Prevalence of ESKAPE Bacteria in Surface Water and Wastewater Sources: Multidrug Resistance and Molecular Characterization, an Updated Review

Abstract

ESKAPE bacteria represent a group of opportunistic bacterial pathogens that display widespread antimicrobial resistance, including resistance to the last-line antibiotics, thereby posing a significant clinical implication globally. Anthropogenic activities, such as wastewater from hospitals, livestock farms, crop fields, and wastewater treatment plants, contribute to the dissemination of antimicrobial-resistant bacterial pathogens into the environment. Surface water sources, including river waters, act as critical points of discharge for wastewater, pollutants, antibiotic-resistant bacteria (ARB), and antibiotic-resistant genes (ARG). These environmental factors, along with others, facilitate the dissemination and survival of ARBs, as well as promote the exchange of ARGs. Therefore, it is crucial to comprehend the current environmental landscape concerning the prevalence and persistence of resistant bacteria, particularly those belonging to the ESKAPE group. This review aims to provide a comprehensive overview of the current dissemination and characterization of ESKAPE bacteria in surface water and wastewater sources.

1. Introduction

Currently, one of the greatest challenges in multidrug resistance (MDR) is the ESKAPE group pathogens. This group includes vancomycin-resistant Enterococcus faecium and methicillin–vancomycin-resistant Staphylococcus aureus classified as high-priority pathogens, as well as carbapenem-resistant Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., classified as critical priority pathogens by the World Health Organization [1,2]. The ESKAPE group comprises the common opportunistic pathogens in nosocomial infections, and they were grouped under the acronym ESKAPE because it reflects their ability to “escape” antibiotics actions, posing a major problem in the selection of effective therapeutic strategies [3,4]. The ESKAPE group is characterized by potential drug resistance mechanisms that fall into several broad categories, including drug inactivation/alteration, modification of drug binding sites/targets, changes in cell permeability leading to reduced intracellular drug accumulation, and biofilm formation [5,6]. These resistance mechanisms can coexist together within a single bacterial cell [7].

Antibiotic resistance is a pressing global health concern intertwined with human, animal, and environmental factors [8]. Infections stemming from antibiotic-resistant bacteria (ARB) are projected to contribute to approximately 10 million deaths worldwide by 2050 [9]. This threat stems from the dissemination of resistant bacteria and their genes among bacterial pathogens and resident microbiota in humans, animals, and the environment, with global-scale effects as recognized by the One Health initiative [10]. While the gut of humans and animals represents a substantial reservoir of resistance and multidrug-resistant (MDR) bacteria [11], the environment is also home to several ubiquitous Gram-negative bacilli that are commonly associated with human and animal infectious diseases [12].

Gram-negative bacteria possessed complex resistance mechanisms mediated by genes encoding extended-spectrum β-lactamase, AmpC, and carbapenemase enzymes, conferring resistance to all beta-lactams, carbapenems, and third-generation cephalosporins. These genes are frequently transferred horizontally via plasmids [13]. Historically, opportunistic organisms have originated from commensal bacteria. However, in addition to their original sources, these bacteria, along with the antimicrobial resistance genes they carry, can also be found in environmental ecosystems such as superficial waters [14]. Over the past decade, there has been a growing interest in understanding the spread of bacterial resistance in the natural environment [15]. This includes resistance to beta-lactams, macrolides, quinolones, sulfonamides trimethoprim, and tetracycline [16], as well as resistance to last-line antibiotics [17] such as carbapenems, and aminoglycosides [18]. Therefore, the objective of this review article is to provide an overview of the current reports about the distribution and characterization of ESKAPE group members reported in wastewater and surface water sources.

2. Multidrug Resistance (MDR)

The multidrug resistance (MDR) is associated with high mortality rates, a decline in treatment efficacy, prolonged infection duration, the spread of resistant pathogens, and substantial medical costs, significantly impacting the effectiveness of antimicrobial agents [9,19]. Currently, the excessive and inappropriate use of antibiotics has greatly contributed to the increased prevalence of MDR bacteria [20,21] resulting in over 700,000 deaths worldwide each year [22].

Furthermore, the resistome refers to all the genes associated with antimicrobial drug resistance. It can be categorized as “intrinsic”, which represents the inherent genetic properties of the bacterium [23]. These genes are encoded in the chromosome and are independent of the previous antibiotic exposure, not related to horizontal gene transfer (HGT) [24]. On the other hand, the “extrinsic” resistome consists of genes acquired through HGT or genetic changes, which can be stably inherited across generations [25]. For instance, certain bacteria exhibit resistance to specific classes of antibiotics [7], including macrolides, tetracyclines, oxazolidinones, β-lactam-β-lactamase inhibitor combinations, as well as last-line antibiotics such as glycopeptides, carbapenems and polymyxins [26] Additionally, globally significant risks are posed by vancomycin-resistance Staphylococcus aureus and Enterococcus spp. which are the most concerning Gram-positive pathogens, while MDR Gram-negative bacteria are progressively developing resistance to all existing antibiotics creating an alarming situation [27].

2.1. Extended-Spectrum β-Lactamases (ESBL) Bacteria in Aquatic Environments

The Ambler classification system categorizes β-lactamase enzymes into four groups (A, B, C, D) based on their central catalytic domain and substrate preference, distinguishing between serine-β-lactamases and metallo-β-lactamases [28]. Class A encompasses the largest cluster of serine-β-lactamases, which act against penicillins, cephalosporins, monobactams, cephamycins, and carbapenems. Enzymes such as bla_TEM, bla_CTX-M, bla_PER, bla_GES, bla_VEB, and bla_KPC are found in all Gram-negative ESKAPE pathogens [29]. The bla_{KPC type} (K. pneumoniae carbapenemase) can be found via a plasmid and represents a prominent example of a class A carbapenemase, playing a crucial role in carbapenem-resistant Enterobacterales bacteria. The bla_{GES type} is inserted into the class 1 integron in a plasmid of P. aeruginosa [30].

Class B are metallo-β-lactamases (MBL), also known as carbapenemases (bla_VIM, bla_NDM, and bla_IMP enzymes), that utilize a Zn²⁺ ion to facilitate substrate hydrolysis. They exhibit limited hydrolytic activity against monobactams and are not inhibited by clavulanic acid or tazobactam [31]. Genes encoding MBLs are typically found on plasmids, and the most common MBL types include IMP types that can be found in all Gram-negative ESKAPE pathogens and VIM types found mostly in P. aeruginosa and A. baumannii and bla_NDM in K. pneumoniae and E. cloacae [32].

Class C enzymes confer resistance to most cephalosporins, and, to a lesser degree, they exhibit hydrolytic activity on penicillins and select monobactams. Plasmid-encoded AmpC-type enzymes are highly expressed in isolates of E. cloacae and P. aeruginosa [33].

Class D enzymes hydrolyze oxacillin. Genes encoding bla_{OXA type} are detected in the chromosomes or plasmids, with bla_OXA-23, bla_OXA-24/40, and bla_OXA-58 being the most prevalent in A. baumannii and bla_OXA-23 and bla_OXA24/40 in P. aeruginosa [34]. Enzymes such as bla_OXA-48-like reported in Enterobacterales and bla_OXA-23, bla_OXA-4/-40, and bla_OXA-58-like in Acinetobacter spp. are carbapenemases [35].

ESBL genes allow bacterial survival in the presence of antibiotics by encoding mechanisms that target various classes of antibiotics [36], particularly β-lactams. These resistance mechanisms are mediated by β-lactamase enzymes that catalyze the hydrolysis of the β-lactam ring, leading to antimicrobial inactivation [37]. Carbapenems, which are broad-spectrum antibacterial agents, have been commonly used to treat ESBL-mediated resistance. However, the overuse of carbapenems has resulted in increased carbapenem resistance and ESBL resistance among Enterobacterales, A. baumannii, and P. aeruginosa, strains in hospital settings [38]. Resistance to last-line antibiotics in environmental or clinical strains of bacterial pathogens frequently leads to pan-resistance [39].

ESBL are enzymes that confer resistance to beta-lactam antibiotics, third-generation cephalosporins, and monobactams. They are often associated with MDR and frequently located on transferable plasmids, allowing for their spread among bacteria and environmental sources, such as surface water [40,41]. Most ESBL members belong to Ambler class A, including bla_GES, bla_VEB, and bla_BEL. However, some other enzymes from class D such as bla_OXA-2 and bla_OXA-10 are also classified as ESBL [42]. ESBL enzymes are commonly found in Gram-negative bacteria such as K. pneumoniae, Enterobacter spp., Acinetobacter sp., and Pseudomonas spp. The ESBLS genes bla_TEM, bla_SHV, bla_CTX-M, and bla_OXA are prevalent in these bacteria [43,44] while bla_CTX-M type ESBL enzymes are known to have environmental origins [45].

In a study conducted by Hassen B., et al. [46] ESBL-producing Escherichia coli and K. pneumoniae were isolated from 105 wastewater and 15 river samples in Tunisia over two years (2017–2018). The water samples were plated on MacConkey agar containing 2 µg/mL of cefotaxime. The study revealed that 25% of wastewater treatment plants (WWTP) and 12% of river water samples tested positive for ESBL-producing Enterobacterales (ESBL-E) strains, with 33 E. coli strains and 4 K. pneumoniae strains identified. Among these strains, 30 out of 37 were determined to be MDR based on Clinical and Laboratory Standards Institute (CLSI 2017) breakpoints. Molecular characterization identified bla_CTX-M-15, bla_CTX-M-1, bla_CTX-M-55, bla_CTX-M-27, bla_SHV, and bla_VEB as the most prevalent ESBL genes produced by the isolates. All K. pneumoniae strains were positive for bla_CTX-M-15 and bla_SHV, and class 1 integrons carrying resistance gene cassettes and IncP plasmids were also detected.

In another study by Hosu M. C., et al. [47], the public health implication of antibiotic resistance genes (ARGs) in P. aeruginosa was investigated in abattoir wastewater and surface water samples from Mthatha, Eastern Cape Province, South Africa, between January and June 2019. Water samples were placed on CHROMagar^TM Pseudomonas agar plates. Out of 36 P. aeruginosa strains detected in wastewater and surface water samples, 20 exhibited a MDR profile based on CLSI 2010 breakpoints, with 60% originating from abattoir wastewater and 40% in from surface water. Additionally, 15 strains were found to carry bla_SHV, bla_CTX-M, bla_TEM, and bla_VIM genes.

The above-mentioned studies conducted by Hassen B., et al. [46] and Hosu M. C., et al. [47] indicate that wastewater sites serve as favored reservoirs for strains of the ESKAPE group, particularly those with MDR profiles and carrying ESBL-associated genes such as bla_CTX-M, bla_SHV, and bla_TEM. ESBL genes are more prevalent than MBL genes for carbapenemase in wastewater than in surface water sources.

2.2. Carbapenem Resistance in Aquatic Environments

Carbapenems are broad-spectrum β-lactam antibiotics that, unlike other β-lactams, are resistant to hydrolysis by β-lactamases and extended-spectrum β-lactamases [48]. Carbapenem resistance in ESKAPE Gram-negative bacteria is mediated by various mechanisms such as enzymatic hydrolysis by carbapenemases [28]. These enzymes are highly transmissible through MGEs such as insertion sequences and plasmids in K. pneumoniae and Enterobacter spp. Strains; transposons, insertion sequences and plasmids in A. baumannii strains and insertion sequences, and plasmids in P. aeruginosa strains [49]. Carbapenemases are classified into A, B, and D (KPC, NDM, VIM, IMP type) in the Ambler classification system and the microorganisms harboring them are often referred to as carbapenemase-producing organisms [50].

Other mechanisms involved in carbapenem resistance include the loss of expression of porin-encoding genes, mutations in chromosomally encoded porin genes, and overexpression of genes encoding efflux pumps [28]. A study conducted by Ehi-Ebomah K., et al. [51] evaluated the occurrence of carbapenem-resistant Enterobacter species harboring bla_NDM-1, bla_KPC, and bla_OXA-48-like genes in various environmental sources in South Africa, including hospital wastewater effluents, wastewater treatment plants final effluents, surface waters (rivers, dams, and canals), irrigation water, farm soil, and vegetable sources. Samples were placed on eosin methylene blue agar plates and then on nutrient agar plates. Out of 243 samples that were analyzed, 142 isolates were identified as Enterobacter spp. with only 115 identified as E. cloacae. The antimicrobial susceptibility test revealed that 85 E. cloacae isolates were resistant to a panel of four carbapenems, with resistance percentages of 80% for doripenem, 75% to meropenem, 72% to imipenem, and 66% to ertapenem. Additionally, among these 85 isolates, 41 harbored carbapenem resistance genes with 29 isolates harboring bla_NDM-1 gene, 9 isolates harboring bla_KPC, and 3 isolates harboring bla_OXA-48-like. The highest occurrence of ARGs in E. cloacae was detected in hospital wastewater effluents at (29%) followed by wastewater treatment plant final effluents (10%), irrigation water, surface water, and farm soils (17%), and vegetables (10%). This study revealed that hospital wastewater effluent is an important reservoir of E. cloacae with carbapenemase genes. However, in comparison to wastewater final effluents, which reported a 10% occurrence, it indicates that treatment remarkably reduces E. cloacae strains. The author attributed these results to a transfer of ARB from hospital wastewater to the farms via the use of surface water for irrigation purposes, nevertheless, this is controversial, since it is known that the hospital wastewater flows into a sewerage network towards the water treatment plants and after this the water flows into the river from which the irrigation canals originate, rather, this bacterial transfer could be attributed to a poor sewage system or some other source of contamination independent of hospital sources.

Another study conducted by Zhang L., et al. [52] aimed to assess the prevalence and characterization of ESBL and carbapenemases-producing bacteria in three distinct sets of samples: untreated hospital sewage samples, treated effluents from hospital wastewater treatment plants (WWTPs), and river water in Sichuan province, China. Given the focus of this review article on the ESKAPE group in relation to water sources, we will delve specifically into the implications of this bacterial group. In August 2019, a total of 104 Gram-negative bacteria isolates were collected. All samples were initially cultivated on MacConkey agar supplemented with cefotaxime (4 µg/mL) or meropenem (2 µg/mL), followed by subculturing on MacConkey agar containing cefotaxime (4 µg/mL). The Enterobacterales groups encompassed 62 strains including 17 strains of K. pneumoniae (27.4%), and 3 strains of Enterobacter spp. (4.8%). Among the remaining isolates, Acinetobacter spp. accounted for 20 strains (18.3%) and Pseudomonas spp. for 6 strains (5.8%). Notably, the most prevalent taxas in untreated wastewater were K. pneumoniae (16.7%) and Acinetobacter spp. (16.7%). Conversely, treated wastewater exhibited a prevalence of resistant strains dominated by Pseudomonas spp. (29.4%) and Acinetobacter spp. (29.4%), while in river water, K. pneumoniae (23.8%) and Acinetobacter spp. (19%) were predominant species. The highest resistance rates were observed against ampicillin and cefotaxime, followed by sulfamethoxazole/trimethoprim, tetracycline, amoxicillin-clavulanic acid, cefoxitin, and ciprofloxacin. Furthermore, an alarming 94.1% of K. pneumoniae strains demonstrated multidrug resistance (MDR), a pattern also evident in Acinetobacter spp. strains based on CLSI 2019 guidelines.

The bla_CTX-M gene was found to be the most prevalent, present in K. pneumoniae (15.94%), Acinetobacter spp. (10.14%), Pseudomonas spp. (7.25%) and Enterobacter spp. (5.80%). The carbapenemases genes bla_NDM were detected in Acinetobacter spp. (34.21%), K. pneumoniae (10.53%), and Pseudomonas spp. (5.26%), while bla_KPC was identified in K. pneumoniae (18.75%), and Enterobacter spp. (12.50%). Notably, Acinetobacter spp. exhibited elevated resistance levels to clinically important antibiotics, with resistance rates of 55% to gentamicin, 85% to cefoxitin, 100% to cefotaxime, 75% for tetracycline, and 65% to trimethoprim/sulfamethoxazole. Overall, the majority strains, including 87.5% of MDR K. pneumoniae strains (with only two presenting bla_NDM-5) were primarily detected in effluent and surface water samples. This study underscores the presence of diverse bacteria within the ESKAPE group, with a substantial number of MDR strains disseminating in untreated wastewater and river water. A comparison between untreated and treated wastewater reveals a significant reduction in strains within the final effluents from hospital WWTPs. However, approximately 50% of the total strains detected in untreated wastewater persisted in river water, suggesting additional pollution sources. Furthermore, it is noteworthy that a considerable proportion of these bacteria endure and survive the wastewater treatment process, underscoring the importance of ongoing monitoring of the distribution of such bacteria, particularly MDR Acinetobacter spp. due to the clinical significance.

2.3. Methichillin Resistance in Aquatic Environments

Methicillin resistance is commonly detected in S. aureus clones worldwide [53]. It is mediated by the mecA or mecC gene, which is located on the staphylococcal chromosomal cassette and encodes a penicillin-binding protein (PBP2a) [54]. PBP2a is a transpeptidase with low affinity for most β-lactams, thereby conferring resistance to all members of the β-lactam class of antibiotics [55]. Methicillin-resistant Staphylococcus aureus (MRSA) has been identified in various settings, including hospitals, communities, environments, and livestock niches [56].

For instance, Ramessar K., et al. [57] reported the spread of MRSA to surface water in Durban, South Africa, resulting from the discharge of improperly treated wastewater effluent. Samples were plated on mannitol salt agar supplemented with cefoxitin. The study identified a total of 80 MRSA isolates, all carrying the mecA gene and exhibiting an MDR profile, (CLSI 2014) from treated effluent and receiving rivers of two WWTPs. Among those isolates, resistance rates of 100%, 98.75%, 96.2–97.5%, and 33.7% were observed for lincomycin, oxacillin, beta-lactams, and vancomycin, respectively. Additionally, 70% of the isolates encoded the blaZ and tetK genes associated with penicillin and tetracycline resistance, while 57.50% of the MRSA isolates carried the hla and sea virulence genes associated with alpha-hemolysin and staphylococcal enterotoxin production. Furthermore, the study identified 51 different resistance patterns, of which 36 were represented by single isolates. This report highlights a significant problem associated with a high number of MDR pathogenic MRSA isolates present in treated effluents, posing a risk to public health due to deficiencies in the wastewater treatment system.

In another study by Silva V., et al. [58] conducted in the Trás-os-Montes and Alto Douro region in Portugal, MRSA was isolated from untreated wastewater of three hospitals: Hospital of Lamego, Hospital of Chaves, and Hospital Vila Real. Among the 96 samples collected from October 2019 to March 2020 (24 samples per site, including treated and untreated wastewater from Hospital Vila Real) were streaked in Oxacillin Resistance Screening Agar Base plates with 3 mg/L of oxacillin, 45 MRSA strains were identified. Out of these, 28 isolates were MRSA while the remaining 17 were coagulase-negative staphylococci. The highest distribution of MRSA was found in the Hospital of Lamego (12.5%) whereas the lowest distribution (7.3%) was observed in Hospital Vila Real which had an on-site WWTP. Based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST 2018) and CLSI criteria, 22 out of 28 MRSA isolates were identified as MDR, with prevalent resistance to cefoxitin and penicillin, and all isolates carried the mecA and blaZ-encoding genes. Furthermore, 78.5% of the isolates exhibited resistance to macrolides and lincosamides due to the presence of ermA, ermB, and ermC genes, while 46.4% showed resistance to aminoglycosides with aph(3′)IIIa and aac(6′)-le-aph(2′)-la genes. The virulence profile revealed that all MRSA harbored the scn gene, a marker of the immune evasion cluster (IEC). Among the MRSA isolates, 23 carried IEC type B genes, while only 1 isolate carried IEC type D genes. Molecular typing identified 26 isolates as ST22-MRSA-IV with agr type I, known as the epidemic clone EMRSA-15 and commonly found in Portuguese hospitals. This report indicates that the presence of MRSA in hospitals wastewater is significantly reduced by WWTPs, although most hospitals lack such facilities. This situation could lead to an increase in the prevalence of MDR staphylococci, as previously mentioned in Silva V., et al. [59] report. In that study, which focused on surface water samples including rivers, streams, irrigation ditches, dams, fountains, and lakes in the Trás-os-Montes and Alto Douro region in Portugal a total of 33 S. aureus coagulase-positive staphylococci (CoPS) and 51 coagulase-negative staphylococci (CoNS) were detected. Four S. aureus were MRSA, all carrying the mecC gene and exhibiting resistance to penicillin and cefoxitin with associated blaZ-SCCmec-IX. These MRSA isolates belonged to agr III and were identified as ST425 t742 and ST130 t843. The remaining 29 S. aureus were susceptible to methicillin but resistant to penicillin and erythromycin due to the presence of blaZ, ermT, and msr genes, respectively. Molecular typing revealed a diversity of 22 different STs, with various clonal complexes represented. The virulence profile of the MRSA isolates showed the presence of genes such as hla, hlb, hld, tst, and eta, with agr type I being the most frequent. Among the 51 CoNS isolates, 94.1% carried the mecA gene and exhibited resistance to penicillin, clindamycin, fusidic acid, tetracycline, aminoglycosides, trimethoprim-sulfamethoxazole, and chloramphenicol, while none were MDR. This report demonstrates significant contamination of lotic surface water, particularly in sewage-affected areas such as rivers, where most CoPS isolates were detected. A comparison with Silva V., et al. [58] study supports the notion that an inadequate WWTP system contributes to significant microbial pollution, including clinically relevant pathogens, in water sources.

2.4. Vancomycin Resistance in Aquatic Environments

In contrast, vancomycin plays a critical and decisive role in treating multidrug-resistant infections caused by Gram-positive bacteria, including S. aureus [60]. Vancomycin resistance is mediated by genes (vanA, vanB, vanD, vanE, vanF, and vanG) that are transferred from enterococcal species [61]. These genes are primarily located on plasmids, carried by transposons, which increases the risk of disseminating vancomycin-resistance medically important susceptible microorganisms such as S. aureus [54]. Among these genes, vanA and vanB are the most commonly found in clinical isolates of E. faecium worldwide [62]. The mechanisms of resistance involve the replacement of D-Ala-D-Ala with D-Ala-D-lac as the terminal amino acids region of Lipid II, which inhibits the activity of penicillin-binding proteins responsible for cross-linking Lipid II into mature peptidoglycan, thereby compromising the integrity of the cell envelope [63]. Methicillin and vancomycin resistance are of significant concern and have been associated with high mortality rates worldwide in the absence of effective containment and treatment options [64].

Matlou D. P., et al. [65] conducted a study to determine the virulence profiles of vancomycin-resistant enterococci isolated from surface and groundwater in the Northwest Province, South Africa. The study analyzed a total of 170 samples (119 groundwater and 51 surface waters) which were placed on Bile Esculin Agar supplemented with vancomycin (16 µg/mL). Among the identified enterococci, 38 were E. faecium, 17 were E. faecalis, and 1 was E. saccharolyticus, with the majority detected in boreholes, dams, and to a lesser extent, in the river. The resistance percentages were highest for nalidixic acid (83.9%), vancomycin (78.6%), and sulphamethoxazole (0–66.7%). Furthermore, 80.4% of the enterococci strains were MDR (guideline unspecified), with 84.4% harboring vanA and 4% harboring vanB-encoding genes. Virulence genes analysis revealed the presence of gel gene in 36.4% of the strains, asa1 in 27.3%, hyl in 6.8%, and esp in 4.5%. This report highlights the significant presence of MDR E. faecium and E. faecalis strains carrying virulence genes. The detection of these pathogenic and MDR enterococci in groundwater and surface water sources indicates a significant pollution source and a concern for public health. Therefore, water monitoring and the implementation of new technology or strategies in the water treatment system are necessary.

Hricová K., et al. [66] conducted a study to determine the prevalence of vancomycin-resistant enterococci (VRE) and antimicrobial residues in wastewater and surface water in Olomouc region, Czech Republic. The study analyzed 144 wastewater samples from various sources including hospitals and veterinary institutes, as well as 36 surface water samples from Morava River between October 2018 and July 2020. The analysis of the antibiotics in water revealed that five out of ten antibiotic residues (ampicillin, clindamycin, tetracycline, tigecycline, and vancomycin) exceeded the predicted no-effect environmental concentrations (PNEC), indicating a likelihood of adverse effects from these antimicrobials in the aquatic environment, particularly in untreated wastewater and to some extent, in effluent from WWTP and surface water. VRE isolates were collected from Brilliance VRE Agar chromogenic plates. The microbiologic analysis identified a total of 18 VRE strains all identified as E. faecium, out of these 180 samples analyzed. Sixteen of these strains were found in wastewater samples, while two were detected in the Morava River. The minimal inhibitory concentration revealed resistance of all VRE strains to ampicillin, penicillin, and erythromycin, with 16 of them also showing resistance to clindamycin and teicoplanin. However, 100% and 90% of the strains were susceptible to linezolid and tigecycline, respectively, according to EUCAST 2021 guidelines. Molecular analysis further revealed that 17 out of 18 VRE strains carried the vanA gene while one strain carried the vanB gene, indicating vancomycin resistance. Additionally, most of the VRE strains possessed virulence genes such as hyl, esp, and esp with hyl genes, with only five strains lacking these genes. Although the detected vancomycin residues in the influent and effluent wastewater of the University Hospital of Olomouc could potentially have a significant environmental effect based on PNEC, the study did not find a direct relationship between vancomycin residues in water and the presence of VRE. The prevalence of enterococci was low, however, given the genomic plasticity of those ESKAPE members, is possible that these VRE strains acquire resistance or become MDR, especially in the presence of lower antibiotic concentrations, as observed with nitrofurantoin, chloramphenicol, linezolid, and erythromycin, where the PNEC values were lower across all sample sites. Nonetheless, the risk of transmission of ARB from the environment to humans primarily relies on bacteria that can colonize the human body, such as the ESKAPE group, rather than ARB that carry ARG but cannot colonize or infect the human body [67].

3. Aquatic Environment as Important Reservoirs of Antibiotic-Resistant Bacteria

Aquatic environments experiencing increased anthropogenic pressure have been recognized as significant reservoirs of antibiotics, ARB, and antimicrobial resistance genes (ARGs) [68]. Municipal wastewater treatment plants (WWTPs) are a pivotal role as they serve as major sinks for antibiotics, ARBs, and ARGs, owing to the excretion of a substantial portion of antibiotics used in human and veterinary medicine through urine and feces [69]. Despite undergoing treatment, antibiotics, ARBs, and ARGs persist within WWTPs [70], ultimately finding their way into rivers and lakes through the discharge of various types of waste, including domestic sewage, pharmaceutical wastewater, and WWTP effluents, resulting in rapid dissemination [71].

The presence of antibiotics in the environment, including soils, hospitals, industries, livestock, agricultural waste, and polluted ecological niches [72], establishes these niches as favorable reservoirs and dissemination pathways for ARB and ARG, which subsequently flow into environmental water, exerting selective pressure [8]. Upon entering the environment, antibiotic residues can adversely affect native biota at multiple trophic levels, primarily due to their chemical and pharmacokinetic properties, as well as the dynamics of environmental microbial communities [73]. This is partly attributed to the water-soluble nature of antibiotics, leading to humans (75%) and livestock (90%) excreting high concentrations of partially degraded or even intact compounds, along with the antibiotics’ half-life (ranging from 3 to 127 days) [74], thereby exacerbating ARG pollution in water [75]. Antibiotics commonly facilitate the selection of ARBs through ARG dissemination, resulting in heightened health and ecological risks via food chains or food distribution networks, even at trace levels [76]. The propagation of ARGs into surrounding environments transforms antibiotic resistance becomes an environmental pollution issue, positioning ARG as a contaminant of emerging concern [77]. The accumulation of antimicrobial agents, detergents, disinfectants, heavy metals, and, other contaminants [78,79] could expedite the occurrence of ARBs and ARGs in different water bodies such as hospital waste effluents, water treatment plants, and tap water [80]. The ESKAPE group may be a relevant component of drug resistance distribution.

3.1. The Distribution of ESKAPE Pathogens to the Environment through Wastewater

ESKAPE group bacteria are commonly found associated with and widely reported in hospital settings worldwide. However, these pathogens also find common environmental reservoirs in soil, water sources (such as surface runoff, drinking water, streams, dams, rivers, sewage, and municipal waste), and plants [81]. Effluents from wastewater treatment plants (WWTPs) and hospital wastewater treatment plants (HWWTPs) serve as significant sources of MDR bacterial populations, including the members of the ESKAPE group [82,83]. Hospital wastewater (HWW) constitutes a complex mixture of chemical and biological substances that are continuously discharged [84]. These discharges, often untreated or inadequately treated, introduce chemical compounds, antibiotics, and ARGs into aquatic environments, posing threats to human health, biota, and the environment [85]. Consequently, WWTPs are considered major hotspots for the exchange of resistance genes between clinical and environmental bacterial isolates [86].

Furthermore, WWTPs represent important sources of ARB and ARGs, as they receive and concentrate municipal wastewater from industries, farms, and hospitals [87]. However, numerous studies indicate that conventional biological treatment processes applied in WWTPs are insufficient in eliminating ARGs, ARBs, and other pollutants (

Table 1. Environmental studies of ESKAPE members detected in wastewater sources.

Author	Location	Sample Type	Origin	ESKAPE	MDR	ESBL	MBL	ARG	VG	Site with More %MDR
Mutuku C., et al. [90]	Pecs, Hungary	Wastewater	Hospital, nursing home, municipal WWTP	K. pneumoniae and E. cloacae	Yes	Yes	Yes	Yes	ND	Hospital wastewater and nursing home
King T. L. B., et al. [91]	South Africa	Wastewater Clinical	Urban/rural hospitals, urban/rural WWTPs, clinic	K. pneumoniae	Yes	Yes	ND	ND	ND	Urban/rural hospitals
Hubeny J., et al. [92]	Poland	Wastewater Surface water	WWTP, river	A. baumannii	Yes	No	Yes	Yes	ND	Wastewater
Surleac M., et al. [93]	Romania	WWWTP and clinical	Influent, effluent (WWTP) and patients	K. pneumoniae	Yes	Yes	Yes	Yes	Yes	Clinical and effluents

Note(s): Multidrug-resistance (MDR); extended-spectrum beta-lactamases (ESBL); metallo-betalactamase (MBL); antibiotic-resistance genes (ARG); Virulence genes (VG); wastewater treatment plant (WWTP); hospital wastewater treatment plant (HWWTP); no data (ND).

) [88]. This enables pathogenic bacteria survive and proliferate within treated wastewater effluents upon their discharge into aquatic and soil environments [89].

Several studies have highlighted hospital wastewater as the primary source reflecting the transmission of ESKAPE group members to the environment through wastewater. These studies investigate the distribution of MDR bacteria, antimicrobial resistance genes, virulence genes, and other contaminants that may facilitate genetic transfer in aquatic ecosystems. For instance, Mutuku C., et al. [90] evaluated the prevalence of β-lactam resistance in enteric bacteria found in urban wastewater in the city of Pecs, Southwest Hungary. The study covered four hospital effluents, one nursing home, two municipal wastewater sites, and three sites within a WWTP during the period 2019–2020. All samples were placed on eosin methylene blue agar-ceftriaxone (2 µg/mL⁻¹) and eosin methylene blue agar-imipenem (8 µg/mL⁻¹). A total of 126 isolates were identified, with E. coli, accounting for 46% of the isolates, K. pneumoniae, for 20.6%, E. cloacae for 7.1%, and the remaining isolates belonging to other species. Most of the isolates were obtained from hospital effluents wastewater and WWTP. The resistance profile revealed that over 80% of the isolates exhibited resistance to third-generation cephalosporins such as ceftriaxone, ceftazidime, cefotaxime, and cefpodoxime, while a smaller proportion showed resistance to carbapenem. Additionally, 70% of K. pneumoniae and 40% of E. cloacae strains were MDR, primarily detected in hospital wastewater, followed by WWTP and municipal wastewater, according to EUCAST 2018 guidelines. Molecular and phenotypic analysis indicated that 85% of K. pneumoniae were ESBL producers, with 49% carrying ESBL genes such as bla_CTX-M-27, bla_TEM-1, bla_OXA-1, as well as other genes associated with resistance to aminoglycoside, chloramphenicol, folate inhibitor, and quinolones. On the other hand, none of the E. cloacae isolates were identified as ESBL producers, but all exhibited resistance to cefoxitin, suggesting possible AmpC strains production. Additionally, all E. cloacae isolates displayed resistance to carbapenems and were identified as MBL producers; however, no carbapenem-resistant genes were detected. According to the author, a significant number of enteric bacteria demonstrated resistance to β-lactams and were identified as ESBL producers with various associated genes, especially K. pneumoniae and E. cloacae strains. In the case of E. cloacae strains, the phenotypic resistance to carbapenems could be attributed to intrinsic mechanisms. Furthermore, all isolates and sites reported a multiple antibiotic resistance index (MAR index) greater than 0.2 indicating a high level of antibiotic contamination at the source. This high contamination may have exerted selective pressure for resistance in the isolated strains.

King T. L. B., et al. [91] conducted a comparative analysis to assess the presence of antibiotic-resistant Klebsiella spp. in urban and rural hospital effluent, urban and rural WWTP, as well as 72 clinical isolates were provided from the National Health Laboratory Service (NHLS) in South Africa between August and November 2017. All samples were transferred on HiCrome Klebsiella selective agar. The study revealed the identification of a total of 145 strains of K. pneumoniae, with the majority originating from urban and rural hospitals and NHLS. The resistance profile analysis indicated a notably high percentage of K. pneumoniae strains displaying resistance in NHLS (94%), followed by rural hospitals (83%), urban hospitals (62%), Darvill WWTP (62%), and Appelsbosch WWTP (50%). Notably, the Appelsbosh WWTP and urban hospitals exhibited a high proportion of resistance to amoxicillin-clavulanic acid, piperacillin-tazobactam, cefotaxime, ceftazidime, cefalexin, while elevated percentages of resistance to ciprofloxacin, norfloxacin, moxifloxacin, gentamicin, and tobramycin were detected in Appelsbosch WWTP, urban hospitals, and rural hospitals. Conversely, a lower proportion of strains showed resistance to ertapenem and doripenem. The MDR profiles asper EUCAST 2018 guidelines, were predominantly observed in urban (21%) and rural hospitals (10%) and Appelsbosh WWTP (50%). Phenotypic testing for ESBL and carbapenemases reveal that only a few strains of Klebsiella spp. Exhibited ESBL production, primarily in urban and rural hospitals. However, NHLS isolates demonstrated a high proportion of ESBL-producing K. pneumoniae strains, which were also resistant to ertapenem, meropenem, doripenem, and imipenem. The report highlighted the significant presence of K. pneumoniae strains in urban and in rural hospital effluents, characterized by a wide range of resistance patterns. Notably, the rural hospital exhibited a high colony-forming unit per milliliter of Klebsiella spp. Additionally, confirmed K. pneumoniae, with a higher quantity of resistant strains compared to the urban hospital, a tertiary-level healthcare facility. These findings suggest that rural hospitals may not always possess modern infrastructure or efficient treatment systems. Consequently, it is imperative to monitor antibiotic overuse in rural areas and consider implementing wastewater sanitization strategies. Additionally, the reported results did not indicate any environmental repercussions by WWTPs due that 50% of MDR K. pneumoniae isolates in Appelsbosh WWTP was represented by only one isolate. However, it would be worthwhile to investigate other bacterium types that pose clinical risks in water sources, such as E. cloacae.

Hubeny J., et al. [92] conducted an extensive investigation into the prevalence of carbapenem resistance genes within Acinetobacter spp. Isolates originating from different segments of a municipal WWTP in the Region of Warmia and Mazury in north-eastern Poland. The study encompassed untreated wasterwater (UWW), activated sludge (AS), and treated wastewater (TWW) sampled during winter (February), summer (June), and fall (September) of 2019. To initiate the analysis, the isolated bacteria were cultured on CHROMagar^TM Acinetobacter chromogenic medium with the inclusion of the MDR supplement CR102 (CHROMagar). Identification of bacterial strains was facilitated through the recA gene, while the presence of carbapenem resistance genes (specifically bla_IMP, bla_IMP-1, bla_VIM, bla_VIM-2, bla_NDM, bla_OXA-23-like, bla_OXA-24-like, bla_OXA-51-like, bla_OXA-58-like) was well as intI1, intI2, intI3 genes and insertion sequences (ISAba1), which influence the overexpression of bla_OXA-23-like and bla_OXA-51-like genes was determined using PCR. A total of 258 isolates categorized as Acinetobacter spp. was procured from both wastewater and river water samples. Among these, 21 isolates were identified as A. baumannii, with 4 isolates originating from UWW, 10 from AS, and 7 from TWW. The months of June and September exhibited the highest number of Acinetobacter isolates. Molecular analysis revealed that Acinetobacter spp. isolated from wastewater were carriers of 9 distinct carbapenem genes emcompasing bla_IMP, bla_IMP-1, bla_VIM, bla_VIM-2, bla_NDM, bla_OXA-23-like, bla_OXA-24-like, bla_OXA-51-like, and bla_OXA-58-like. Additionally, the intI1, intI2, and intI3 genes were detected. Notable, the gene bla_OXA-51-like was observed as the most prevalent amongst the identified carbapenem resistance genes. Regarding the resistance profile of A. baumannii, it was found that only one strain harbored three distinct resistance genes (bla_IMP, bla_OXA-51 and bla_IMP-1). Among the A. baumannii isolates, a total of 18 exhibited the presence of bla_OXA-51 genes, however, only 7 isolates were confirmed to possess ISAba1/bla_OXA-51 in wastewater, encompassing TWW, while the ISAba1/bla_OXA-23 complex was not detected. This significant finding accentuated the notable abundance of carbapenemase genes, along with the presence of int genes within Acinetobacter spp. Isolates, predominantly during the summer and fall seasons. This seasonal trend could potentially be attributed to weather conditions that favor the proliferation of these genes, distinguishing it from the A. baumannii isolates. These outcomes might suggest that antibiotic resistance might potentially transcend across different bacterial species, particularly those more prevalent within the environment, by promoting the genetic exchange mechanisms [92]. Moreover, it is important to point out that a considerable number of these bacteria persist and survive the wastewater treatment process, emphasizing the importance of continued monitoring of the distribution of such bacteria, particularly MDR Acinetobacter spp. due to the clinical relevance that represents.

Surleac M., et al. [93] conducted a genomic characterization of MDR K. pneumoniae strains collected from three clinical units and three receiving WWTPs (influent and effluent) in Southern Romania between December 2018 to June 2019. A total of 178 K. pneumoniae strains were recovered from antibiotic-enriched media: ChromID ESBL agar, ChromID OXA-48 and ChromID CARBA. The prevalence of MDR strains was 92.3% of clinical strains, 87.5% of effluent strains, and 82.3% from influent strains were determined according to CLSI 2018 breakpoints. Genotypic analysis of β-lactamases encoding ARGs revealed the most prevalent and widely distributed genes to be bla_SHV, bla_OXA, bla_TEM, and bla_CTX-M. In clinical samples, the most frequent genes were bla_CTX-M-15, bla_OXA-1, bla_SHV-106, bla_TEM-1, bla_OXA-48, and bla_TEM-150. In influent samples, bla_CTX-M-15, bla_OXA-1, bla_OXA-48, bla_OXA-10, bla_NDM-1, bla_CMY-4, bla_SHV-145, and bla_TEM-1 were the most common genes detected. In effluent: bla_CTX-M-15, bla_OXA-48, bla_OXA-1, bla_TEM-1, bla_SHV-158, bla_SHV-187 and bla_KPC-2 were prevalent. Additionally, a significant number of virulence genes (VGs) were detected in influent strains, while their presence in the effluent and clinical samples was lower (40%-50%). Among the frequently detected virulence genes were fim genes (associated with adherence), ent, fep, fyu, irp, and ybt genes (associated with iron acquisition), and omp genes (related to secretion system-T6SS-III). The report highlights the significant dissemination of ARGs (ESBL and carbapenemase genes) and VGs from the hospital to the environment through K. pneumoniae strains studied. These results underscore the urgent need for wastewater monitoring, as it represents a crucial reservoir with substantial selection pressure and environmental risks. Previous studies have shown that untreated hospital wastewaters serve as important niches that harbor a diversity of bacteria, antibiotic residues, and other contaminants exerting selective pressure. Although municipal WWTPs are also considered significant niches, a comparison between municipal WWTPs and hospital WWTPs indicates that municipal WWTPs receive a lower quantity of bacteria and other contaminants, than those found in untreated and treated hospital wastewater. However, the effectiveness and technology employed in the treatment system are crucial factors that contribute to water quality. Nevertheless, members of the ESKAPE groups, including K. pneumoniae, had demonstrated the ability to survive and persist during the treatment process and in the environment. However, the prevalence and associated health risks in the environment are not yet well understood, as very few studies have explored the correlation between virulence profiles and resistance profiles.

3.2. ESKAPE Distribution in Surface Water Sources

Aquatic systems can be divided into estuarine/marine water and fresh water. Fresh water is found in both surface and subsurface reservoirs [94,95]. Surface water can further be classified into two categories: lentic and lotic water [96]. Lotic water refers to flowing water bodies such as rivers, streams, springs, and creeks, while lentic water is stagnant water bodies such as lakes, ponds, swamps, or bogs [97,98].

Aquatic environments are important pathways for bacterial dissemination [99]. Runoff from WWTPs, industries, hospitals, and farms flows into the natural environment, allowing MDR bacteria to spread from their anthropogenic sources to natural ecosystems. As a result, these ecosystems become reservoirs of ARB and ARGs (

Table 2. Environmental studies of ESKAPE members detected in surface water sources.

Chracteristics	Reports
Authors	Barthley P. S., et al. [102]	Freitas D. Y., et al. [103]	Mathys D. A., et al. [104]	Suzuki Y., et al. [105]	Azuma T., et al. [106]
Location	Brazil and United States	Brazil	United States	Philippines	Japan
Sample type	Surface water	Surface water	Treated wastewater and surface water	Wastewater and surface water	Influent and effluent wastewater, and surface water
Origin	Brazillian rural river and urban lake—American lake and WWTP	Amazonian lake	Metropolitan- rural WWTPs and open waterways-ocean waters	Hospital sewage and rivers	Hospital effluent, WWTP influent-effluent and river
ESKAPE members	K. pneumoniae and E. cloacae	A. baumannii, E. cloacae and K. pneumoniae	K. pneumoniae, E. cloacae, and P. aeruginosa	K. pneumoniae and E. cloacae	All
MDR	Yes	Yes	ND	ND	Yes
ESBL	Yes	Yes	Yes	Yes	Yes
MBL	Yes	No	Yes	Yes	Yes
ARG	Yes	Yes	Yes	Yes	No
VG	No	No	ND	ND	No
MGE	plasmid	Integrons class 1 and insertion sequences	Plasmids	Plasmids	ND
Type of pollution	Fecal and metals	Normal	ND	ND	ND
Site with more %MDR	Brazilian and United States urban lakes	Lake	ND	ND	Hospital effluents and WWTPs influents

Note(s): Multidrug-resistance (MDR); extended-spectrum beta-lactamases (ESBL); metallo-betalactamase (MBL); antibiotic-resistance genes (ARG); virulence genes (VG); wastewater treatment plant (WWTP); no data (ND).

) [100,101]. This transfer of resistance genes poses risks to human health, as susceptible pathogenic bacteria associated with humans can acquire theses resistance genes and become resistant.

Several reports have highlighted the role of surface waters as potential reservoirs of antibiotic resistance, fecal contamination, antibiotic resistance genes, and Enterobacterales. Bartley P. S., et al. [102] conducted a study in 15 locations around an urban lake and 10 locations along a rural river system in Northeast Brazil (July 2015), comparing them with five locations in an urban lake and sewer system in Northeast Ohio in the United States (October 2015). The study revealed a high degree of coliforms (Coliscan Easygel media) in both the Brazilian and US urban lakes, with a significant presence of antibiotic-resistant bacteria (ARB). However, Enterobacterales resistant to fluoroquinolones, aminoglycosides, trimethoprim/sulfamethoxazole, and cephalosporines were more frequently identified in an urban lake in Brazil compared to other locations. Water samples were subjected to screening for plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrS, and aac(6′)-Ib-cr) as well as carbapenemase genes (bla_NDM, bla_SPM, bla_IMP, bla_OXA-48, bla_KPC, and bla_VIM) using PCR. Among the detected genes in the water samples, bla_OXA-48 was the most prevalent, being present in 100% of the samples from the southern shore and 43% of those from the northern shore of the Brazilian urban lake. Furthermore, the study employed a microarray assay based on match ligation-mediated amplification, hybridization, and detection to screen for various types of β-lactamase genes (bla_KPC, bla_NDM, and bla_CTX-M-1, bla_CTX-M-15, bla_CTX-M-2, bla_CTX-M-8, and bla_CTX-M-25) in ESBL-positive Enterobacterales strains. Additionally, PCR was used to detect the presence of carbapenemasa genes (bla_OXA-48-like, bla_VIM, and bla_IMP).

The report also identified 40 Enterobacterales, with K. pneumoniae (10 strains) and E. cloacae (2 strains) ranking as the second and third most prevalent bacterial species, respectively. The susceptibility testing, following CLSI 2015 guidelines, revealed that 89% of the K. pneumoniae strains exhibited ESBL production and MDR, with the concurrent presence of also bla_CTX-M, bla_TEM, and bla_SHV ESBL genes, as well as plasmid-mediated quinolone resistance (PMQR) gene aac(6′)-Ib-cr. Among the E. cloacae strains, resistance was observed solely against trimethoprim/sulfamethoxazole, polymyxin B, and aminoglycoside. Notably, the genes bla_KPC, bla_OXA-48, bla_NDM, bla_IMP, and bla_VIM genes were not detected in any of the Enterobacterales strains. These findings strongly indicate that urban sites exhibit substantial contamination levels, potentially stemming from significant human fecal contamination, which could be linked to factors suchs as the prevalence of septic tanks in 43% of households and the prominence of small-scale agriculture among residents. This pervasive contamination presents a notable health hazard due to the diverse recreational activities undertaken in the area. Furthermore, the fecal contamination is likely a contributing factor to the elevated prevalence of antimicrobial resistance and genetic exchange.

Another study by Freitas D. Y., et al. (2019) focused on Lake Água Preta, located in the Amazonia region within the Utinga State Park in Pará, Brazil. The aim of the study was to evaluate the occurrence and diversity of third-generation cephalosporin-resistant Gram-negative bacteria. A total of 33 isolates were collected (MacConkey agar medium supplemented with cefotaxime 8 µg/mL⁻¹) from 18 samples at six selected sites in October 2014. The report indicated that water quality parameters, both physicochemical and microbiological, were within the limits set by legislation. Among the Gram-negative bacteria identified, three isolates of A. baumannii were found to be MDR profile according to CLSI 2017 guidelines, as well as resistance to heavy metals such as zinc, cadmium, cobalt, and arsenic. One MDR E. cloacae strain and five MDR K. pneumoniae strains carrying bla_TEM, and bla_SHV ESBL genes, as well as the intl1 gene, were also identified. None of the strains were carbapenem-resistant. Additionally, four MDR strains of Pseudomonas spp. were detected. Despite the water quality parameter being within normal limits, the presence of heavy metal-resistant A. baumannii and other bacteria indicates bacterial contamination and contributes to antibiotic resistance. Furthermore, it is worth noting that a higher number of ESKAPE bacteria were detected in a confined surface water body, all of which were MRD, ESBL producers, and possessed MGEs. This suggests that in stagnant lentic water, contaminants may persist for longer periods, leading to the detection of strains with MGEs, whereas in flowing loti water, the presence of such strains may be less pronounced.

Mathys D. A., et al. [104] reported the role of WWTPs in the dissemination of carbapenemase-producing bacteria (CPB) in surface waters. Samples were collected from treated effluent, and upstream/downstream surface water from 50 WWTP in 48 states across the United States in July and December 2016. All samples were processed in MacConkey broth modified with 0.5 µg/mL of meropenem and 70 µg/mL of zinc sulfate, with the broth then inoculated onto similarly enriched MacConkey agar. Among the 50 WWTPs sampled, 30% (15 WWTPs) yielded 30 CPB strains with clinically relevant genotypes. Most of these strains belonged to Enterobacterales, primarily K. pneumoniae and E. cloacae, and were predominantly found in metropolitan areas, both downstream and upstream of the WWTPs. Additionally, only one E. cloacae was found in the effluent of a WWTP in a rural area. These bacteria carried carbapenemase genes (bla_KPC, bla_NDM), ESBL genes (bla_TEM, bla_SHV, bla_CTX, bla_OXA, bla_CARB, bla_KPC, and bla_MIR), and multiple plasmids several incompatibility groups. Interestingly, a higher number of CPBs were detected in WWTPs effluents that employed chlorination compared with those using ultraviolet light treatment in metropolitan zones. However, no significant difference was observed in the proportion of CPB between WWTP effluents and upstream or downstream surface water samples. These findings demonstrate a diverse mixture of CPB species with resistance to carbapenems, ESBL, and plasmids. Nevertheless, it is important to note that only 30 strains clinically relevant strains from the ESKAPE group were identified among the CPB. The efficacy of the treatment technology used in WWTPs is another crucial aspect, with ultraviolet light treatment appearing to be more advantageous than chlorination. Finally, the study suggests that other WWTPs, combined stormwater/sewer overflows, or sanitary sewer overflows may contribute to the proportion of CPBs detected in surface water, highlighting the presence of additional pollution sources.

Suzuki Y., et al. [105], conducted a study to assess the prevalence and genetic characteristics of carbapenemase-producing Enterobacterales in seven hospital sewage systems (four of which had sewage treatment) and ten river water sites in the Philippines in August 2016 and August 2018. A total of 83 samples were collected and plated on CHROMagar mSuperCARBA, and 124 carbapenemase-producing Enterobacteriaceae were identified (according to CLSI 2015 guidelines). Among them, 11 K. pneumoniae isolates (seven strains in hospital and four strains in rivers) were detected, carrying genotypic carbapenemase genes (bla_KPC and bla_NDM) as well as an ESBL gene (bla_CTX). Additionally, nine E. cloacae strains were identified, with seven strains found in hospitals and two strains in rivers. These E. cloacae strains exhibited genotypic resistance to carbapenemase (bla_NDM and bla_IMI) and ESBL gene (bla_CTX). The most prevalent carbapenemase gene detected was NDM, and all of them harbored plasmids with different incompatibility groups. The study observed a higher proportion of ESKAPE members and other Enterobacterales in hospital sewage compared to river water. However, the report indicated that K. pneumoniae isolates, primarily belonging ST147 (carriying bla_NDM-1 and bla_NDM-7), and one belonging to ST11 (bla_KPC-2) constitute major carbapenemase-producing clones. Furthermore, six E. coli isolates, belonged to CC10, were found to carry various carbapenemase genes including bla_NDM (comprising bla_NDM-1, bla_NDM-5, and bla_NDM-7), bla_KPC-2, and bla_OXA-48-like (encompassing bla_OXA-48 and bla_OXA-181). These genes were detected both in clinical settings and in the environment, possibly suggesting that some hospital and domestic sewage is directly discharged into municipal sewage systems, thereby leading to the presence of carbapenemase-producing Enterobacterales (CPE) in river water. Similar observations of E. coli CC10 have been documented in the Danube Riber, Yamato Riber, and sewage in Pakistan. Moreover, the presence of bla_NDM-1, associated with ST147, and bla_KPC-2, linked to ST11 in K. pneumoniae, has also been also reported in clinical settings in Germany and China. Once again K. pneumoniae and E. cloacae as members of the ESKAPE group, were prevalent in both water sources and exhibited clinically relevant genotypic resistance to carbapenems. This highlights the concern regarding the persistence of these strains in the environment and the potential for genetic transfer. Another important aspect is the lack of treatment systems in some hospitals, which could contribute to an increased proportion of persistent clinically relevant strains in the environment and facilitate genetic transfer.

Azuma T., et al. [106], conducted a study focused on characterizing the ability of sunlight to inactivate ARB and antimicrobial-susceptible bacteria present in wastewater and river water within various locations, including hospital effluents, WWTP influent and effluent, WWTP secondary effluent, and the Yodo River Basin in an urban area of Japan. A total of 24 samples were collected from six sites, four times in different seasons in 2018 and 2019. The study analyzed six classes of ARB suggested by Word Health Organization (WHO): carbapenem-resistant Enterobacterales (CRE), ESBL-producing Enterobacterales (ESBL-E), MDR Acinetobacter (MDRA), MDR P. aeruginosa (MDRP), methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) using selective chromogenic media (chromID CARBA, chromID ESBL, chromID MRSA, chromID VRE New, CHROMagar MDRA, and CHROMagar MDRP). The results showed that Hospital effluents and WWTP influents had higher bacterial concentrations compared to WWTPs effluents treated with chlorination, WWTP secondary effluent, and river water. The bacterial concentration in WWTPs effluents with chlorination and river water exhibited a similar range. In decreasing order, the classes of bacteria with the highest concentrations were CRE, ESBL-E, MDRA, MDRP, MRSA, and VRE. In surface water, the classes with the highest bacteria concentration were CRE, ESBL-E, MDRA, VRE, and MDRP in that. Regarding the inactivation of ARB, the study found that ozonation of WWTPs effluents was an effective option for disinfecting pathogenic microorganisms, elimination of organic pollutants, physiologically active substances, pharmaceuticals, and personal care products. Additionally, the study observed the inactivation of ARB by sunlight, with different bacteria showing varying times for >90% removal. CRE, ESBL-E, MDRA, MDRP, and VRE, were almost completely inactivated after 5 h of sunlight exposure. This study directly focused on the ESKAPE group in aquatic sources, assessing the relevance of different technologies in wastewater treatment processes and their prevalence in surface water. The findings indicated that untreated wastewater from hospitals and WWTPs harbored pathogenic bacteria with carbapenem and ESBL resistance profiles, predominantly from Enterobacteriaceae family. The effectiveness of the treatment process system used was associated with the proportion of bacteria present in surface water. Both sunlight and chlorine disinfection demonstrated similar results in ARB inactivation during the flow of water into the river. However, ozonation appeared to be a better option for inactivating both pathogenic and no pathogenic bacteria. The prevalence of CRE in surface water, despite efforts to remove it, is a critical issue to consider due to the pathogenic nature and resistance profiles of these strains. Addressing this issue requires improvement strategies, technology, infrastructure maintenance, and monitoring in wastewater treatment. However, the health risk posed by these strains in the environment is not clear yet. Therefore, is necessary to complement this information with studies involving cases of infections acquired from environmental water sources by members of the ESKAPE group.

According to the findings from the above-mentioned studies, it is evident that comprehensive monitoring of antibiotic-resistant bacteria and their respective resistance genes in aquatic environments is almost important at present. Efficient management of sewage, particularly in healthcare facilities, and increased awareness among healthcare professionals and the public regarding the growing threat of antibiotic resistance in the environment should be emphasized. Furthermore, future research focused on understanding the dynamics of bacterial persistence, scenarios of genetic transfer, and ecological equilibrium within water body niches is still necessary. Addressing these issues appropriately would contribute to the preservation of the efficacy of current antibiotics essential for the treatment of significant bacterial pathogens, while also safeguarding human and environmental health.

3.3. Mexico: Overview of ESKAPE Bacteria and Their Distribution in Water Sources

In Mexico, there is currently a lack of research on the prevalence and behavior of the ESKAPE group in aquatic environments. However, several environmental studies have been conducted to investigate the dominant bacterial populations and to characterize bacteria with specific traits in water bodies. Some of these studies have reported the presence of enteric bacteria that coincide with members of the ESKAPE group, as summarized in

Table 3. Environmental studies of ESKAPE bacteria in Mexico.

Characteristics	Reports
Authors	Galarde-López M., et al. [107]	Tapia-Arreola A. K., et al. [108]	Fuentes M. D., et al. [109]	Lendo-Hernández H. M., et al. [110]	Delgado-Gardea M.C.E., et al. [111]
Location	Mexico City	Estado de Mexico, Queretaro, Michoacan, Guanajuato and Jalisco	United States–Mexico border	Tepic, Nayarit, Mexico	Ocampo, Chihuahua, Mexico
Sample type	wastewater (treated—untreated)	Surface water	Surface water	Surface water and clinical	Surface water
Origin	HWWTP	Lerma River Basin and Lake Chapala	Río Grande	Mololoa River	River, waterfall, and stream
ESKAPE strains	K. pneumoniae	K. pneumoniae	K. pneumoniae	E. faecium and E. fecalis	E. cloacae and K. pneumoniae
MDR	Yes	Yes	Yes	Yes	Yes
ESBL	Yes	Yes	Yes	Yes	ND
MBL	Yes	Yes	Yes	Yes	No
ARG	Yes	Yes	Yes	Yes	ND
VG	No	Yes	No	Yes	ND
MGE	ND	ND	Integrons 1 and 2	No	No
Type of pollution a	ND	ND	Water quality	Mercury, cadmium, chromium	Water quality

Note(s): Hospital wastewater treatment plant (HWWTP); no data (ND); multidrug-resistance (MDR); Extended-spectrum beta-lactamases (ESBL); metallo-betalactamase (MBL); antibiotic-resistance genes (ARG); virulence genes (VG); mobile genetic elements (MGE); ^a type of pollution or analysis performed.

and discussed below.

One such study conducted by Galarde-López M., et al. [107], aimed to determine the presence and persistence of carbapenemase-producing Klebsiella spp. in raw wastewater and treated wastewater from two tertiary hospitals: the Regional High Specialty Hospital of Ixtapaluca (HRAEI) and the National Institute of Oncology (INCAN) WWTPs at Mexico City Metropolitan Zone (February 2020). These WWTPs are equipped with a pretreatment process, involving the removal of coarse waste, followed by aeration, clarification, and sedimentation, and finally, disinfection and exposure to ultraviolet light. The water samples were plated on HiCrome^TM ECC Agar plates. The study identified a total of 30 Klebsiella spp. isolates, with 26 strains identified as K. pneumoniae and four as K. oxytoca. The distribution of K. pneumoniae in HRAEI was 7 in raw wastewater and 7 in treated wastewater, while in INCAN, it was 11 in raw wastewater and 8 in treated wastewater. Susceptibility testing using the minimal inhibitory concentration method (CLSI 2021) revealed that 18 Klebsiella spp. were resistant to ampicillin-sulbactam, ceftazidime, and ceftriaxone, while 16 of 17 strains were resistant to piperacillin-tazobactam, cefoxitin, doripenem, ertapenem, imipenem, cefepime, and ciprofloxacin. Notably, 80% of K. pneumoniae strains from treated wastewater in both hospitals exhibited resistance to cephalosporins and carbapenems. Furthermore, 81.2% of carbapenem-producing Klebsiella spp. were identified as ESBL producers, with the detection of bla_KPC and bla_OXA-48-like genes. It is important to highlight that 81.25% of Klebsiella spp. Carried the bla_KPC gene. The focus of this report was on carbapenemase-producing Klebsiella spp. strains isolated from treated and untreated wastewater in two WWTPs employing advanced technology. Despite the relatively low quantity of strains obtained, K. pneumoniae was found to be the most prevalent species, and its abundance remained significant even after wastewater treatment. Another noteworthy finding is the high prevalence of resistance to cephalosporins, penicillin with inhibitors, and carbapenem, indicating that at least 80% of K. pneumoniae isolates were ESBL producers. Although the report specifically mentions resistance mechanisms, it is possible that 16 strains may be MBL producers, given their resistance to imipenem, doripenem, and ertapenem.

Tapia-Arreola A. K., et al. [108] conducted a study aimed at determining the frequency of antimicrobial-resistant Gram-negative bacteria along the Lerma River basin and Lake Chapala through five states of Mexico (Estado de Mexico, Queretaro, Michoacan, Guanajuato, and Jalisco) in February 2021. The samples were enriched in Luria Bertani broth and plated onto CHROMagar^TM ESBL and CHROMagar^TM mSuperCARBA. A total of 59 isolates were collected, with K. pneumoniae being one of the most noteworthy strains detected in six sites out of 20 sampled sites. Other identified strains included six species of Pseudomonas spp. and Acinetobacter spp. (excluding A. baumannii). In general, the report indicated that out of 59 strains, 58 exhibited resistance to β-lactam antibiotics, with high resistance frequencies observed against cefotaxime and ceftazidime. Additionally, 44% of the isolates showed resistance to carbapenems (imipenem and meropenem), followed by fluoroquinolones. K. pneumoniae (10.2%) was the third most prevalent strain exhibiting MDR according to CLSI 2021 criteria, preceded by Citrobacter freundii (13.6%) and Serratia marcescens (11.9%). The molecular screening revealed the presence of various resistance-associated genes, including bla_CTX-M, qnrB, and bla_OXA which are associated with resistance to β-lactams, fluoroquinolones, and carbapenems, respectively. Furthermore, a high diversity of virulence genes was detected, which K. pneumoniae shared with C. freundii and E. coli. This report highlights a high prevalence of MDR strains, with K. pneumoniae once again being one of the most prevalent species and exhibiting a high percentage of resistance to β-lactams and carbapenems. This observation suggests the possible presence of ESBL and MBL producers among the isolates. Moreover, K. pneumoniae possesses clinical significance due to the high frequency and diversity of virulence genes identified. Additionally, the presence of Pseudomonas spp. was notable, with P. putida detected at a frequency of 8.6% exhibiting MDR, although P. aeruginosa was not found. P. putida is also another clinically relevant species within the Pseudomonas genus that could contribute to the acquisition of infections through environmental sources.

Fuentes M. D., et al. [109] conducted a pilot test to assess the concentration of antibiotic residues, the presence of mobile genetic elements, genes encoding ESBL, and MDR bacteria in the Rio Grande. Fifteen water samples were collected along a 26 km stretch the river, encompassing three sites: El Paso, TX (site 1), an area associated with dairy, cotton, cattle, and farming products processing; Sunland Park, NM (site 2), a zone impacted by effluents from Sunland Park WWTP; and Juarez, Mexico (site 3), a region known for high recreational activities during February, April, July, September, and December 2017. The results revealed fecal contamination levels exceeding the standard set by the Texas Commission of Environmental Quality, with a concentration of 126 MPN/100 mL. ESBL genes were detected in 73% of the samples, with bla_TEM in 60% and bla_CTX-M in 47% of the samples. Class 1 integrons were detected at 47% of the sample sites, while class class2 integrons were found at 20% of the sample sites. Samples were plated in bile esculin azide agar, modified mTEC agar, mEndo agar and mannitol salt agar. A total of 310 isolates were collected, and 28 isolates were selected for molecular analysis of ESBL genes. Among these isolates, 14.3% carried the bla_SHV gene, 46.4% bla_TEM, 14.3% carried bla_SHV in conjunction with bla_TEM and 3.6% carried bla_TEM along with bla_CTX-M. The overview of isolates revealed that 91 isolates exhibited resistance to at least two synergistic antibiotic combinations, 101 isolates were resistant to at least four individual antibiotics, and 21 isolates displayed resistance to 20 or more individual antibiotics, primarily observed at sites 1 and site 3. K. pneumoniae was the second most prevalent strain identified, characterized by the presence of bla_TEM, and bla_SHV genes, as well as carbapenem-resistance. Bacteria identification and antimicrobial susceptibility patterns was carried out by MicroScan autoSCAN-4 automated bacterial identification system employing MicroScan panels NBPC 34 and PBPC 20 panel. This report not only highlights the presence of various prevalent enterobacterial isolates but also underscores the abundance of ESBL genes and MGE in surface water sources, both exposed and unexposed to wastewater. These findings suggest horizontal gene transfer (HGT) among the environmental microbiome. Among the 28 MDR enterobacteria identified with ESBL genes and carbapenem resistance, K. pneumoniae was the second most frequently identified strain after E. coli. This report unveils a substantial selection pressure resulting from the presence of β-lactam genes and MGE in the Rio Grande, contributing to the resistant profiles observed in enterobacteria. Hence, these findings may indeed signify a matter of public health significance, potentially given rise to gastrointestinal and opportunistic infections, particularly on those locales designated for recreational purposes. Furthermore, considering the Rio Grande´s status as a transitional zone bridging two borders and accommodating diverse activities, an ongoing monitoring of the microbiome, with particular emphasis on clinically pertinent bacterial strains, becomes imperative. To provide more comprehensive insight, it is advisable to complement such investigations with an analysis of virulence profiles.

Lendo-Hernández H. M., et al. [110] conducted a study in the Mololoa River, located in the city of Tepic, Nayarit, Mexico with a specific focus on E. faecium and E. fecalis. A total of 48 samples were collected from four polluted sites along the Mololola River in 2011 were plated on agar KF plates. The findings revealed that all the isolated strains demonstrated tolerance to heavy metals, with resistance observed up to concentrations of 325 µM of mercury, cadmium, and chromium. Microbiological analysis indicated the presence of E. fecalis strains exhibiting resistance to β-lactams, penicillin with inhibitors, oxacillin, linezolid, and imipenem, which are considered last-line antibiotics. Furthermore, these strains displayed MDR and carried various virulence genes (aceI, efaA, asa1, gelE, and esp), exhibiting a higher prevalence compared to E. faecium. Notably, these strains were isolated from locations near municipal wastewater discharge points, and trash confinement areas with contamination from industrial and municipal sources [110]. Similar to the findings of Fuentes M. D., et al., this report also highlights the high tolerance of isolates to heavy metals. This factor is significant as it provides selective pressure on bacteria, facilitating the development of resistance mechanisms due to the antimicrobial properties of heavy metals. Additionally, the detection of enterococci with a wide range of resistance and virulence genes close to polluted wastewater sources indicates significant pollution origin directly impacting surface water sources with pathogenic enterococci. Moreover, it suggests the possible presence of bacterial pathogens in the river water. Consequently, it is imperative to implement improved wastewater treatment systems, repair drainage, and sewage infrastructure, and closely monitor bacterial pathogens in the water. Furthermore, although Delgado-Gardea M. C. E., et al. [111] falls outside the scope of reports published within the last 5 years, it provides valuable information concerning the characterization of ESKAPE group in environmental water sources Basaseachi Falls National Park, Ocampo, Chihuahua, Mexico. The study involved the collection of 49 surface water samples from 13 selected sampling sites along the Basaseachi waterfall and its main rivers were collected in 2013. Samples were plated on Salmonella and Shigella agar, TCBS agar, and MacConkey Agar. The results indicated that the surface water exceeded Mexican standards for total and fecal coliform counts, with a concentration of 3.5 MPN/100 mL. Among the 33 Gram-negative isolates identified, the majority were E. coli followed by E. cloacae and K. pneumoniae. E. cloacae exhibited resistance to penicillin with inhibitors, second-, third-, and fourth-generation cephalosporins, aztreonam, and a single isolate demonstrated MDR according to AutoScan-4 system and CLSI 2016 guidelines. In this case, most of the sample sites exhibited fecal pollution that exceeds the established by Mexicans. This observation could be directly associated with human activities or a deficient water treatment system in the area. Moreover, there was a low quantity diversity of bacteria identified. Regarding E. cloacae it is not specified whether the same strain or the three strains are resistant to all the antibiotics previously listed, which would the potential ESBL production. Therefore, further information is needed to clarify this aspect. Overall, most of the reports conducted in Mexico have identified contamination sources near garbage dumps, recreational areas, crop fields, and wastewater treatment plants, indicating direct contamination by human activities. Moreover, poor water quality contributes to exerting selective pressure and facilitates the exchange of genetic material. The most frequently detected ESKAPE strains were K. pneumoniae, E. cloacae, and E. faecium all of which exhibited MDR profiles and resistance to carbapenems. The persistence of these strains in surface waters poses a significant microbiological risk with the potential to impact public health. It is evident that there is a lack of sufficient reports in Mexico focusing on the ESKAPE groups of bacteria in water sources. Given that Mexico is currently experiencing high migratory movements and population growth, it is anticipated that these reported figures could increase and diversify. Therefore, it is strongly recommended to enhance the strategies employed for wastewater treatment, improve sewage infrastructure, and closely monitor ESKAPE bacteria.

4. Conclusions

First and foremost, ESKAPE bacterial pathogens pose a significant health risk in hospital settings. However, the emerging issue of their transmission from clinical environments to the wider environment must be closely monitored. Untreated wastewater from hospitals has been observed to harbor a high quantity of MDR strains, potentially contributing to HGT. Conversely, treated wastewater from hospital WWTPs has shown a significant reduction in bacterial load when equipped with efficient technology. Similarly, municipal WWTPs are sources of abundant bacterial load, but efficient treatment processes can effectively remove most bacteria, although a small proportion of ESKAPE members may persist and flow into surface waters.

Inadequate wastewater treatment plays a crucial role in surface water pollution by wastewater, allowing the survival of numerous antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) during the treatment process, thus turning surface waters into an important reservoir for the exchange of ARGs and the emergence of ARB pathogens. Moreover, surface water serves as a vehicle for dissemination, as humans can acquire these pathogens through direct or indirect contact. On the other hand, efficient wastewater treatment processes, such as ultraviolet light treatment, have demonstrated satisfactory results by significantly reducing bacterial load in large urban areas and mitigating dissemination risks. However, various environmental factors, including human activities, contribute to the persistence of ARB pathogens, emphasizing the need for continuous monitoring of surface and residual water bodies, as well as the implementation of water sanitation strategies. Although this phenomenon is more pronounced in urban areas where wastewater flow into deep surface waters or bodies with larger volumes, cases reflecting adverse effects have been observed, particularly when effluent wastewater from deficient WWTPs leads to high bacterial loads in surface water, potentially leading to the of pathogenic MDR bacteria.

Lastly, K. pneumoniae, E. cloacae, E. faecium, and E. faecalis have been identified as the most prevalent and persistent MDR members of the ESAKPE group in wastewater and surface water, capable of surviving water treatment processes. Their presence in aquatic environments exerts selective pressure in the presence of various contaminants, antibiotic residues, resistance, and virulence genes, making them a significant concern. However, the distribution and prevalence of the entire ESKAPE group in different aquatic environments remain challenging to determine due to limited studies. Furthermore, it is important to acknowledge the contribution of the ESKAPE group to the overall microbiome of aquatic bodies. Additionally, based on the literature reviewed, an increase in other Gram-negative strains associated with clinical infections and possessing MDR and virulence profiles has been observed. Therefore, it is recommended to closely monitor the distribution of the ESKAPE group and other clinically relevant strains in the environment.