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Article

Characterization of Escherichia coli Isolates in Recreational Waters: Implications for Public Health and One Health Approach

by
Lúcia Gomes
1,
Adriano A. Bordalo
1,2 and
Ana Machado
1,2,*
1
Laboratory of Hydrobiology and Ecology, Instituto de Ciências Biomédicas Abel Salazar (ICBAS—UP), University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
2
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR—UP), University of Porto, Novo Edifício do Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal
*
Author to whom correspondence should be addressed.
Water 2024, 16(18), 2695; https://doi.org/10.3390/w16182695
Submission received: 11 August 2024 / Revised: 12 September 2024 / Accepted: 21 September 2024 / Published: 23 September 2024
(This article belongs to the Section Water and One Health)
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Abstract

:
Escherichia coli is commonly found in the gastrointestinal tract of warm-blooded animals and is routinely used as an indicator of fecal contamination in recreational waters. While most E. coli are commensal, some can harbor pathogenic potential, posing a major public health risk. This study investigated the sources of fecal contamination in recreational waters through the characterization of E. coli isolates. Presumptive E. coli detection was performed using selective and differential media, confirmed by PCR, and followed by characterization. E. coli were detected in all studied aquatic environments, with most isolates belonging to phylogenetic groups D1 (24%, non-human mammals) and B1 (20%, birds), suggesting animals as primary contaminants sources. Among E. coli isolates, 35% were identified as diarrheagenic E. coli (DEC), with enterotoxigenic (34%) and enterohemorrhagic (26%) being the most prevalent pathotypes. Furthermore, 85% of all isolates and 86.5% of DEC isolates exhibited multi-drug resistance, with highest rates displayed in phylogenetic subgroups A1 (human and non-human mammals) and B1. This study highlights the importance of the One Health approach encompassing a human, animal, and environmental holistic health perspective to effectively manage public health strategies and ensure the safety of beachgoers.

1. Introduction

Worldwide, the use of water for recreational purposes has been increasing, and activities in coastal waters are popular with both residents and tourists [1]. Recreational activities have a relevant social-economic impact on leisure, tourism, trading, and health promotion [2,3]. However, direct contact with water can also be a privileged route of exposure to potential pathogenic microorganisms, including those that are naturally present in water, which can lead to waterborne diseases [4].
Pathogens causing waterborne diseases are primarily introduced into recreational waters through fecal contamination, particularly near urban areas [5]. Sources of fecal contamination include improperly treated sewage, stormwater discharges, agricultural practices, surface runoff, influx from polluted rivers, ship discharges, and wild or domestic animals [4,6].
Currently, the European Union uses the levels of two Fecal Indicator Bacteria (FIB), Escherichia coli (E. coli) and intestinal enterococci (IE), to screen the recreational quality of water bodies and estimate the health risks for users [7,8]. This classification system is based on findings from various epidemiological studies that have shown a correlation between FIB levels in recreational water and the incidence of gastrointestinal illnesses [9,10].
E. coli is the most common facultative anaerobic bacterium inhabiting the digestive systems of warm-blooded animals, including humans [11]. Furthermore, research indicates that E. coli can survive in the environment without a host, becoming a natural part of bathing water, sand, soil, and algae [12,13]. Nonetheless, some strains can acquire pathogenetic or toxigenic virulent factors, becoming virulent for both humans and animals [14,15,16]. Therefore, the high prevalence of E. coli is a potential risk to both humans and animals and is associated with high rates of morbidity and mortality [17,18].
E. coli exhibits a clonal population structure in which strains can be assigned to one of the main phylogenetic groups: A, B1, B2, or D [19,20]. These groups differ in their host association, presence of virulence factors and persistence in the non-host environment [21]. Escobar-Páramo et al. [22] and Carlos et al. [19] investigated the frequency of specific phylotypes in humans, non-human mammals, and birds. These studies revealed a higher frequency of phylogenetic groups A and B2 in humans, A and B1 in non-human mammals, and D and B1 in birds. Based on the combination of the genetic markers described in Escobar-Páramo et al. [22], Carlos et al. [19] further detailed the phylogenetic groups, subdividing them into A0, A1, B1, B22, B23, D1, and D2.
While most E. coli strains are of commensal nature, a subset of these strains is capable of being pathogenic for humans and animals. Pathogenic E. coli can be divided into two main groups: intestinal pathogenic E. coli and extra-intestinal pathogenic E. coli. Intestinal pathogenic E. coli, also known as diarrheagenic E. coli (DEC), are primarily responsible for gastrointestinal diseases. Based on the phenotypic traits and virulence factors, DEC strains can be further classified into enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), and diffusely adhering E. coli (DAEC) [20,23,24]. Two of the five major DEC pathotypes, ETEC and EPEC, rank among the top-10 causes of diarrhea worldwide. In 2016, ETEC was estimated to cause 220 million reported cases and 51,186 deaths annually, while EPEC accounted for 14 million cases and 12,337 deaths each year [25]. Phylogenetically distinct from commensal and intestinal pathogenic E. coli groups, avian pathogenic E. coli (APEC) strains are categorized into the extra-intestinal pathogenic E. coli subgroup [26] and have been suggested as potentially foodborne zoonotic pathogens [27,28,29].
Antibiotic resistance is a major challenge in modern medicine, recognized as one of the greatest threats to human health in the 21st century [30]. In addition to human use, antibiotics and antimicrobials play a crucial role in crop and animal livestock efficiency [11]. Antibiotic misuse and overuse have contributed to the growing resistance of E. coli to antibiotics [31]. As a result, treating infections caused by E. coli is becoming increasingly challenging, as many strains are now resistant to first-line antibiotics and even to newer generations of antimicrobial agents [32,33].
E. coli ranks third in the list of twelve antibiotic-resistant priority pathogens described by the World Health Organization (WHO) [34]. E. coli antimicrobial resistance is considered a major public health concern since these bacteria can easily share resistance genes with other bacteria, acting as both donors and recipients [33]. Furthermore, E. coli prevalence in different organisms and environments, coupled with the ability to circulate between them, makes these bacteria recognized contributors to the dissemination of antibiotic resistance genes in natural environments [35,36,37].
Strategies to control and prevent the spread of enteric pathogens in recreational water include epidemiological studies, routine water monitoring, microbial source tracking, and public health communication and policy. To address the multifaceted issues of E. coli in aquatic environments, including pathogenicity, genetic diversity, and antibiotic resistance, adopting the One Health approach, a WHO initiative, that focuses on the interconnected health of people, animals, and ecosystems is essential.
This study sought to investigate the profile of E. coli strains retrieved from recreational waters in northwest (NW) Portugal, exploring the source contribution to the fecal contamination occurrence and the potential implications for public health.

2. Materials and Methods

2.1. Study Design and Sample Collection

In this study, recreational waters (estuarine and coastal beaches) and discharged effluents (raw sewage and treated wastewater) were analyzed to evaluate the fecal contamination in the urban northwest (NW) Portugal area (Figure S1).
A total of 30 surface water samples from 23 sampling sites were collected at ebb tide, using 0.5 L sterile plastic bottles, against the current to minimize the potential contamination by the operator. The samples were kept in the dark in a refrigerated ice chest and processed within 4 h of collection. Key environmental parameters (temperature, conductivity, salinity, pH, and turbidity) were measured in situ using a multiparameter YSI 6000 probe (YSI Incorporated, Yellow Springs, OH, USA).

2.2. E. coli Identification and Characterization

Water samples (from 1 mL of a 1:1000 dilution up to 100 mL, depending on the sample contamination) for microbial enumeration were filtered in triplicate onto sterile cellulose nitrate membranes (0.45 μm pore size, 47 mm diameter, Sartorius, Göttingen, Germany). Fecal coliforms and E. coli were assayed in mFC Agar (mFC, Difco, Le Pont de Claix, France, thermo-tolerant coliforms ISO 9308-1 [38]) at 44.5 °C, and Chromocult Coliform Agar (CHR, Merck, Darmstadt, Germany, ISO 9308-1) at 35 °C, respectively. Typical colonies were evaluated after 24 h of incubation, and the results were expressed as CFU/100 mL. On average, for each sample, 15 presumptive E. coli colonies were isolated and subcultured from the selective media to Luria–Bertani (LB) (Merck, Darmstadt, Germany) and stored at −80 °C in LB broth containing 25% glycerol for further identification and characterization.
E. coli DNA was isolated by the boiling method [39] through cell disruption and release of intracellular DNA. Briefly, an overnight culture (1 mL) was centrifuged for 10 min at 13,200 RPM. The supernatant was discarded, and the pellet was resuspended in 1 mL of nuclease-free water and incubated 15 min at 100 °C. The suspension was then centrifuged at 2000 RPM for 3 min, and the supernatant was transferred into a new sterile microtube, diluted 1:100, and used as a template for PCR.
E. coli confirmation was performed by PCR based on the 23S rRNA gene [40] and the uidA housekeeping gene [41]. Details, including primer sequences, expected amplicon sizes, annealing temperatures, and extension times, are given in Table S1. PCR reactions were performed using Go Taq® G2 Green Master Mix (Promega Corporation, Madison, WI, USA) in a 25 µL reaction volume containing each primer at the concentration displayed in Table S1 and 5 µL of DNA template. Vibrio cholerae CIP 62.13 was used as a negative control, E. coli ATCC 25922 was used as a positive control, and a reaction mixture with all reagents except the DNA template was used as a no-template control.
Agarose gel electrophoresis (2%) in 1X TAE buffer (0.04 mol/L Tris-acetate, 0.001 mol/L EDTA [pH 8.0]) was used to separate the PCR products, which were then stained with GreenSafe and viewed under UV light.
To assess their potential as pathogens, all E. coli isolates identified as positive underwent a comprehensive screening for virulence genes. The phylogenetic group determination was assayed by multiplex PCR of the genes chuA, yjaA, and TspE4C2 [42] (Table S1). The isolates were grouped into four phylogenetic groups based on the presence or absence of these genes: A (chuA−, TspE4C2−), B1(chuA−, TspE4C2+), B2 (chuA+, yjaA+), and D (chuA+, yjaA−). Following the method described by Escobar-Páramo et al. [22], isolates within each group were further differentiated into subgroups based on additional variations in the targeted genes: A0 (chuA−, yjaA−, TspE4.C2−), A1 (chuA−, yjaA+, TspE4.C2−), B1 (chuA−, yjaA−, TspE4.C2+), B22 (chuA+, yjaA+, TspE4.C2−), B23 (chuA+, yjaA+, TspE4.C2+), D1 (chuA+, yjaA−, TspE4.C2−), and D2 (chuA+, yjaA−, TspE4.C2+).
DEC pathotypes were assigned based on the presence of DEC virulence markers assessed by PCR (Table S1), following the criteria described by [43]. E. coli isolates lacking the DEC virulence markers were defined as commensal E. coli. Avian pathogenic E. coli-associated genes (APEC) were screened using primers and conditions described in Table S1.

2.3. Antibiotic Susceptibility Evaluation

The antibiotic susceptibility of each E. coli isolate was evaluated by the Kirby–Bauer disk diffusion method, according to the standard test method for antimicrobial disc susceptibility method [44,45]. To ensure a standardized bacterial concentration for testing, the inoculum density was adjusted to 0.5 McFarland turbidity standard using a spectrophotometer (λ = 625 nm). The adjusted inoculum was then plated on Mueller–Hinton agar (Liofilchem, Roseto degli Abruzzi, Italy) and incubated at 37 °C for 18 h.
The isolates were tested against 22 antibiotics (Oxoid, Hants, UK) belonging to 9 different classes: Aminoglycosides—Amikacin 30 µg, Gentamicin 10 µg, Kanamycin 30 µg, Tobramycin 10 µg, Streptomycin 10 µg; Carbapenems—Ertapenem 10 µg, Imipenem 10 µg; Cephalosporins—Cefotaxime 5 µg, Cefoxitin 30 µg, Ceftazidime 30 µg, Cephalothin 30 µg; Fluoroquinolones—Ciprofloxacin 5 µg, Nalidixic acid 30 µg; Macrolides—Erythromycin 15 µg; Miscellaneous agents—Chloramphenicol 30 µg, Nitrofurantoin 300 µg, Rifampicin 5 µg, Trimethoprim—sulfamethoxazole 25 µg; Monobactams—Aztreonam 30 µg; Penicillins—Amoxicillin—Clavulanic acid 30 µg, Ampicillin 10 µg; and Tetracyclines—Tetracycline 30 µg.
The inhibition zone diameter was measured and interpreted according to the EUCAST guidelines for the order Enterobacterales as resistant or susceptible [45]. In the case of the absence of EUCAST guidelines, the CLSI guidelines were followed [44]. Isolates with intermediate susceptibility were categorized as being susceptible. The strain Escherichia coli ATCC 25922 was used as quality control.

2.4. Data Analysis

The percentages of the pathotypes and the phylogenetic groups of E. coli isolates were examined using descriptive statistics.
The antibiotic resistance profiles of E. coli isolates were analyzed through a presence/absence matrix in the Primer 6 software package (version 6.1.11; [46]), which was used as input data to evaluate the similarity between isolates. Hierarchical clustering was conducted using the Bray–Curtis similarity method and the group average linkage method. The analysis employed a 5% significance level, with 1000 mean permutations and 999 simulations.

3. Results

In this study, a characterization of pathogenic antibiotic-resistant E. coli in recreational waters was conducted to understand their potential public health impact. Microbiological and physico-chemical analysis results are summarized in Table S2. Overall, coastal sampling sites exhibited lower fecal contamination levels compared to estuarine sites, which are located in areas with greater anthropogenic impact.
A total of 362 fecal coliform colonies were isolated from the different aquatic environments screened. PCR analysis with species-specific primers targeting both the 23S rRNA gene and the uidA housekeeping gene confirmed 75% (272/362) of the isolates as E. coli.
The isolates revealed high diversity being distributed across all four phylogenetic groups and seven respective subgroups (Figure 1). Regardless of the contamination level of the samples, the majority of the isolates belonged to the phylogenetic groups A and D, accounting for 33% (90/272) and 31% (84/272) of the isolates, respectively. The subgroups showing the lowest isolates frequency were the subgroups B22 (3%, 7/272) and D2 (7%, 18/272). Samples with lower E. coli concentrations showed a slightly lower frequency of subgroup B23 (humans) but a higher prevalence of subgroups A1 (non-human mammals) and D2 (non-human mammals and birds).
PCR screening for diarrheagenic genetic traits identified markers in 35% (96/272) of the E. coli isolates among the different aquatic environments evaluated (Table 1), with higher prevalence in isolates (43%) from estuarine water samples. ETEC E. coli was the higher frequency pathogenic type within this study, with 34% (33/96) of the isolates belonging to this pathotype. On the other hand, enterohemorrhagic E. coli (EHEC) was detected in 26% (25/96) of the isolates analyzed in estuarine waters, associated with animal origin (Figure 2). Sampling sites with lower fecal contamination revealed a slightly lower percentage of virulence genes.
Hybrid pathogenic pathotypes, harboring virulence factors from more than one DEC type, were detected in 10% (10/96) of the DEC isolates (Table 2).
While all the APEC-associated genes evaluated in this study have been successively identified, fyuA (44%, 120/272), ompT (39%, 106/272), and iss (36%, 98/272) genes exhibited the highest detection frequencies (Figure 3).
All 272 E. coli isolates exhibited resistance to at least one of the twenty-two antibiotics tested from nine different classes (Table 3). A total of 85% (232/272) (Figure 2 and Figure 4) of the isolates were classified as multi-drug resistant, meaning that resistance to at least one antibiotic from three or more different classes was observed. The high rate of multi-drug resistant isolates was observed regardless of the level of fecal contamination in the water sample. E. coli isolates displayed higher levels of resistance to miscellaneous agents and macrolides. Additionally, these isolates exhibited lower resistance to carbapenems and monobactams.
Rifampicin (99.2%) and erythromycin (98.5%) were the antibiotics to which a higher number of E. coli isolates were revealed to be resistant, while no isolates showed resistance to imipenem or nitrofurantoin (Table 3 and Figure 4). The isolates belonging to phylogenetic subgroups A1 (17/272) and B1 (17/272) showed the highest virulence and multi-drug resistance rates. Of the DEC isolates, 86.5% (83/96) exhibited multi-drug resistance (Figure 2). Hierarchical cluster analysis of E. coli isolates based on antibiogram profiles revealed a high degree of similarity among isolates, with all isolates clustering together with around 50% similarity, regardless of the pathotype or aquatic environment associated (Figure 4). Nevertheless, it was possible to identify clusters of E. coli isolates exhibiting higher resistance to specific antibiotic classes. Clusters A and B displayed higher resistance to penicillins, while cluster B also contained isolates with increased fluoroquinolone resistance (Figure 4).

4. Discussion

E. coli is a common intestinal bacterium found in warm-blooded animals, which can be readily detectable in various environmental conditions, making it a reliable, widely used indicator of fecal contamination [47]. E. coli can survive and persist in the aquatic environment, where antibiotic-resistant genes were known to be transferred horizontally through plasmid-mediated integrons from resistant to non-resistant bacteria [48]. Recreational waters have been identified as an important reservoir of antibiotic-resistant E. coli [48]. According to the GBD 2016 Diarrhoeal Disease Collaborators report [25], enterotoxigenic E. coli (ETEC) is one of the leading bacterial causes of diarrhea. ETEC is estimated to be responsible for approximately 220 million cases worldwide, affecting around 75 million children under five and leading to an annual death toll of between 18,700 and 42,000 people. The detection of E. coli responsible for diarrheal diseases has been employed extensively on clinical samples for epidemiological and diagnostic purposes. Comparatively, less research has been conducted to understand the distribution, survival, and pathogenicity of E. coli in surface water environments [49,50].
The ubiquitous presence of E. coli found across all recreational sampling sites studied, albeit at varying isolation rates, warranted further characterization. Despite the public health significance of E. coli in recreational waters, information on the phylogenetic distribution of the bacteria in the Atlantic temperate area of Portugal remains scarce. The present study identified a higher frequency of phylogroups A and D, similar to the findings of Pereira et al. [51] in the Tagus estuary (Lisbon region), using the Clermont triplex PCR method. Even so, the isolates belonging to groups B1 and B2 were also detected in both studies. The E. coli strains were isolated from recreational water sites impacted by human activity, which is consistent with previous research demonstrating a relationship between the B2 phylogroup and anthropogenic contamination [19].
Although all phylogenetic E. coli subgroups were found, a higher frequency of phylogenetic groups B1 and subgroup D1, which are typically linked to non-human mammal and bird origin, was detected, compared to human-associated subgroups B22 and B23. This predominancy suggests that animals may be a relevant contributor to the fecal contamination in recreational waters, a source often ignored by decision-makers. Moreover, the phylogenetic subgroups found in this study agree with the results of other recent studies performed in recreational waters worldwide [12,24,52].
Among the DEC isolates identified, ETEC was the dominant pathotype. This finding aligns with previous research by Ferdous et al. [24] and Alfinete et al. [53]. The detection of ETEC is worthy of concern since the heat-liable (LT) and heat-stable (ST) enterotoxin genes encoded on ETEC plasmids can be transmitted to other bacteria and increase the risk of severe diarrhea [54].
This study identified a concerning prevalence of EHEC, one of the most important zoonotic pathogens recognized under One Health, primarily associated with cattle [55] and linked to bloody diarrhea and potential kidney failure [56]. EHEC was detected in slightly over a quarter of the E. coli isolates from recreational waters, surpassing the prevalence reported in similar studies in the United States (14%), South Africa (3%), and Georgia (0.2%) [57,58,59].
In both developed and developing countries, EPEC strains have emerged as a major cause of watery diarrhea, which is often accompanied by dehydration, fever, and vomiting [60]. In this study, 12.5% of the isolates from recreational waters were identified as atypical EPEC (aEPEC). However, the same was not observed in South Africa, where aEPEC was the dominant pathotype detected [59]. This discrepancy could be attributed to geographical variations and potential differences in human activity impacting the water bodies. Distinguishing between typical and atypical EPEC is important due to the different disease associations. While tEPEC primarily affects infants, aEPEC has been reported to cause infections in both children and adults [61].
This study also identified 10% and 6% of the E. coli isolates as EIEC and EAEC, respectively. While EIEC primarily causes dysentery in humans and animals [62], EAEC is one of the major causes of DEC-associated food and waterborne enteric infections, particularly affecting immunocompromised individuals and children [63]. These results are similar to those reported by Ferdous et al. [24] in Bangladesh. However, the same was not observed in a South African study by Alfinete et al. [53], where EAEC was the second-most common pathotype found in the water, and EIEC was not detected.
Hybrid pathotypes were observed in 10% of DEC isolates, harboring variable combinations of virulence genes. The occurrence of these pathotypes may be explained by the ability of E. coli to acquire virulence genes through the mechanism of conjugation on mobile genetic elements (plasmids) [64]. The same trend was found in other studies from Bangladesh [24] and South Africa [53,59]. In the present study, 2% of the DEC isolates were identified as belonging to the EHEC/ETEC hybrid pathotype, which has previously been associated with diarrheal disease in both humans and animals [65]. Additionally, other hybrid pathotype combinations were detected, including EAEC/aEPEC (2%), aEPEC/ETEC (1%), and EAEC/EHEC (2%).
ETEC hybrid isolates combining ETEC and EHEC belonged to different phylogenetic subgroups (B23 and D1), ETEC/aEPEC (phylogenetic subgroup A1), and ETEC/EAEC (phylogenetic subgroup A1) were identified in this study. These results suggest that the ETEC strains may have a genetic background flexibility that allows them to acquire genes from other pathotypes, enhancing their infectious potential and ability to adapt to various ecological settings, including recreational waters [66]. Similar behavior has also been reported in hybrid isolates EHEC/EAEC (phylogenetic subgroups A1 and D1) and aEPEC/EAEC (phylogenetic subgroups A1 and D1) from a South Africa assessment [59]. The emergence of these virulent strains raises public health concerns due to the potential to cause severe diarrhea outbreaks [65].
Pathogenicity genes found in avian pathogenic E. coli (APEC) include iss, cnf1, and ibe10, which enhance survival in the bloodstream, cause cell death, and facilitate invasion of brain blood vessels [67]. In this study, the iss gene, which contributes to APEC’s ability to survive in the bloodstream, was detected in 36% of the E. coli isolates, suggesting the potential contribution of animals, such as birds, to fecal contamination in recreational waters. Moreover, since some APEC strains can be zoonotic [68], the results highlight the potential health risk to beachgoers.
E. coli have been isolated from aquatic environments in previous studies, with highly diverse antibiotic resistance profiles reflecting geographical determinants, sources of fecal contamination, and exposure to antibiotics [30,49,51,69,70,71,72]. This study detected high levels of antibiotic resistance among E. coli isolates from recreational waters, regardless of the overall fecal contamination in the water sample. All the isolates exhibited resistance to at least one of the antibiotics tested, and a high multi-drug resistance rate of 85% was observed. Rifampicin and erythromycin showed the highest antibiotic resistance levels, with percentages over 98%. These antibiotics are frequently used in both human medicine (e.g., leprosy, tuberculosis, pneumonia, skin conditions, dental abscesses, and sexually transmitted infections) and veterinary medicine (e.g., bacterial infections and gastrointestinal motility problems), potentially contributing to the widespread resistance observed in this study. In contrast, minimal resistance was detected for newer-generation antibiotics, such as nitrofurantoin, imipenem, and aminoglycosides, which are typically reserved for severe infections and in Portugal, only used in humans in hospital settings. The same was observed in the Portuguese Tagus estuary [51]. In contrast, a study by Bolukaoto et al. [59] in Bangladesh reported a much higher rate (70%) of resistance to imipenem, a broad-spectrum carbapenem antibiotic. This difference emphasizes the potential geographic variation in antibiotic resistance patterns since imipenem is often more economically advantageous in the context of developing nations [73].
The prevalence of antibiotic resistance among the E. coli phylogenetic groups was analyzed. Isolates belonging to phylogenetic groups A and D exhibited a higher prevalence of resistance to individual antibiotics, while multi-drug resistance was more frequent in groups A and B1. Conversely, group B2 isolates displayed lower resistance levels and a lower incidence of multi-drug resistance, which aligns with previous research suggesting their greater susceptibility to antibiotics [74]. Moreover, 86.5% of the DEC isolates displayed multi-drug resistance, with phylogenetic group B1 and subgroup A1 showing the highest multi-drug resistance rates. These findings align with previous research that indicated group B1 was more prevalent in environments with human impact [75,76].
A limitation of our study was the lack of regional validation for the phylogenetic group contamination source association method [19,22]. Microbial source tracking methods require such validation in new locations to ensure the selected microbial targets are present in the intended hosts and are exclusive to fecal sources.

5. Conclusions

Understanding the origin, virulence traits, and antibiotic resistance profile of pathogens that occur in aquatic environments is fundamental for assessing the potential risks for human infection. Our findings suggest humans are exposed to pathogenic antibiotic-resistant E. coli through recreational waters, potentially leading to infections. Moreover, our results suggest that animals are major contributors to the fecal contamination of recreational waters. The diversity of E. coli strains found in this study, which ranged from commensal inhabitants of the gastrointestinal tract to DEC capable of causing intestinal or extra-intestinal disorders, denote the strains genetic flexibility and ability to adapt to new ecological settings. The detection of hybrid pathotypes, virulence traits, and antibiotic resistance emphasizes the role of recreational waters as reservoirs and transmission enhancers, facilitating the dissemination of genetic traits that can lead to the emergence of more severe and treatment-resistant diseases. Mitigating fecal contamination in recreational waters, especially when dealing with antibiotic-resistant E. coli and significant contributions from animals, requires a multifaceted approach. This can include regular monitoring using advanced microbial source tracking techniques to help identify specific contamination sources, enhancing wastewater treatment, and improving waste management by installing animal waste disposal stations and enhancing sewage treatment facilities. Additionally, establishing vegetative buffer zones around water bodies can filter runoff and reduce contamination. Reducing seagull populations through habitat modification, scare tactics, or reproductive control methods and managing trash to minimize food sources is pivotal in areas where they contribute significantly to fecal contamination. Public education campaigns are essential for encouraging responsible behavior among users. This study highlights the importance of adopting the One Health approach to effectively manage public health risks associated with recreational waters and ensure safety for users.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w16182695/s1, Table S1: List of primers and PCR conditions used in the present work. Table S2: Identification, physico-chemical parameters and fecal contamination indicators for each sampling site evaluated. Figure S1. Location of the sampling sites in the NW Portugal identified by aquatic environment. References [77,78,79,80,81,82,83,84,85] are cited in the supplementary materials.

Author Contributions

Conceptualization, A.A.B. and A.M.; methodology, L.G. and A.M; validation, A.A.B. and A.M.; formal analysis L.G. and A.M.; investigation, L.G. and A.M.; resources, A.A.B. and A.M.; data curation L.G. and A.M.; writing—original draft preparation, L.G. and A.M.; writing—review and editing, A.A.B. and A.M.; supervision, A.A.B. and A.M.; project administration, A.A.B. and A.M.; funding acquisition, A.A.B. and A.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Project Forbath (E! 115737) under the EUREKA EUROSTARS EU Partnership Program for Innovative SMEs, and by ANI through national funds. This work was also partially supported by the project ATLANTIDA (NORTE-01-0145-FEDER-000040) and by the Project BeachSafe (PTDC/SAU-PUB/31291/2017), co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds. CIIMAR is supported by FCT/UIDB/04423/2020 and UIDP/04423/2020.

Data Availability Statement

All data generated or analyzed during this study are included in this published article [and its Supplementary Information files]. Notwithstanding, the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

The authors gratefully acknowledge Rui Oliveira and Claúdio Azevedo for their assistance during sample collection campaigns.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Distribution of E. coli isolates by phylogenetic groups and respective subgroups (n = 272). Each color represents a different phylogenetic group, and the variations in color intensities represent subgroups within the same group.
Figure 1. Distribution of E. coli isolates by phylogenetic groups and respective subgroups (n = 272). Each color represents a different phylogenetic group, and the variations in color intensities represent subgroups within the same group.
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Figure 2. Percentage of E. coli isolates resistant to different antibiotic classes (n = 272). Treemap showing the distribution of E. coli isolates with virulence genes (pathotypes) and multi-drug resistance grouped according to phylogenetic subgroup (n = 195). Enteroaggregative E. coli (EAEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC).
Figure 2. Percentage of E. coli isolates resistant to different antibiotic classes (n = 272). Treemap showing the distribution of E. coli isolates with virulence genes (pathotypes) and multi-drug resistance grouped according to phylogenetic subgroup (n = 195). Enteroaggregative E. coli (EAEC), enterohaemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC).
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Figure 3. Percentage of APEC-associated genes detected among the E. coli isolates studied (n = 272).
Figure 3. Percentage of APEC-associated genes detected among the E. coli isolates studied (n = 272).
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Figure 4. Hierarchical cluster analysis and heat map of the antibiotic resistance profile of E. coli isolates (n = 272) according to the aquatic environmental source. Isolates with 100% of similarity were clustered together. Thicker vertical lines divided the 9 different antibiotic classes used. From left to right: Aminoglycosides—Amikacin (AK) 30 µg, Gentamicin (CN) 10 µg, Kanamycin (K) 30 µg, Tobramycin (TOB) 10 µg, Streptomycin (S) 10 µg; Carbapenems—Ertapenem (ETP) 10 µg, Imipenem (IPM) 10 µg; Cephalosporins—Cefotaxime (CTX) 5 µg, Cefoxitin (FOX) 30 µg, Ceftazidime (CAZ) 30 µg, Cephalothin (KF) 30 µg; Fluoroquinolones—Ciprofloxacin (CIP) 5 µg, Nalidixic acid (NA) 30 µg; Macrolides—Erythromycin (E) 15 µg; Miscellaneous agents—Chloramphenicol (C) 30 µg, Nitrofurantoin (F) 300 µg, Rifampicin (RD) 5 µg, Trimethoprim—sulfamethoxazole (SXT) 25 µg; Monobactams—Aztreonam (ATM) 30 µg; Penicillins—Amoxicillin—Clavulanic acid (AMC) 30 µg, Ampicillin (AMP) 10 µg; and Tetracyclines—Tetracycline (TE) 30 µg.
Figure 4. Hierarchical cluster analysis and heat map of the antibiotic resistance profile of E. coli isolates (n = 272) according to the aquatic environmental source. Isolates with 100% of similarity were clustered together. Thicker vertical lines divided the 9 different antibiotic classes used. From left to right: Aminoglycosides—Amikacin (AK) 30 µg, Gentamicin (CN) 10 µg, Kanamycin (K) 30 µg, Tobramycin (TOB) 10 µg, Streptomycin (S) 10 µg; Carbapenems—Ertapenem (ETP) 10 µg, Imipenem (IPM) 10 µg; Cephalosporins—Cefotaxime (CTX) 5 µg, Cefoxitin (FOX) 30 µg, Ceftazidime (CAZ) 30 µg, Cephalothin (KF) 30 µg; Fluoroquinolones—Ciprofloxacin (CIP) 5 µg, Nalidixic acid (NA) 30 µg; Macrolides—Erythromycin (E) 15 µg; Miscellaneous agents—Chloramphenicol (C) 30 µg, Nitrofurantoin (F) 300 µg, Rifampicin (RD) 5 µg, Trimethoprim—sulfamethoxazole (SXT) 25 µg; Monobactams—Aztreonam (ATM) 30 µg; Penicillins—Amoxicillin—Clavulanic acid (AMC) 30 µg, Ampicillin (AMP) 10 µg; and Tetracyclines—Tetracycline (TE) 30 µg.
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Table 1. Single pathotypes and associated virulence genes detected in the E. coli isolates studied.
Table 1. Single pathotypes and associated virulence genes detected in the E. coli isolates studied.
Pathogenic TypeGene Detectionn (%)
Commensal-176 (65%)
DEC-96 (35%)
 ETECeltB6 (6%)
estA27(28%)
 EHECvt111 (12%)
vt23 (3%)
vt1 + eaeA3 (3%)
vt2 + eaeA8 (8%)
 aEPECeaeA12 (13%)
 EIECipaH10 (10.5%)
 EAECpCVD6 (6%)
Hybrid-10 (10.5%)
Note(s): DEC—diarrheagenic E. coli; ETEC—enterotoxigenic E. coli; EHEC—enterohaemorrhagic E. coli; aEPEC—atypical enteropathogenic E. coli; EIEC—enteroinvasive E. coli; EAEC—enteroaggregative E. coli.
Table 2. Hybrid pathotypes and associated virulence genes detected in the diarrheagenic E. coli isolates studied (n = 96).
Table 2. Hybrid pathotypes and associated virulence genes detected in the diarrheagenic E. coli isolates studied (n = 96).
Pathogenic TypeGene Detection Combinationn
EHEC/ETECvt1 + eltB1
vt1 + estA1
aEPEC/ETECeaeA + estA1
EAEC/ETECpCVD + estA1
EAEC/EAECpCVD + vt12
EAEC/aEPECpCVD + eaeA2
EHECvt1 + vt2 + eaeA2
Table 3. Antibiotic susceptibility of the E. coli isolates studied (n = 272).
Table 3. Antibiotic susceptibility of the E. coli isolates studied (n = 272).
Class of AntibioticAntibioticCodeDisk Content (µg)E. coli Isolates
ResistantSusceptible
AminoglycosidesAmikacinAK309263
GentamicinCN105267
KanamycinK303269
StreptomycinS10142130
TobramycinTOB106266
CarbapenemsErtapenemETP104268
ImipenemIPM100272
CephalosporinesCefotaximeCTX54268
CefoxitinFOX305267
CeftazidimeCAZ301271
CephalothinKF30150122
FluoroquinolonesCiprofloxacinCIP515257
Nalidixic acidNA3052220
MacrolidesErythromycinE152666
Miscellaneous AgentsChloramphenicolC3015257
NitrofurantoinF3000272
RifampicinRD52702
Trimethoprim-sulfamethoxazoleSXT2564208
MonobactamsAztreonamATM303269
PenicillinsAmoxicillin—Clavulanic acidAMC30124148
AmpicillinAMP1092180
TetracyclinesTetracyclineTE3046226
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Gomes, L.; Bordalo, A.A.; Machado, A. Characterization of Escherichia coli Isolates in Recreational Waters: Implications for Public Health and One Health Approach. Water 2024, 16, 2695. https://doi.org/10.3390/w16182695

AMA Style

Gomes L, Bordalo AA, Machado A. Characterization of Escherichia coli Isolates in Recreational Waters: Implications for Public Health and One Health Approach. Water. 2024; 16(18):2695. https://doi.org/10.3390/w16182695

Chicago/Turabian Style

Gomes, Lúcia, Adriano A. Bordalo, and Ana Machado. 2024. "Characterization of Escherichia coli Isolates in Recreational Waters: Implications for Public Health and One Health Approach" Water 16, no. 18: 2695. https://doi.org/10.3390/w16182695

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