Comprehensive Assessment of Multidrug-Resistant and Extraintestinal Pathogenic Escherichia coli in Wastewater Treatment Plant Effluents

Multidrug-resistant (MDR) Escherichia coli poses a significant threat to public health, contributing to elevated rates of morbidity, mortality, and economic burden. This study focused on investigating the antibiotic resistance profiles, resistance and virulence gene distributions, biofilm formation capabilities, and sequence types of E. coli strains resistant to six or more antibiotic classes. Among 918 strains isolated from 33 wastewater treatment plants (WWTPs), 53.6% (492/918) demonstrated resistance, 32.5% (298/918) were MDR, and over 8% (74/918) were resistant to six or more antibiotic classes, exhibiting complete resistance to ampicillin and over 90% to sulfisoxazole, nalidixic acid, and tetracycline. Key resistance genes identified included sul2, blaTEM, tetA, strA, strB, and fimH as the predominant virulence genes linked to cell adhesion but limited biofilm formation; 69% showed no biofilm formation, and approximately 3% were strong producers. Antibiotic residue analysis detected ciprofloxacin, sulfamethoxazole, and trimethoprim in all 33 WWTPs. Multilocus sequence typing analysis identified 29 genotypes, predominantly ST131, ST1193, ST38, and ST69, as high-risk clones of extraintestinal pathogenic E. coli. This study provided a comprehensive analysis of antibiotic resistance in MDR E. coli isolated from WWTPs, emphasizing the need for ongoing surveillance and research to effectively manage antibiotic resistance.


Introduction
Multidrug-resistant (MDR) bacteria pose a significant threat to public health [1].MDR is characteristically defined as resistance to three or more classes of antimicrobial agents [2].MDR bacteria are the predominant etiological determinants responsible for therapeutic failure in the context of infectious diseases, thereby engendering increased duration and magnitude of morbidity, mortality rates, and economic burden on healthcare systems [3].MDR bacteria are commonly linked to infections acquired in healthcare settings, and some species have increasingly become the common cause of community-wide infection [1].
Wastewater treatment plants (WWTPs) play a crucial role as reservoirs and origins of antibiotic-resistant bacteria, as well as antibiotic resistance genes that govern their resistance [4].The infiltration of antibiotics and their metabolites into wastewater primarily stems from the overuse of antibiotics, improper disposal methods, and the excretion of pharmaceuticals by humans and animals [5].Additionally, pharmaceutical compounds and resistant bacteria can infiltrate wastewater treatment systems through the discharge of hospital, industrial, and residential wastewater, which is eventually released into the environment.Research on WWTPs as crucial sites for the spread of antibiotic and multidrug resistance has been ongoing [6].
Escherichia coli is a prominent pathogenic micro-organism that causes various infections, including increasingly severe cases linked to antimicrobial-resistant strains that contribute to rising morbidity and mortality rates [7].E. coli plays a crucial role in the dissemination of antibiotic resistance and serves as a valuable indicator of transmission pathways because of its widespread distribution, cohabitation in ecological niches similar to those of other enteric pathogens, and potential transmission through shared routes [8].Although E. coli is intrinsically susceptible to most clinically relevant antimicrobial agents, it possesses a remarkable capacity to accumulate resistance genes through horizontal gene transfer [9]; this allows it to acquire diverse genetic elements, such as plasmids, integrons, and transposons, thereby gaining a broad spectrum of antibiotic resistance genes and developing multidrug resistance [10].Currently, the increasing prevalence of multidrug resistance in E. coli is a global concern affecting human and veterinary medicine [9].MDR E. coli strains may experience selective pressure in human, animal, and environmental settings, leading to the emergence of high-risk MDR E. coli lineages.These high-risk clones exhibit enduring fitness and widespread dissemination, associated with significant variability in resistance and virulence genes [10].
Extraintestinal pathogenic E. coli (ExPEC) lineages are the primary causative agents of a majority of extraintestinal infections in humans worldwide, leading to substantial direct healthcare expenses and societal burdens.Although ExPEC strains encompass a multitude of lineages, only a specific subset is responsible for most infections [11].Prior research has demonstrated that strains affiliated with phylogroups A and B1 are typically commensal, whereas those associated with phylogroups B2, D, and E exhibit characteristics consistent with those of extraintestinal pathogenic strains [12,13].The predominant approach for discerning clonal complexes or lineages associated with ExPEC is multilocus sequence typing (MLST) [11].A multitude of sequence types (ST)s and clonal complexes (CC) have been characterized based on the genetic markers of E. coli.The most commonly documented lineages include ST131, ST69, ST10, ST405, ST38, ST95, ST648, ST73, and ST1193, which were identified in infections associated with healthcare and community settings [10].Thus far, investigations on MDR ExPEC strains have predominantly focused on isolates derived from human samples, while there is a scarcity of comprehensive studies simultaneously assessing MDR ExPEC using culture-based methods with effluents from WWTPs.
The objective of this study was to evaluate the antibiotic resistance of strains isolated from WWTP effluent, particularly those that exhibited resistance to six or more antibiotic subclasses.Additionally, we aimed to ascertain the prevalence of antibiotic resistance and virulence genes within these strains, as well as evaluate their biofilm-forming ability.Furthermore, we aimed to identify the predominant sequence type through MLST analysis.

Isolation and Identification of E. coli
In 2021, effluent samples were collected from 33 WWTPs in Korea.To isolate pure strains of E. coli from these samples, a 100-µL aliquot of the wastewater effluent was streaked onto CHROMagar™ orientation agar (CHROMagar, Paris, France) plates using a disposable bacterial loop.Following an incubation period of 24 h at 35 • C, we observed pink colonies and subsequently transferred them onto MUG Nutrient Agar (Difco Laboratories, Sparks, MD, USA) plates for species identification.After 24 h of incubation, fluorescent colonies were selected using ultraviolet illumination.To confirm that the isolated strains were E. coli, we performed either 16S rRNA sequencing or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) [14].

Phenotyping of Antibiotic Resistance
The phenotypic profiles of the E. coli isolates were assessed using the microdilution method, which is a common technique for testing antimicrobial susceptibility.Bacterial suspensions adjusted to a 0.5 McFarland standard were introduced into a 96-well microtitration plate (KRNV5F, Daejeon, South Korea), which is commercially available for use in the livestock industry and contains 16 antimicrobial agents.Following a 1-day incubation at 35 • C, the minimum inhibitory concentration (MIC) was determined either through visual inspection or using an automated reader.This reader greatly simplifies reading microdilution tests and ensures accurate recording of results by effectively identifying microbial growth within the wells.The MIC endpoints were determined based on the predefined breakpoints for the 16 antimicrobial agents in the panel according to the guidelines established by the Clinical Laboratory Standard Institute [15] and the European Committee on Antimicrobial Susceptibility Testing [16].The antibiotic resistance rate was calculated as a percentage, representing the number of strains exhibiting values above the MIC breakpoint for each antibiotic divided by the total number of strains.

Determination of Phylogroups
Phylogenetic characterization of all E. coli isolates (designated A, B1, B2, C, D, E, and F) was performed using quadruplex PCR with four specific phylogenetic markers, chuA, yjaA, TspE4.C2, and arpA.This approach closely followed the procedure established by Clermont et al. [28].Additionally, secondary PCR was performed to differentiate between groups E and C, targeting the arpA and trpA regions, respectively.Furthermore, groups B2 and F were subdivided into a newly identified group designated G using a triplex PCR assay that encompassed the trpA, cfaB, and ybgD markers, in accordance with the methodology proposed by Clermont et al. [29].

Multilocus Sequence Typing Analysis
Seven housekeeping genes, adk, fumC, gyrB, icd, mdh, purA, and recA, were amplified using the recommended primers [30].The products were purified using a QIAquick PCR Purification Kit (Qiagen Inc., Hilden, Germany) and sequenced by the Macrogen Sequencing Service (Macrogen, Seoul, Republic of Korea).Allele numbers for the seven gene fragments of each isolate were determined via comparative analysis with the corresponding alleles accessible at on https://pubmlst.org/(accessed on 4 October 2023).The allelic numbers and their corresponding genotype designations were allocated by the curator on the MLST website.

Biofilm Analysis
Biofilm formation assays were performed as described previously [31][32][33], with some modifications.Bacterial strains were cultured in Muller Hinton Agar and standardized to a density of 0.5 McFarland units in 0.85% NaCl medium.A 10-µL aliquot of each suspension was diluted 1:20 in 190 µL of Luria-Bertani (LB) medium in 96-well plates.Following incubation at 37 • C for 24 and 48 h, the plates underwent triple washing with 0.85% NaCl medium, and the remaining adherent bacteria were fixed with 200 µL of methanol per well.After air drying, the wells were stained with 0.1% crystal violet for 20 min.Subsequently, the wells were rinsed with distilled water and air dried.The crystal violet dye bound to the attached cells was resuspended in 200 µL of ethanol, and optical density (OD) was measured at 550 nm.
Biofilm formation was categorized as negative, weak, moderate, or strong.The cut-off value (ODc) was defined as the mean OD value above three standard deviations of the negative control.All experiments were performed in triplicate, and the results were averaged.The OD values measured at 595 nm for the negative controls served as the ODc, following a previously described method [31][32][33], with slight modifications.The biofilm-forming ability was classified as follows: OD < ODc, nonbiofilm producers; ODc < OD < 2 × ODc, weak producers; 2 × ODc < OD < 4 × ODc, moderate producers; and OD > 4 × ODc, strong producers.

Determination of Antimicrobial Residue
The 16 antimicrobial residues in the wastewater samples were determined as follows: Initially, each sample (500 mL) was pretreated via filtration through a 0.2-µm PVDF filter.Subsequently, 900 µL of the filtered sample was transferred into amber autosampler vials.To this, 100 µL of a 1% acetic acid solution, 40 mg/mL of ethylene diaminetetra-acetic acid disodium salt dihydrate (Na 2 EDTA), and 10 µL of 10 ng/mL isotopically labeled standards were added.The resulting pretreated sample (200 µL) was then subjected to high-performance liquid chromatography coupled with tandem mass spectrometry [34].The presence of residual antibiotics and their average concentration in WWTPs from 33 sampled facilities have been delineated in the results.

Statistical Analysis
To examine the relationship between phenotypes and genotypes, we conducted a chisquare test.This statistical test was chosen to determine if there is a significant association between the categorical variables under study.All statistical analyses were performed with a significance level set at p < 0.05.Specifically, the chi-square test results were considered statistically significant if p < 0.001.
We screened 14 virulence genes, and the results are presented in Table 1.fimH had the highest prevalence (97.3%), followed by fyuA, traT (both at 59.5%), and kpsMTII (43.2%).Notably, ipaH, stx1, and stx2 were not detected.In the context of functional categorization, analysis of the screened virulence genes revealed that the genes associated with bacterial adhesion, fimH, and iron acquisition, fyuA, were prevalent in most strains.Additionally, traT, associated with serum resistance, was detected in over 50% of the strains, whereas east1 was associated with toxin and hemolysin production.

Assessment of Biofilm Formation Capability
Assessment of biofilm-forming proficiency demonstrated that only 31% of the examined samples were capable of forming biofilms.Within this subset, 27%, 1%, and 3% possessed a weak, moderate, and strong ability to form biofilms, respectively (Figure 3b).

Residual Antibiotic Measurement
We investigated the residual concentrations of 18 antibiotics during the dry and wet seasons, focusing on effluents from 33 WWTPs.The results are presented in Table 3.Seven antibiotics (ciprofloxacin, sulfamethoxazole, trimethoprim, ceftazidime, tetracycline, cefepime, and meropenem) were detected in the dry-season samples, whereas six antibiotics were detected in the wet-season samples, except for meropenem.Ciprofloxacin, sulfamethoxazole, and trimethoprim were detected at all 33 WWTP effluent sampling points.

Discussion
In this study, we investigated the antibiotic resistance rates of 918 E. coli strains isolated from WWTP effluent.We focused on the strains exhibiting resistance to six or more antibiotic classes.To our knowledge, this study is the first comprehensive analysis of antibiotic resistance in MDR E. coli isolated from WWTPs using culture-based methods, encompassing analyses of antibiotic resistance genes, virulence genes, biofilm formation ability, and MLST analysis for E. coli typing.Overall, E. coli isolates from the WWTPs exhibited antibiotic resistance in the following order: ampicillin, nalidixic acid, tetracycline, sulfisoxazole, and streptomycin.In the case of MDR strains, the major antibiotics with high resistance rates were nearly identical, albeit with different orders.MDR strains demonstrated increased resistance to most antibiotics.Moreover, our investigation revealed concurrent resistance, mirroring earlier research outcomes [35], wherein every chloramphenicol-resistant strain exhibited concomitant resistance to tetracycline and ampicillin.Notably, in contrast to the typical pattern observed for most antibiotics, colistin and meropenem did not manifest an inclination toward increased resistance rates in MDR strains.This suggests a potentially independent mechanism of colistin-and meropenem-induced resistance compared with that of other antibiotic classes.Recent studies have highlighted the significant resistance rates of E. coli strains to various antibiotics, underscoring an urgent concern regarding antibiotic resistance in WWTPs.In South Africa, E. coli exhibited a substantial resistance rate of 92.2% to sulfamethoxazole and an MDR rate of 81.11% in WWTPs [36].In Japan, ampicillin-resistant E. coli showed the highest prevalence among antibiotic-resistant strains, followed by those of levofloxacin, cefotaxime, ceftazidime, and tetracycline in WWTPs [37].
Bacteria carrying extended-spectrum beta-lactamases (ESBL) commonly possess one of the three gene types bla TEM , bla CTX-M , and bla SHV , thereby enhancing their ability to develop resistance against β-lactam antibiotics [38].Moreover, ESBL-producing E. coli are more inclined to exhibit resistance to multiple drugs than E. coli that do not produce ESBL [39].In the present study, the majority of MDR E. coli strains possessed bla TEM and bla CTX-M , with the presence of bla SHV notably lacking.Within the CTX-M groups, CTX-M-1 and CTX-M-9 emerged as the prevalent types, consistent with trends observed in previous studies conducted in Korea [40].One significant observation was the predominant detection of CTX-M-8 in MDR E. coli strains associated with phylogroup D. Specifically, CTX-M-8 was detected in 100% of the ST38 strains, which are ExPEC lineages.This emphasizes the importance of understanding the genetic factors that determine antibiotic resistance in distinct bacterial lineages.
Correlation analysis between phenotype and genotype showed significant associations between cephalosporins, such as cefoxitin, ceftazidime, and ceftiofur, and the bla TEM and bla CMY-2 genes, and between nalidixic acid and the qnrS gene, consistent with previous research findings [41,42].In the case of streptomycin resistance, it has been found to be closely associated not only with strA and strB but also with tetA, tetB, and bla TEM .Conversely, cases where the phenotypic characteristics of E. coli antibiotic resistance did not align with the genotypic features were frequently observed, consistent with previous research [43].This phenomenon is explained by the increased frequency and sustained presence of MDR isolates, even when antibiotic selection pressure is absent, due to the influence of co-selection mechanisms [43].
Beyond examining antibiotic-resistance mechanisms through phenotypic and molecular analyses, assessing bacterial virulence profiles has emerged as a paramount concern in contemporary microbiological research.Virulence in E. coli is orchestrated through mechanisms, such as adhesion, toxin synthesis, the synthesis of polysaccharide capsules, iron acquisition via siderophores, invasion, and the production of additional factors designed to target immune cells [38].We established that 97.3% of MDR E. coli strains harbored fimH, whereas 59.9% harbored ibeA and traT.The potential surface virulence factor fimH is common in E. coli, and fimH mediates cell adhesion, thereby assisting in the formation of bacterial biofilms [44].Virulence genes exhibited a slightly higher prevalence in isolates from hospital-acquired infections than in isolates from community-acquired infections, with fimH (71.8%) and fyuA (68.2%) being the most commonly distributed virulence genes [45].In the present study, MDR E. coli displayed a high prevalence of fimH, which is associated with cell attachment.However, the ability to form biofilms was mostly absent or weak.Specifically, 69% of MDR E. coli formed no biofilm, whereas approximately only 3% formed strong biofilms.This finding contrasts with recent assessments of biofilm formation in fish processing facilities [46], where the majority of organisms showing moderate to strong biofilm-forming capabilities were identified as MDR.Conversely, a recent investigation targeting uropathogenic E. coli uncovered an inverse correlation between biofilm formation and antibiotic resistance [47].This implies a cost of resistance to bacterial cells, suggesting that strains with lower resistance may rely on biofilms to enhance survival, as demonstrated in our study.This may explain why the biofilm formation capability of MDR strains was low in this study.
In a recent review by Kocsis et al. [10], the dissemination of extraintestinal pathogenic high-risk international clones of E. coli was extensively discussed, with a particular focus on major clones, such as ST131, ST1193, ST38, ST10, ST69, ST73, ST405, ST410, and ST457.Our study confirmed that MDR bacteria from WWTPs were mainly ExPEC strains, and 44.6% of the isolated E. coli strains, which were resistant to six or more antibiotic classes, were linked to the high-risk international clones highlighted by Kocsis et al.Furthermore, as reported by Wang et al. [48], MLST analysis confirmed the predominant association between human-derived E. coli strains and ST1193, ST73, ST648, ST131, ST10, and ST1668.Additionally, a Danish study [49] revealed that 38% of ESBL-producing E. coli isolates from patients were epidemic MDR E. coli ST131 within the B2 group.Consequently, we deduced that the predominant MDR E. coli isolated from the WWTPs correlated with human-derived strains.However, this study did not specifically focus on hospital wastewater; instead, it encompassed the entirety of domestic wastewater generated in urban areas.Therefore, there are some limitations to considering this solely due to human-derived pathogenic E. coli.Thus, further research spanning from hospital wastewater to the influent of WWTPs is deemed necessary for precise source tracing.
ExPEC strains are generally assigned to phylogroups B2 and D [50].Moreover, these strains survive in municipal wastewater treatment processes, particularly those associated with ST131 ESBL-producing E. coli [51,52].ST131 and ST1193 within phylogroup B, and ST69 and ST38 within phylogroup D, were predominant in our study.ST131, as documented in studies conducted across Brazil, Nigeria, and Austria, exhibits significant pathogenic potential, MDR, and involvement in infections affecting humans and livestock [53].ST131 also exhibits a heightened propensity for biofilm formation and the manifestation of antibiotic resistance [54].ST1193 is a newly recognized worldwide MDR clone with high-risk potential, significantly contributing to community-onset urinary and bloodstream infections.Since 2012, the prevalence of ST1193 has been increasing worldwide, with reports of it replacing ST131 in certain regions [55].ST38 and several other ExPEC lineages have emerged as a predominant strain in recent years and have been consistently isolated from extraintestinal infections worldwide [56].
Recent findings indicated that ExPECs, including clinically significant strains of urinary pathogenic E. coli, are present in treated wastewater effluents and demonstrate a specific adaptation to endure wastewater treatment procedures [51].Therefore, we may have observed a high proportion of ExPECs among the MDR bacterial strains isolated from WWTPs.MDR ExPECs pose a potential public health risk that necessitates continuous monitoring.Further research is needed to manage antibiotic resistance within WWTPs.Such efforts are expected to play a crucial role in minimizing the impact of antibiotic resistance on public health.
The findings of this study highlighted the critical need for enhanced strategies to combat antibiotic resistance in wastewater treatment plants.The identification of high-risk MDR E. coli strains, such as ST131, ST1193, ST38, and ST69, underscores the urgency of implementing advanced treatment technologies and stricter regulatory measures.Currently, research is underway to effectively reduce antibiotic resistance and resistance genes using advanced technologies, such as advanced oxidation processes, membrane filtration, and UV disinfection [57], and such research should continue in a preventive capacity in the future.Moreover, routine monitoring and surveillance programs must be established to track the evolution and dissemination of antibiotic-resistant bacteria and antibiotic-resistant genes within WWPTs.Stakeholders, including policymakers, public health officials, and WWTP operators, can leverage these results to inform targeted inventions and policy frameworks aimed at mitigating the spread of antibiotic resistance.By adopting a multifaceted approach that combines technological advancements, stringent regulations, and continuous monitoring, it is possible to significantly curb the proliferation of AMR and safeguard public health.

Conclusions
This study represents the first comprehensive analysis of antibiotic resistance in MDR E. coli isolated from WWTPs using culture-based methods.Through examination of antibiotic resistance genes, virulence genes, biofilm formation ability, and E. coli typing, we confirmed a close association between MDR E. coli from WWTPs and human-derived strains, particularly extraintestinal pathogenic E. coli, such as ST131, ST1193, ST38, and ST69.These findings emphasize the crucial importance of managing antibiotic resistance in WWTPs and underscore the necessity for ongoing monitoring and further research to mitigate the public health impact.

Table 1 .
Prevalence of virulence genes in multidrug-resistant E. coli.

Table 1 .
Prevalence of virulence genes in multidrug-resistant E. coli.

Table 2 .
Profiles of extraintestinal pathogenic sequence types from wastewater treatment plants.

Table 2 .
Profiles of extraintestinal pathogenic sequence types from wastewater treatment plants.

Table 3 .
Residual antibiotic concentrations in the effluent samples from wastewater treatment plants during dry and wet seasons.