PCR amplification. It was found that the quinolone and the beta-lactamase resistance genes aac(6′)-Ib-cr and bla_OXA, including gene bla_OXA-101, were prevalently detected in urban WWTP wastewater.

Rodriguez-Mozaz et al. [22] investigated the presence of ARGs in HWW and urban MWW of Girona, Spain, using an RT-PCR assay. ARGs such as qnrS, tetW, sul1, bla_TEM and ermB conferring resistance against antibiotics such as quinolones, tetracycline, sulfonamides, beta-lactam and macrolides were detected in significant amounts in both HWW and urban MWW. Similarly, qPCR was employed to elucidate and compare the composition of ARGs from a WWTP present in six hospitals and three rivers in Kathmandu Valley, Nepal. The results revealed that the resistomes of all sample sources were composed of ARGs qnrS (quinolone resistance), bla_NDM-1 (beta-lactam resistant), sul1 (sulfonamide resistant), bla_CTX-M (beta-lactam resistant) and tetB (tetracycline resistant) [19]. It was reported that the ARG patterns observed in river water and WWTPs were almost similar. The river water samples reported to contain the sulI gene and bla_NDM-1 gene as the most and least abundant ARGs, whereas in the HWW, the carbapenem resistant bla_CTX-M was found to be the most predominant in followed by sulI, bla_NDM-1, qnrS and tetB.

Hutinel et al. [67] further outstretched qPCR for the detection of ARGs in HWWs and MWWs in Gothenburg, Sweden for a period of five years. The authors stated that the following ARGs, namely mcr-1, mcr-3, mcr-4, mcr-5, sul4 and gar, were prevalent across all samples, whereas the genes optrA and cfr(A) were detected especially in HWW. In addition, the most abundant genes in both HWW and MWW were found to be mcr-3 and mcr-5, which were relatively more abundant in MWW.

3.4. Regional Variation of ARGs in MWW

The AMR resistome of various MWW samples have been assessed in Asian countries (India, China, Singapore and South Korea), European countries (Poland, Portugal, Sweden, Spain, Ireland, Germany, Cyprus, Finland and Norway) and North American countries (Canada and USA). Based on these studies, the MWW of developing countries such as India, China and Iran and developed countries such as Portugal and Poland displayed large quantities of ARGs ranging from 10² to 10¹⁰ copies/mL [11,15,66,76,78], whereas the MWW samples from countries like Japan, South Korea, Singapore and Sweden [52,55,67,79] showed a moderate concentration of ARGs (from 10³–10⁶ copies/mL). The lowest amounts of ARGs (ranging from 10¹–10³ copies/mL) were detected in MWW samples tested from Canada and Northern European countries like Spain, Ireland, Germany, Cyprus, Finland and Norway [14,17].

Of these countries, the Indian MWW samples primarily comprises genes resistant towards antibiotics such as tetracycline (tetA, tetB), macrolide-lincosamide-streptogamin(MLS) (ermB, ereA) and beta-lactams (bla_OXA and bla_TEM) and multidrug (mexA) in ARG carriers such as P. aeruginosa, E. coli, and Acinetobacter species [15]. Conversely, in Chinese MWW samples, the vancomycin (VanA, VanB), sulfonamide (sul1, sul2) and beta-lactams resistant genes were more prevalent in MWW carrier organisms like S. aureus, K. pneuominae and Enterococcus species [74,78]. ARGs such as aac(6′)-Ib-cr, qnrS2, (quinolone resistance) bla_TEM (beta-lactam), ermB(macrolide) and sulI (sulfonamide) were documented to be predominant in urban cities in Portugal [66]. On the other hand, in Iran, the most abundant ARGs were Gentamicin resistance (aac(3)-I), Chloramphenicol resistance (cmlA1) and Ceftazidime resistance (ctx-m-32) genes [11]. In Poland, the MWW samples were reported to contain antibiotic resistant organisms such as Methicillin-resistant Staphylococcus aureus and Vancomycin-resistant Enterococci species [76]. The ARGs such as optrA, mcr, cfr, sul4, and gar were most prevalent in Swedish MWWs, where S. aureus, Enterococcus species and E. coli are the major ARG carriers [67]. In addition, the Japanese MWW samples were found to contain a vast quantity of sul1, qacE, and blaCTX-M, which confer resistance against sulfonamides, beta-lactam and multidrug, which are the most predominant ARGs [79]. ARGs such as bla_TEM, bla_OXA (beta-lactam resistance) and tetM (tetracycline resistance) were the most abundant ARGs in MWW samples of South Korea and Singapore [52,55]. Lastly, Canada and northern European countries like Spain, Ireland, Germany, Cyprus, Finland and Norway exhibited relatively lower levels of aadA, mecA, sul1, ermB, tetC, cmxA, bla_oxa, quacEdelta1, ermF and tetQ, where the most abundant ARGs were carried by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecium, and Streptococcus spp in MWW samples [17].

4. Mitigation Methods for Disinfecting AMR Pathogens and Genes in Wastewater Treatment

In general, wastewater treatment (WWT) involves the separation of heavier suspended solids and biological organic matter by simple gravity sedimentation/floating in a tank [81], followed by the removal of remaining organic matter (suspended, colloidal and dissolved solids) with the aid of diverse microorganisms, with varying degree of aeration [82] and further removal of traces of finer suspended solids and excess nitrogen and phosphorus that adequately promote unwanted plant growth [81,83]. Additionally, the disinfection of viruses, bacteria and other harmful microorganisms using chlorination, UV treatment, ozone treatment and membrane filtration in the treated wastewater would be the final stage [84].

Although these conventional treatment methods are widely applied for the removal of AMR pathogens in wastewater, the production of toxic by-products (a cause of secondary pollution in wastewater), lower treatment efficiency and high energy consumption are the major setbacks [85]. For instance, UV radiation-based inactivation, albeit being effective, is constrained by the lower penetration of the depth possible because of the regrowth of ARGs due to an incomplete inactivation. This method achieved moderate ARG reduction ranging from 1 to 3 log reductions [86,87,88]. Chlorination-based disinfection can be used to achieve ARG log reduction, i.e., 2–4 log units, but this method has threat of producing harmful by-products such as haloacetic acids and trihalomethanes [89,90]. Conversely, ozonation offers enhanced efficiencies ranging from 2 to 6 log reduction in ARGs, but these methods are limited due to their high operational cost and the formation of toxic by-products such as bromate [90,91,92,93]. Membrane bioreactors involve the combination of biological filtration and physical filtration and achieve a 2–6 logs removal of ARGs. But these methods are hindered by membrane fouling and are dependent on operational conditions [16,94,95].

Therefore, advanced oxidation processes such as photocatalysis, Fenton processes, and electro-oxidation processes that disinfect the pathogenic microorganisms or their genes at molecular level have been suggested for the effective removal of microorganisms or their AMR genes from WWT [96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111]. Photocatalysis offers a variable range of ARG reduction from a 0.2 to 6 log reduction. When combined with other methods such as UV and ozonation, it achieves a superior ARG removal compared to UV or ozonation alone [99,100,101,102,109]. On the other hand, Fenton processes are capable of achieving approximately up to a 6 log reduction or the inactivation of ARGs without the formation of harmful by-products [87,105,107,111,112]. The electro-oxidation methods are unique in their ability to consistently achieve significant reduction efficiency up to a 5 log reduction with a reduced dependence on chemical additives [103,113,114]. These modern techniques not only enhance the overall efficacy of ARG removal but also mitigate the environmental risks associated with traditional methods. By reducing harmful by-products and offering greater flexibility in treatment conditions, advanced processes like photocatalysis, the Fenton process and electrochemical oxidation represent a significant advancement in wastewater treatment technologies [115]. Table 3 consolidates the methods demonstrated for ARG reduction in wastewater.

Table 3. The degree of reduction in ARG concentration by various methods of wastewater treatment.

S. No	Sample Type	Target ARGs	Method of Wastewater Treatment	Reduction in ARGs	References
1	Urban MWW	blaTEM, qnrS and sulI	Photocatalytic ozonation (TiO2 rings and LED light)	Reduced to 10 gene copies/mL	[109]
2	MWW	mecA	Photocatalysis (TiO2/H2O2 based and under UV radiation)	5.8 log	[102]
		ampC		4.7 log
3	WWTP	Sul1	Photocatalytic ultrafilteration	~98%	[101]
		Sul2
		floR
4	Wastewater	ampC	Photocatalysis (Graphene based TiO2/Solar radiation)	Complete removal	[100]
5	WWTP effluents	tetA	Photocatalysis (Ag/AgBr/g-C3N4 nano composites/Visible light)	49%	[96]
		tetM		86%
		tetQ		69%
6	Domestic WW	i (sulI, sulII and tetQ)	Photocatalysis (with constructed wetlands)	1.27–1.72 log	[97]
		e (sulI, sulII and tetQ)		0.23–0.65 log
7	WWTP	sul1, tetX and tetG	Fenton oxidation (UV/H2O2)	2.58 to 3.79 logs	[116]
8	Urban MWW	blaOXA, blaCTX-M, sul1 and tetM	Solar Fenton process	Reduced to ~10 copies/mL	[106]
9	Wastewater effluents	Intracellular tetA	Photo-Fenton process	3.79log	[105]
		Intracellular blaTEM-1		2.19log
		Extracellular tetA and Extracellular blaTEM-1		~6log
10	HWW	qnrA, qnrB, qnrS, qnrD, aac(6′)-Ib-cr, qepA	Electro-peroxone and sequencing batch reactor treatment	-	[117]
11	MWW	tetA and Sul1	Electrochemical oxidation (Blue TiO2 nanotube/under UV lights)	2.1−2.3 for long sequence target 1.3−1.8 for short sequence target	[114]
12	MWW	tetG , floR and sul1	Electro-oxidation using a Magneli phase Ti4O7 anode	99%	[104]
13	MWW	sulI and sulII	EMBF bioreactor	(2/3)rd compared with conventional biofilm membrane	[103]
14	Wastewater	sulI	Electro-oxidation	4.8 log	[118]
		tetA		1.8 log

Table 3. The degree of reduction in ARG concentration by various methods of wastewater treatment.

S. No	Sample Type	Target ARGs	Method of Wastewater Treatment	Reduction in ARGs	References
1	Urban MWW	blaTEM, qnrS and sulI	Photocatalytic ozonation (TiO2 rings and LED light)	Reduced to 10 gene copies/mL	[109]
2	MWW	mecA	Photocatalysis (TiO2/H2O2 based and under UV radiation)	5.8 log	[102]
		ampC		4.7 log
3	WWTP	Sul1	Photocatalytic ultrafilteration	~98%	[101]
		Sul2
		floR
4	Wastewater	ampC	Photocatalysis (Graphene based TiO2/Solar radiation)	Complete removal	[100]
5	WWTP effluents	tetA	Photocatalysis (Ag/AgBr/g-C3N4 nano composites/Visible light)	49%	[96]
		tetM		86%
		tetQ		69%
6	Domestic WW	i (sulI, sulII and tetQ)	Photocatalysis (with constructed wetlands)	1.27–1.72 log	[97]
		e (sulI, sulII and tetQ)		0.23–0.65 log
7	WWTP	sul1, tetX and tetG	Fenton oxidation (UV/H2O2)	2.58 to 3.79 logs	[116]
8	Urban MWW	blaOXA, blaCTX-M, sul1 and tetM	Solar Fenton process	Reduced to ~10 copies/mL	[106]
9	Wastewater effluents	Intracellular tetA	Photo-Fenton process	3.79log	[105]
		Intracellular blaTEM-1		2.19log
		Extracellular tetA and Extracellular blaTEM-1		~6log
10	HWW	qnrA, qnrB, qnrS, qnrD, aac(6′)-Ib-cr, qepA	Electro-peroxone and sequencing batch reactor treatment	-	[117]
11	MWW	tetA and Sul1	Electrochemical oxidation (Blue TiO2 nanotube/under UV lights)	2.1−2.3 for long sequence target 1.3−1.8 for short sequence target	[114]
12	MWW	tetG , floR and sul1	Electro-oxidation using a Magneli phase Ti4O7 anode	99%	[104]
13	MWW	sulI and sulII	EMBF bioreactor	(2/3)rd compared with conventional biofilm membrane	[103]
14	Wastewater	sulI	Electro-oxidation	4.8 log	[118]
		tetA		1.8 log

4.1. Photocatalysis

Photocatalysis has been widely employed for the removal and inactivation of ARGs in wastewater. In WWT, during photocatalysis, reactive oxygen species (ROS) are generated in the presence of light, which results in the increased permeability of the cell membrane and the oxidation of cell contents such as DNA, RNA and proteins, resulting in the inactivation of microorganisms or their AMR genes [119,120]. Due to its enhanced efficacy and stability, the production of nontoxic by-products and affordability, TiO₂-based photocatalysis has been the most popularly applied photocatalysis method for the removal of ARGs from WWT plants [96,97,99,100,101,102,109]. For instance, Moreira et al. [109] employed a photocatalytic ozonation process in combination with TiO₂-coated Raschig rings (in the presence of LED light) for the removal of ARGs from urban MWW in comparison with the single ozonation method. The authors reported that this method was effective for the removal of ARGs such as bla_TEM, qnrS and sulI conferring resistance against antibiotics such as beta-lactam, quinolones and sulfonamides. The efficiency of this method for ARG degradation was found to be ~98% under UV irradiation. In another study, Koraolia et al. [100] aimed for the elimination of ARGs such as sul1, ampC, mecA and ermB associated with resistances against sulfonamide, ampicillin, Methicillin and macrolide from urban wastewaters using a graphene-based TiO₂ composite photocatalyst in the presence of a solar radiation. The results revealed that this method efficiently removed the ampC gene, but the ARGs were detected to be persistent throughout the treatment process. Likewise, a combination of H₂O₂/TiO₂-based photocatalysis under UV irradiation was employed for nullifying the AMR genes (mecA and ampC) within their hosts MRSA and P. aeruginosa. respectively. Their study resulted in a reduction of 5.8 log values and 4.7 log values of mecA and ampC, respectively. In the presence of TiO₂-based photocatalysis under UV radiation (120 mJ/cm²) and the addition of H₂O₂, it showed an enhanced efficiency in controlling AMR genes [102]. Ren et al. [101] had incorporated TiO₂ into the Polyvinylidine fluoride ultra-filtration membrane for the removal of ARGs such as floR (Florfenicol resistance), sulI and sulII (sulfonamide resistance) from wastewater treatment plants. The authors documented that the efficiency of this method for ARG degradation was found to be ~98% under UV irradiation. Like TiO₂ and their composites, Ag/AgBr/g-C₃N, nano composites had also been employed in degrading tetracycline-resistant genes from waste-water under visible light irradiation-based photocatalysis, and the ARG removal efficiencies of the nanocomposites for tetA, tetM and tetQ were found to be 49%, 86% and 69%, respectively [96]. Felis et al. [98] extended the study for the determination of the performance of TiO₂-based photocatalysis along with solar light-driven photolysis for the removal of ARGs from wastewater. The study revealed the enhanced efficiency of photocatalysis (83.8%) compared to solar-driven photolysis (80%) for the removal of ARGs such as from wastewater. More recently Chen et al. [97] combined a photocatalytic treatment system with a constructed wetland for WWT and assessed its efficiency in removing ARGs such as sul1, sul2 (sulfonamide resistance) and tetQ (tetracycline resistance) from urban MWW. The results showed that the combined treatment with photocatalysis and constructed wetlands significantly reduced both intracellular (1.27–1.72 log values) ARGs and extracellular ARGs (0.23–0.65 log values) from wastewater samples.

4.2. Fenton

Fenton oxidation processes are highly efficient and relatively cheap processes that have emerged as promising methods for the elimination of ARGs from wastewaters. In the Fenton oxidation process, a OH free radicals (OH•) or a singlet oxygen (¹O₂) produced from the reaction between H₂O₂ and Fe²⁺/Fe³⁺ (constituents of Fenton reagents) lyse cells in an acidic medium [106,110]. Due to its enhanced efficiency, operational ease and low inherent toxicity, Fenton oxidation has been employed as an advanced method for wastewater treatment [111]. Zhang et al. [121] evaluated the performance of Fenton oxidation process in WWT plants and found that it efficiently reduced the concentration of ARGs such as sul1, tetX and tetG (e.g., 2.58 to 3.79 logs of ARGs) in wastewater effluents after treatment with UV/H₂O₂.

In addition, solar-driven Fenton oxidation linked with membrane bioreactors were tested for the removal of ARGs such as sulI, ermB and ampC from wastewater samples [112], which were found to reduce the total DNA concentration in the wastewater sample by 97%. Similarly, Serna Galvis et al. [87] validated the performance of solar-driven photo-Fenton process for the removal of the carbapenem-resistant gene, i.e., bla-_KPC from HWW. It was observed that total degradation bla-_KPC was achieved through this process in 5 h. The solar photo-Fenton process (combined with granular activated carbon treatment) was also extended to assess its potential for eliminating ARGs from urban wastewater effluents. The method effectively degraded ARGs such as bla_OXA, bla_CTX-M, (beta-lactam resistance) qnrS, (quinolone resistance) sul1 (sulfonamide resistance) and tetM (tetracycline resistance) in the wastewater samples [106]. Yet another study by Ahmed et al. [107] studied the enhanced removal of the antibiotic resistant E. coli and ARGs such as tetA and bla_TEM by a Photo- Fenton process under visible LED light and neutral pH. Similarly Ahmed et al. [105] designed a modified photo-Fenton system with ethylenediamine-N,N -di-succinic acid (EDDS) and investigated its performance for the degradation of ARGs from wastewater effluents. This process was found to degrade both intercellular (iARGs) and extracellular ARGs (eARGs), including itetA (3.79 log value), ibla_TEM (2.19 log reduction), etetA and ebla_TEM (~6 log reduction each).

4.3. Electro-Oxidation

Electro-oxidation is another method that generates hydroxyl free radicals from catalysts in the presence of high-voltage electrodes. As described in the photocatalysis and Fenton process, the free radicals generated through the electro-oxidation process also oxidize the cellular and genomic components, resulting in the inactivation and removal of ARGs [113,122]. For instance, Zheng et al. [117] assessed the reduction in quinolone-resistant ARGs such as qnrA, qnrB, qnrS, qnrD, aac(6′)-Ib-cr and qepA from HWW through pre-treatment with electroperoxone followed by a sequencing batch reactor treatment. The maximum reduction in ARGs was reported from samples subjected to 75 min of electroperoxone treatment. Similarly, Wang S et al. [114] employed an electrochemical oxidation process with a blue TiO₂ nanotube array as the photoanode under UV radiation and validated its efficiency in the removal of ARB and ARGs. This set up was found to possess enhanced efficiency for the removal of ARGs such as tetA and sul1 (carried on a plasmid) and for inactivation of E. coli strains that were resistant to the antibiotics sulfamethoxazole and tetracycline. Wang et al. [123] investigated the inactivation of MDR Salmonella typhimurium and removal of ARGs such as tetG, floR and sul1 through electro-oxidation using a Magneli phase Ti₄O₇ anode. This method was found to reduce the ARGs contained in the pathogen by ~99%. Li et al. [103] evaluated the performance of an electrochemical membrane biofilm (EMBF) reactor for the removal of sulfonamide resistance genes such as sulI and sulII from wastewater effluents. The % abundance of ARGs found in wastewater after treatment with EMBF reactor was found to be reduced by one-third when compared with that of the conventional membrane biofilm reactor. Ni et al. [118] designed a CeO₂@CNT-NaClO-based electrified membrane and validated its efficiency in the removal of ARGs from wastewater through the electro-oxidation process. The results showed that the abundance of ARGs such as sulI and tetA genes were reduced by 4.8 and 1.8 log values in the wastewater sample after treatment with the CeO₂@CNT-NaClO-based electrified membrane. Figure 3 summarizes these three mitigation methods, unraveling the mechanism behind the mitigation effect.

The various ARGs reported in hospital wastewater and municipal wastewater were reviewed and the gravity of the situation was assessed. The existing methods for disinfecting the AMR pathogens and genes in these ecosystems were reviewed. Most of these methods are the conventional ones that possess certain limitations. Their ARG treatment efficiencies in real-scale WWTPs were observed to exhibit a mere 2–3 logs reduction, and in some cases, no reduction at all [124]. Additionally, variable responses to the treatment were also noticed, indicating that only certain types of ARGs were inactivated [124]. Increasing the usage doses of UV light, ozone and chlorine to maximize the efficiency will lead to huge energy costs and an excessive release of hazardous chemical residues. Combining multiple techniques is also a recommendation that will enhance and push the individual performances to break limitations. Adsorption is one of the most explored and studied mitigation technique with respect to wastewater treatment. In spite of the fact that adsorption is one of the most facile, cost-effective, easily scalable and robust techniques, its applicability in the removal of ARB/ARGs from the aquatic environment has been sufficiently put to use. Only a few reports have been published on the utility of adsorption techniques for ARB/ARG reduction [125,126,127]. This review prompts more investigations in this aspect in order to harness the full potential of this technique in ARG remediation.

With the advancement of nanotechnology, the use of nanomaterials for wastewater treatment has become an attractive option. Few authors used nanosilver and silver ions to eliminate ARGs from wastewater and other authors report the use of Titanium dioxide (TiO₂) nanoparticles through photocatalysis, combined with the use of chlorination and UV, to inactivate ARGs in WW [128]. According to Maillard and Hartemann, (2013) [124], colloid silver, metallic silver and salt silver inhibit antibiotic resistance genes in water. Adaora et al. have reviewed the ability of surface-modified silica dioxide nanoparticles and sulfidated nanoscale zero-valent iron in the uptake and degradation of ARGs in wastewater. Nanoscale zero-valent iron (nZVI) has gained more attention for eliminating contaminants/pollutant in the last two decades. It is relatively inexpensive, eco-friendly and has a strong reducing capacity (Fe²⁺ + 2e⁻ → Fe(s), E⁰ = − 0.44 V) [129]. It is able to bring about physical damage and oxidative stress to bacteria [130,131] through the FeO core simultaneously oxidizing to iron oxides and hydroxides [132]. As a nanoscale material, S-nZVI may provide a cost-effective way for ARG removal [133].

Very few nanomaterials amidst the vast ocean of nano particles have been reported or demonstrated against ARGs. This is a huge gap concerning this area of research. While nanotechnology has always had plenty to offer, the restricted use of nanoparticles in an aspect ideally close to human welfare and the environment is seen as a lacuna that calls for immediate attention. Options such as carbon-based nanomaterials, like carbon nanotubes, could effectively remove pathogens from WW due to their antimicrobial properties [134,135]; thus, they are promising materials for targeting ARGs owing to their remarkable pore structure, which facilitates effective adsorption and retention. The application of the wide range of options that carbon nanomaterials have to offer, including buckyballs, nanotubes, graphene nanosheets, grapheme, GO, carbon nanodots, and the like, is essential.

Paruch et al. [128] have evaluated the application of novel nanomaterial-based micro/nanoparticles and polymer films for ARG applications. Polymer-based composite nanomaterial options are also versatile options, yet only a couple of reports exist on their application to ARGs. Lipopolysaccharide (LPS)-imprinted polymer films (LPS-MIP) and quaternary ammonium-functionalized-kaolin microparticles (QAS-K), which were designed for specifically targeting GNB and GPB, respectively, were demonstrated for their utility against ARGs [128]. This polymeric nanocomposite was deployed for ARG mitigation in a wastewater treatment plant. It could successfully lead to the specific recognition and capture of whole bacteria [136,137,138] and was effective against ARGs [139].

Furthermore, the lack of quality data and efforts in removing antibiotic-resistant bacteria and their genes compared to the abundance of data on the prevalence or incidences of these substances in wastewater is something that was noticed through the survey performed to compile this review. On the whole, it is high time that well-structured and strategized mitigation methods are investigated and real-time solutions are provided. Given the fact that so many lethal ARGs are floated into the environment, more than mitigation efforts, careful monitoring and stringent measures need to be enacted to stop the release of these ARGs into the environment in the first place.

5. Conclusions

This review highlighted the abundances of different types of antibiotic resistance in two major reservoirs of ARGs, i.e., hospital and municipal wastewater, which is a foreseen threat to the human lives across the globe. Mitigation methods, gaps in the applied technologies and future prospects have been discussed. The development of newer techniques for wastewater treatment with enhanced efficiency is an urgent requirement that needs better understanding about the survival strategies and the interaction between the constituents of antibiotic resistomes in hospital and municipal wastewaters. More fundamental and applied research in this direction will prove beneficial.