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Article

Prevalence and Resistance Patterns of Campylobacter spp. and Arcobacter spp. in Portuguese Water Bodies

by
Igor Venâncio
1,
Inês Martins
1,
Rodrigo M. Martins
1,
Mónica Oleastro
2 and
Susana Ferreira
1,*
1
RISE-Health, Department of Medical Sciences, Faculty of Health Sciences, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
2
National Reference Laboratory for Gastrointestinal Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal
*
Author to whom correspondence should be addressed.
Water 2025, 17(18), 2767; https://doi.org/10.3390/w17182767
Submission received: 23 July 2025 / Revised: 4 September 2025 / Accepted: 15 September 2025 / Published: 18 September 2025
(This article belongs to the Special Issue Evaluation of Microbiological Indicators for Water and Wastewater Treatment and Reuse)
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Abstract

Campylobacter spp. and Arcobacter spp. are recognized etiological agents of gastroenteritis worldwide. While poultry is their best-known reservoir, human exposure can also occur via environmental pathways, particularly through contaminated water sources, which play a significant role in their transmission dynamics. In addition to their pathogenicity and widespread environmental prevalence, increasing antibiotic resistance has contributed to the global emergence of multidrug-resistant strains, hindering effective treatment. Here, the distribution and antibiotic resistance potential of Campylobacter spp. and Arcobacter spp. isolates collected from water bodies in Portugal were investigated. Water samples were collected from rivers, their tributaries, and springs, at 25 sites over a six-month period. Campylobacter spp. were isolated from 13.3% of the samples, whereas Arcobacter spp. were detected in 57.6% of the samples. Of the 27 isolated Campylobacter isolates, 44.0% were resistant to at least one antibiotic, while only one strain exhibited a multidrug-resistant (MDR) phenotype. In contrast, 98.9% of the 177 Arcobacter isolates were resistant to at least one antibiotic, with 15.8% classified as MDR. These findings contribute to the surveillance of Campylobacter spp. and Arcobacter spp., highlighting the critical role of aquatic environments in their epidemiology and supporting the need to incorporate waterborne transmission pathways into integrated surveillance and control strategies within the One Health framework.

1. Introduction

Clean water is fundamental to human, animal, and environmental health, embodying the core principles of the One Health approach. Despite significant efforts and progress by leading international authorities on public health and water quality, particularly in controlling and preventing infectious diseases, the World Health Organization estimates that 1.4 million people die each year due to inadequate drinking water, sanitation, and hygiene, with diarrheal disease accounting for most of the attributable burden. Noteworthy, most of these deaths are in low- and middle-income countries, and children under five represent a significant part of them [1].
Microbial contamination of water, including bacteria, viruses, protozoa, and helminths, contributes to increased degradation of the aquatic environment and can arise from interconnected human, animal, and environmental factors, such as agricultural runoff, wastewater discharge, domestic and wildlife interactions, and climate-change-driven disruptions [2,3]. Among these, fecal contamination of raw water, combined with increased proximity of waterfowl to humans, livestock, and poultry, can threaten water safety and potentially facilitate the spread of diseases across species and ecosystems. Contaminated water from diverse sources may act as a reservoir for bacterial pathogens, thereby amplifying the hazard [2,4]. Many waterborne bacterial pathogens primarily target the gastrointestinal tract and are subsequently shed in the feces of infected humans and animals, a transmission pattern characteristic of species within the Campylobacter and Arcobacter genera [2,5,6]. Although the ecology of Campylobacter and Arcobacter in aquatic environments have been characterized globally, their prevalence and impact in Portugal remain poorly understood [7,8].
Campylobacter and Arcobacter genera, both members of the Campylobacterales order, encompass closely related species characterized as fastidious Gram-negative organisms but ubiquitous in nature [9]. In the public health sphere, Campylobacter spp. is consistently ranked as the most reported cause of bacterial gastrointestinal infection worldwide, with some cases of campylobacteriosis being followed by postinfectious autoimmune disorders, such as reactive arthritis and Guillain-Barré syndrome [10,11,12]. Similarly, Arcobacter species can cause gastroenteritis as well as bacteremia in humans, and, in addition, reproductive diseases, such as abortion, are reported in animals [5,13]. Due to similar clinical manifestations, infections caused by Arcobacter spp. are often misdiagnosed as campylobacteriosis [5,14]. Within the Campylobacter genus, Campylobacter jejuni and Campylobacter coli are the most common causative agents of infections, although other non-C. jejuni/C. coli species have also been implicated in cases of gastroenteritis [15,16]. Among the Arcobacter genus, Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii are the most relevant species of public health concern [13]. While infections caused by these enteropathogens are not usually life-threatening, their infective dose can be low, and infections can induce severe discomfort [17].
Foods of animal origin and contaminated water are widely regarded as the main source of Campylobacter and Arcobacter infection [4,5,18]. Water has been implicated in Campylobacter spp. and Arcobacter spp. infectivity cycles either directly through drinking, domestic use, food production, or recreational purposes, or indirectly by colonization of livestock [19,20,21]. Environmental waters can act as reservoirs for these bacteria [22]; even though Campylobacter spp. and Arcobacter spp. survival outside the host may be limited and vary among strains, both bacteria can adapt and persist in aquatic environments, also supported by the ability to transition into a viable but non-culturable state [22,23,24,25,26,27]. Compared with Campylobacter spp., Arcobacter species are more versatile, with the ability to thrive in aerobic conditions and at lower temperatures, allowing it to persist in different hosts and environments for longer periods [5,13,28]. Therefore, these bacteria are commonly found in diverse water sources, including lakes and rivers, spring water, seawater, sewage effluent, agricultural runoff, treated wastewater, and surface water, and have been detected in groundwater and inadequately treated drinking water supplies [8,29,30,31,32,33].
Waterborne outbreaks caused by Campylobacter’ and Arcobacter’ have been reported worldwide, with epidemiological associations between their persistence in various water bodies and disease cases documented in either developed or developing countries [21]. Although Campylobacter is recognized as associated with waterborne outbreaks [34], the role of water as a source for sporadic infections might still be underrecognized [20].
In addition to their pathogenic potential and widespread environmental distribution, Campylobacter spp. and Arcobacter spp. also represent a significant public health concern due to their increasing antimicrobial resistance. Although most cases are self-limiting, antibiotic treatment is advised in patients with severe and chronic symptomatology, when infections are extraintestinal or in post-infectious scenarios, and is usually administered to immunocompromised individuals [5,6,12,13]. In those cases, antibiotic therapy includes fluoroquinolones and macrolides as first-line antibiotics, while carbapenems are recommended for severe Campylobacter spp. systemic infections [35]. Less consensual fluoroquinolones, tetracyclines, and aminoglycosides antibiotics classes are reported by some authors for treatment of Arcobacter spp. infections [13]. However, the resistance rates of these bacteria to various empirical antibiotics have hindered the effectiveness of antibiotic therapy [15,35,36,37,38,39,40,41,42,43]. Aquatic environments are of particular concern, as they frequently contain high levels of antibiotics and resistance genes, and connect different ecosystems, thereby facilitating the global dissemination of resistance. These water matrices can also function as reservoirs and transmission pathways for enteropathogens, allowing them to acquire or develop resistance [44,45,46].
Although water is globally recognized as an important transmission route in the epidemiology of campylobacteriosis and arcobacteriosis, studies in Portugal remain scarce, leaving significant knowledge gaps regarding their prevalence and antimicrobial resistance in aquatic environments. This study aims to address these gaps by investigating the occurrence and distribution of these pathogens, as well as characterizing their antibiotic resistance profiles.

2. Materials and Methods

2.1. Water Sample Collection

Sampling was conducted between October 2019 and July 2020 at 25 locations within the municipalities of Covilhã, Fundão, and Belmonte, Portugal (Figure 1). Over this period, water samples were collected during six-monthly sampling events for Campylobacter spp. and five for Arcobacter spp. At each site, a water sample was collected into sterile flasks of 1 L and 200 mL volumes and transported under refrigeration conditions to the laboratory, where they were processed on the same day. A total of 150 water samples were collected from the Zêzere River, its tributaries, and nearby spring water sources.

2.2. Campylobacter spp. and Arcobacter spp. Isolation

In the laboratory, two different protocols for water samples enrichment and isolation were employed to recovery of Campylobacter spp. and Arcobacter spp. from water samples considering the distinct physiological growth requirements of these genera. For Campylobacter spp., the method described by Jokinen et al. (2012) was utilized with some modifications [47]. Briefly, 1 L of water sample was filtered through a 0.45 µm pore diameter membrane (Ahlstrom, Barenstein, Germany), and then the filter was incubated in 25 mL of Bolton Broth (BB, Oxoid, UK) supplemented with 5% (v/v) defibrinated horse blood and Bolton Selective Supplement (Oxoid, Hampshire, England). After incubation, for 48 h at 37 °C under microaerophilic conditions (5% O2, 10% CO2 and 85% N2), 20 μL of enrichment broth was applied on mCCDA plates (CCDA selective Supplement, Oxoid, England), subsequently incubated at 42 °C in microaerophilic conditions for 48 h. After this period, at least three characteristic colonies were selected and subcultured in Blood Agar (BA, Oxoid, England) plates for 24 h at 42 °C in microaerophilia. Then, at least three colonies were transferred into two new BA plates, with one incubated in microaerophilia and the other in aerobiosis, at 37 °C for 24 h. All colonies that did not show growth in aerobic conditions were then subcultured for further analysis.
Similarly, for the isolation of Arcobacter spp. and according to Isidro et al. (2020) [38], after filtering 200 mL of water samples with sterile nitrocellulose membranes with pores of 0.22 µm (Advantec, Tokyo, Japan), these were incubated with 9 mL Arcobacter Broth (AB, Oxoid, England) supplemented with CAT Selective Supplement (Oxoid, England) at 30 °C for 48 h in aerobic conditions. Afterward, 200 μL of enrichment broth was applied onto a 0.45 μm pore diameter membrane filter, placed onto a BA plate supplemented with 5% (v/v) defibrinated horse blood, and allowed to passively filter at room temperature for 30 min. After removal of the filter, the plates were incubated at 30 °C for 24 h to 72 h under aerobiosis. As before, in each sample that showed typical colonies, at least three colonies were subcultured into BA plates, which were incubated for 24 h at 30 °C under aerobic conditions.
All selected colonies that tested positive for oxidase activity (Oxidase Test, Liofilchem, Teramo, Italy) and exhibited Gram-negative rod morphology were preserved at −80 °C in Brain Heart Infusion broth (BHI, Liofilchem, Italy) supplemented with 20% of glycerol for subsequent molecular identification.

2.3. Molecular Detection and Identification for Campylobacter spp. and Arcobacter spp.

Presumptive Campylobacter spp. and Arcobacter spp. colonies from the culture plates were identified at the genus level by PCR methods. DNA was extracted using boiling lysis method on isolates suspension. Molecular identification of Campylobacter spp. at the genus level was performed using the genus-specific PCR described by Linton, Owen, and Stanley (1996) [48]. Subsequently, all isolates positive for Campylobacter spp. were analyzed using Matrix-Assisted Laser-Desorption/Ionization Time-of-Flight Mass Spectrometry MS (MALDI-TOF, VITEK® MS, bioMérieux, Marcy l’Etoile, France) for species identification at the Gastrointestinal Infections Reference Laboratory of the National Institute of Health Doctor Ricardo Jorge. For isolates likely to belong to Arcobacter spp., these were first identified using the genus-specific PCR described by Harmon and Wesley (1996) [49], followed by identification at the species level using multiplex PCR (mPCR) previously described by Houf et al. (2000) [50], and mPCR proposed by Douidah et al. (2010) [51]. In cases of uncertainty, the atpA gene was sequenced to support species-level classification, in accordance with [52]. All the primers used for PCR techniques are designated in Table S1. Reference strains C. jejuni NCTC 11168, A. butzleri LMG 10828, A. cryaerophilus LMG 10829, and A. skirrowii LMG 6621 were used as positive controls.

2.4. Isolate Genotyping

Genetic diversity of isolates was evaluated by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR), using the primers described by Houf et al. (2002) [53] (Table S1). The isolates were cultured on BA plates, for 48 h at 37 °C in microaerophilic atmosphere for Campylobacter spp., and at 30 °C in aerobiosis for Arcobacter spp. isolates. DNA was extracted as described by Houf et al. (2002) [53], and used at a concentration of 25 ng/µL. After amplification, 10 µL of each product were analyzed by electrophoresis in a 2% agarose gel at 120 V for 2 h 15 min, stained and then visualized under UV light to identify the distinct profiles.

2.5. Antibiotic Susceptibility Testing

All confirmed water isolates with a distinct genetic profile obtained by ERIC-PCR were subject to antibiotic susceptibility testing using the disk diffusion method and the agar dilution method for Campylobacter spp. and Arcobacter spp. isolates, respectively. For Campylobacter spp. isolates, Müeller-Hinton Agar (MHA, Oxoid, UK) plates supplemented with 5% (v/v) defibrinated horse blood and β-Nicotinamide adenine dinucleotide (20 mg/L) (Sigma-Aldrich, St Louis, MO, USA) were inoculated with isolates suspensions adjusted to 0.5 McFarland. After, antibiotic disks representing β-lactams (ampicillin (10 µg)), macrolides (erythromycin (15 µg)), aminoglycosides (gentamicin (10 µg)), fluoroquinolones (ciprofloxacin (5 µg)), and tetracyclines (tetracycline (30 µg)) (Oxoid, UK) were placed on the plates and incubated microaerophilically at 37 °C for 24 h. For Arcobacter spp. isolates, the minimum inhibitory concentration (MIC) for the same five antibiotics belonging to different classes was determined, with antibiotic dilutions ranging from 256 to 0.06 μg/mL (ampicillin (NZYTech, Lisboa, Portugal), ciprofloxacin (Sigma-Aldrich, USA), erythromycin (Sigma-Aldrich, USA), gentamicin (Sigma-Aldrich, USA), and tetracycline (Sigma-Aldrich, USA)) incorporated into MHA plates supplemented with 5% (v/v) defibrinated horse blood, which were inoculated with 2 μL of each isolate suspension adjusted to 0.5 McFarland of turbidity and then diluted to 107 CFU/mL. After inoculation, plates were incubated for 48 h at 30 °C under microaerobic conditions. According to previous reports [38,54,55,56], no specific breakpoints have been established for Arcobacter spp. or for non-jejuni/non-coli Campylobacter species. Therefore, isolates were classified as susceptible or resistant using the MIC interpretative criteria provided in the EUCAST Clinical Breakpoint Tables (v. 15.0, January 2025) [57]. When no breakpoint was available, the following approach was applied: for non-jejuni/non-coli Campylobacter species, the breakpoints defined for C. jejuni (zone diameters) were used; for Arcobacter spp., molecular cutoff for ampicillin and ciprofloxacin were applied, while erythromycin’ breakpoints defined for C. coli were adopted due to their better correlation with previously reported molecular resistance markers; and when none of these criteria were suitable, breakpoints established for Enterobacterales were considered.

3. Results

Overall, Campylobacter spp. was detected in 13.3% (20/150) of the water samples (Table 1). Detection was highest in river samples (20.0%), followed by tributaries (16.7%), with spring water showing the lowest prevalence (6.7%). Among the 20 positive samples, C. jejuni was the most frequently isolated species (40.0%), followed by C. coli (35.0%), C. lari (20.0%) and C. upsaliensis (5.0%). C. coli was predominantly recovered from river samples, while C. jejuni was more commonly isolated from tributaries and spring water. C. upsaliensis was identified in only one sample from a river tributary, therefore considering this water source the one with the greatest species diversity. Analysis of the recovery rates from the 25 sampling sites over the six-month study period suggested a seasonal pattern, with the highest prevalence observed during the autumn and winter months (17%; October, November, December, and February) and the lowest during spring and summer (6%; March and July) (Figure 2).
All the positive isolates were analyzed by ERIC-PCR, and 27 distinct genetic profiles were obtained. Antimicrobial susceptibility testing of the 27 Campylobacter isolates against antibiotics from different classes revealed that all strains were susceptible to gentamicin and erythromycin. However, resistance was observed in 7.4% of the isolates to ciprofloxacin, 18.5% to tetracycline, and 33.3% to ampicillin. (Table 2). Among the different water sources, no ciprofloxacin resistance was detected in Campylobacter isolates recovered from river samples, while only a single isolate, C. jejuni from a river tributary, exhibited multidrug resistant (MDR) phenotype. Nevertheless, over 44.0% of the Campylobacter spp. isolates displayed resistance to at least one antibiotic class.
The overall prevalence of Arcobacter spp. species in the 125 water samples collected was 57.6% (72/125), with the highest frequency of detection observed in river samples (100.0%), followed by its tributaries (74.0%) and spring water (20.0%) (Table 3). Of the 72 positive water samples, A. butzleri was the most frequently isolated species (98.6%), followed by A. cryaerophilus (15.3%) and A. skirrowii (1.4%). Notably, A. butzleri and A. cryaerophilus were co-isolated from 10 samples, while A. butzleri and A. skirrowii from one sample of spring water. A. butzleri was the most commonly isolated species across all water sources, followed by A. cryaerophilus. Considering the recovery rates per sampling month a similar proportion was obtained during the autumn and winter months (October, November and February) (58%) and the spring and summer months (March and July) (56%) (Figure 2).
From the positive samples, 177 distinct genetic profiles were obtained by ERIC-PCR. When tested for susceptibility, 92.7% and 83.6% of the isolates exhibited resistance to ampicillin and tetracycline, respectively. In contrast, lower resistance rates were observed for ciprofloxacin (11.9%) and erythromycin (11.3%), with gentamicin showing the lowest resistance at just 5.1% (Table 4). Overall, with the exception of tetracycline, A. cryaerophilus showed higher resistance rates to the antibiotics tested compared to A. butzleri isolates. Among water sample sources, similar resistance rates were found between river and its tributaries. However, a higher percentage of the spring water’s isolates were resistant to ampicillin (100.0%), contrasting with ciprofloxacin and gentamicin, for which no resistance was observed among these isolates. A total of 15.8% of all isolates exhibited a MDR profile, including 14.5% of A. butzleri, 27.3% of A. cryaerophilus, and the single A. skirrowii isolate, being resistant to three or more classes of antibiotics. Nevertheless, a 98.9% mono-antibiotic resistance rate was obtained for Arcobacter spp. isolates, and 85.3% of all isolates exhibited resistance to antibiotics from two or more different classes.
Overall, Arcobacter spp. were detected at a higher prevalence (57.6%) than Campylobacter spp. (13.3%), with river samples consistently showing the highest contamination rates for both bacterial genera. A. butzleri emerged as the most prevalent Arcobacter species, while C. jejuni and C. coli were the dominant Campylobacter species. Regarding antimicrobial resistance, Campylobacter isolates demonstrated generally lower resistance rates than Arcobacter isolates, with the latter also showing higher MDR (15.8% vs. 3.7%).

4. Discussion

Campylobacteriosis remains endemic in developing countries, with notification rates rising since the post-pandemic period in the European Union. Also, and despite efforts by health authorities, a recent trend toward stabilization in human infections has been stated [59,60]. While consumption and handling of poultry meat is considered a major transmission route of human campylobacteriosis [18], human exposure to Campylobacter spp. may also occur through environmental pathways [22,61]. Evidence suggests that environmental sources, in particular water, may account for an additional 5.0–10.0% of human campylobacteriosis cases. However, the contribution of water as a transmission route is likely underrecognized and underestimated in current epidemiological assessments [62,63]. Water functions as a major transmission vehicle for Campylobacter spp., facilitating the circulation of infections among humans, poultry, wild birds, and domestic animals. This is particularly relevant in aquatic environments that support agricultural, recreational, cultural, and animal-related activities, where cross-species transmission and environmental contamination are more likely to occur [19,20,21,22]. Although Arcobacter spp. have been recognized globally as an emerging enteropathogen, its epidemiology, significance and transmission to humans and animals are not as thoroughly studied or established to those of Campylobacter spp. [21]. Conversely, the role of water in the transmission of Arcobacter infections is increasingly acknowledged [30]. In this context, the present study investigated the prevalence and antibiotic resistance profiles of Campylobacter spp. and Arcobacter spp. strains isolated from waterbodies in the central interior region of Portugal.
The overall prevalence of Campylobacter species in water samples (13.3%) is in accordance with recent studies, where prevalences between 4.4% and 15.4% were reported [64,65,66,67]. Still, a high variability of isolation rates has been reported across regions and countries for waters of different types, and prevalence rates over 50.0% have been described [29,68]. The differences observed may be attributed to variations in detection methods, sample size, local anthropogenic influences, and the physicochemical properties of the waterbody [69]. This challenge is further amplified when culturing fastidious bacterial pathogens such as Campylobacter spp., as they can rapidly transition into a viable but non-culturable state (VNBC), resulting in the underestimation of their true prevalence in environmental samples [70,71]. Consequently, Campylobacter spp. is rarely detected directly from water samples during outbreak investigations, primarily due to the delay between the initial contamination event and subsequent environmental sampling [31]. At the species level, C. jejuni was the most commonly isolated species, as previously reported [64,65,66,67], possibly due to its enhanced tolerance to environmental stress conditions compared to C. coli [31,68,72]. Moreover, the methods employed may preferentially detect the most commonly isolated culturable taxa, thereby underrepresenting the overall biodiversity present.
Regarding Arcobacter spp., although high isolation rates have also been reported in samples from various sources, the overall prevalence observed in this study (57.6%) was higher than that reported in several other studies conducted in recent years [73,74,75,76]. As with Campylobacter spp., the recovery of Arcobacter spp. from environmental samples is influenced not only by the ecological characteristics of the water sources and regional geographic and climatic differences, but also by the organism’s fastidious nature. Its ability to transition into VBNC state further complicates detection and contributes to potential underestimation in prevalence studies [70,71]. Importantly, the absence of standardized detection methods for Arcobacter spp., particularly in water samples, may contribute to the variability in reported prevalence rates across studies [21]. Despite some degree of inter-study variation, the overall prevalence observed in this investigation, as well as the prevalence in each type of water body, aligns with previously reported trends. In a review and meta-analysis on the Arcobactereaceae prevalence in aquatic environments, a pooled prevalence of 69.2% was reported, with higher detection rates in seawater (78.0%), surface water (64.5%), and groundwater (39.6%), being the prevalence lower in processing water (34.3%) and water intended for human consumption (3.2%) [8]. These findings align with the pattern documented in the present study, and underscore concern not only regarding the potential risks associated with recreational use of these water sources, but also their possible impact on the food chain. In terms of species diversity, A. butzleri, A. cryaerophilus, and A. skirrowii have previously been isolated from water samples, exhibiting similar proportional distributions to those observed in this study. In fact, Venâncio et al. (2022) presented in their meta-analysis, that A. butzleri remained the most frequently isolated species from water samples, showing the highest overall prevalence (58.3%) and being consistently recovered from all investigated water samples [8]. A. cryaerophilus followed, with an overall prevalence of 42.5%, and is commonly reported as the second most prevalent Arcobacter species in water. In contrast, A. skirrowii exhibited a much lower overall prevalence (12.7%), and was not detected in some categories of water samples analyzed [8]. While the identification methodologies used may have limitations in reliably differentiating all Arcobacter species, this study employed an integrated approach combining culture-based techniques with molecular methods, with a particular focus on the three species most frequently associated with human disease [21,77].
Human infections caused by Campylobacter spp. display a marked seasonality, with peak incidence typically observed from late spring through early autumn [59,60]. Consequently, fluctuations in Campylobacter presence in waterbodies are expected, mirroring variable risks of waterborne infections [68]. Nonetheless, in the present study, Campylobacter species were detected in environmental waters throughout the year, even the winter months. While some studies have demonstrated higher recovery rates during summer [59,65], or no significant seasonal variation [68], others have documented increased prevalence during autumn and winter compared to spring or summer [61], fully supported by the results obtained in this investigation. This finding is consistent with observations showing that favorable conditions such as high relative humidity, rainfall, lower UV radiation and lower temperatures are positively correlated with the prolonged survival of Campylobacter spp. in environmental water during autumn and winter months [78]. Nevertheless, these observations should be interpreted with caution, as the data does not cover the entire national territory and may not fully capture regional variability. In addition, although sampling was conducted across different seasons, the results should be regarded as indicative trends rather than conclusive evidence of seasonal patterns.
Arcobacter infections are infrequently reported and often underdiagnosed or misclassified [79], making it difficult to draw conclusions about seasonality in human arcobacteriosis. In water bodies, Arcobacter spp. prevalence can exhibit seasonal variations, often influenced by factors such as temperature, rainfall, and water quality, with higher detection rates observed during warmer months [80]. In the present study, no evident seasonal trend was noticed, aligning with other studies [75]. This may be primarily attributed to the greater environmental adaptability of Arcobacter spp. in comparison to Campylobacter spp. [5,13,28].
Campylobacter spp. and Arcobacter spp. resistant to antibiotics used in clinical and veterinary practice have been isolated from water samples [15,38]. Given the importance of monitoring antimicrobial resistance in isolates circulating within aquatic environments for the effective surveillance of campylobacteriosis and arcobacteriosis, the antibiotic resistance profiles of the strains recovered from water samples were evaluated. Resistance to ampicillin, tetracycline, and ciprofloxacin was observed among both Campylobacter and Arcobacter isolates, albeit in varying proportions. The ciprofloxacin (6.7%) and tetracycline (20.0%) resistance rates identified in Campylobacter isolates in this study are consistent with those reported in previous investigations involving water-derived isolates [67]. However, most earlier studies have documented higher resistance levels across all tested antibiotics, highlighting possible regional and temporal variability in antimicrobial resistance patterns [66,81,82]. Regarding Arcobacter, although MDR rates are generally higher than those found in the current study, the trend of a higher resistance rate for ampicillin (92.7%), followed by ciprofloxacin (11.9%) and erythromycin (11.3%), and a lower resistance rate for environmental isolates to gentamicin is supported by recent studies [39,40,73,74,75]. The absence of defined breakpoints for non-jejuni/non-coli Campylobacter species represents a limitation in accurately assessing their resistance profiles. Similarly, although molecular cut-offs have been proposed for resistance to ampicillin and ciprofloxacin in A. butzleri [58], the unimodal MIC distribution reported for tetracycline and erythromycin, as well as the low number of gentamicin-resistant isolates described by some authors [58,83], further complicate the interpretation of resistance data. In addition, some studies have identified resistance mechanisms associated with erythromycin MICs ≥ 8–16 µg/mL [38,84,85]; however, no clear resistance breakpoint has yet been defined. Altogether, these aspects highlight the need for additional studies involving larger strain collections to overcome these limitations, and potentially correlating phenotypic and genomic information.
While Campylobacter and Arcobacter resistance to β-lactams appears widespread in water sources, the comparatively lower resistance rates to other tested antibiotics may reflect a combination of factors. These could include reduced anthropogenic contamination in the study region or potential bacterial fitness costs associated with certain antimicrobial resistance profiles, both of which are supported by recent evidence but warrant further study [24,85,86,87]. Although the relatively low number of Campylobacter isolates limits direct comparison with Arcobacter spp., the latter exhibited higher antimicrobial resistance rates. Although the resistance rates observed in this study are lower than the critical resistance levels frequently reported for priority antibiotics, particularly in isolates from human clinical samples [15], the resistance profiles identified nonetheless represent a significant public health concern. This data is even more alarming when thought of from the perspective of the One Health framework. As an interface between human and animal activities, water bodies serve as reservoirs and hotspots for the horizontal transfer of antimicrobial resistance genes among microbial communities. These aquatic environments, rich in diverse bacterial populations and mobile genetic elements, enhance opportunities for gene exchange, promoting the spread of resistance traits among commensal and pathogenic species. This dynamic increases the risk of resistance determinants being acquired by human and veterinary pathogens, thereby intensifying the public health threat from environmental sources s [44,45,46]. Additionally, the selective pressure imposed by the presence of sub-inhibitory antibiotic concentrations along with other co-selective agents such as heavy metals and biocides in water bodies further drive the development and spread of antimicrobial resistance in these settings [44,45,46].

5. Conclusions

In conclusion, this study provides valuable insights into the environmental surveillance of Campylobacter spp. and Arcobacter spp., emphasizing their persistence in aquatic ecosystems and the associated risks to human and animal health in Portugal. The widespread presence of these bacteria across various water sources, combined with significant resistance to clinically relevant antibiotics, underscores the complex ecological behavior and public health implications of these enteropathogens in aquatic environments. Given the strong interconnection between environmental reservoirs and both human and animal health, the findings of this study highlight the need to adopt a One Health approach that fully integrates the role of water in addressing the spread, impact and associated challenges of these enteropathogens. This knowledge can contribute to improving water quality and safety monitoring, as it provides data on pathogen occurrence and resistance patterns. Such information can inform future research, support risk assessment, and guide the design of targeted interventions aimed at mitigating contamination sources and tracking emerging resistance trends.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w17182767/s1, Table S1: Primers used in this study, including target genes, nucleotide sequences, annealing temperatures and expected amplicon sizes.

Author Contributions

Conceptualization, M.O. and S.F.; methodology, I.V., I.M. and R.M.M.; formal analysis, I.V., I.M. and R.M.M.; writing—original draft preparation, I.V. and I.M.; writing—review and editing, M.O. and S.F.; supervision, M.O. and S.F.; funding acquisition, M.O. and S.F. All authors have read and agreed to the published version of the manuscript.

Funding

This work was developed within the scope of the CICS-UBI projects UIDB/00709/2020 and UIDP/00709/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES.

Data Availability Statement

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

Acknowledgments

Inês Martins is recipient of a doctoral fellowship within the scope of the research fellowship contract signed with FCT, I.P. with the reference 2024.06414.BDANA.

Conflicts of Interest

The authors declare no conflicts of interest.

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Water 17 02767 g001
Figure 1. Representation of the 25 study areas for water sampling within the municipalities of Covilhã, Fundão, and Belmonte, Portugal. • Zêzere river, • Tributaries, • Spring water. Adapted from https://www.google.pt/maps (accessed on 1 October 2019).
Figure 1. Representation of the 25 study areas for water sampling within the municipalities of Covilhã, Fundão, and Belmonte, Portugal. • Zêzere river, • Tributaries, • Spring water. Adapted from https://www.google.pt/maps (accessed on 1 October 2019).
Water 17 02767 g001
Water 17 02767 g002
Figure 2. Distribution of positive samples based on the identification of Campylobacter spp. and Arcobacter spp. isolates during each sampling month.
Figure 2. Distribution of positive samples based on the identification of Campylobacter spp. and Arcobacter spp. isolates during each sampling month.
Water 17 02767 g002
Table 1. Prevalence of Campylobacter species among water sources, and species distribution.
Table 1. Prevalence of Campylobacter species among water sources, and species distribution.
Sample SourceN. of Positive Samples/N. Total of Samples (%)N. of Species/N. of Positive Samples (%)
C. jejuniC. coliC. lariC. upsaliensis
River6/30 (20.0%)0/6 (0.0%)4/6 (66.7%)2/6 (33.3%)0/6 (0.0%)
River tributary10/60 (16.7%)6/10 (60.0%)2/10 (20.0%)1/10 (10.0%)1/10 (10.0%)
Spring water4/60 (6.7%)2/4 (50.0%)1/4 (25.0%)1/4 (25.0%)0/4 (0.0%)
Total20/150 (13.3%)8/20 (40.0%)7/20 (35.0%)4/20 (20.0%)1/20 (5.0%)
Table 2. Distribution of antibiotic resistance of Campylobacter species isolated from water bodies according to species.
Table 2. Distribution of antibiotic resistance of Campylobacter species isolated from water bodies according to species.
AntibioticN. of Resistant Isolates/N. of Total Isolates (% Resistance)
C. jejuniC. coliC. lariC. upsaliensisTotal
n = 8n = 9n = 9n = 1n = 27
Ampicillin1/8 (12.5%)0/9 (0.0%)7/9 (77.8%)1/1 (100.0%)9/27 (33.3%)
Ciprofloxacin1/8 (12.5%)1/9 (11.1%)0/9 (0.0%)0/1 (0.0%)2/27 (7.4%)
Erythromycin0/8 (0.0%)0/9 (0.0%)0/9 (0.0%)0/1 (0.0%)0/27 (0.0%)
Gentamicin0/8 (0.0%)0/9 (0.0%)0/9 (0.0%)0/1 (0.0%)0/27 (0.0%)
Tetracycline1/8 (12.5%)3/9 (33.3%)1/9 (11.1%)0/1 (0.0%)5/27 (18.5%)
MDR profile1/8 (12.5%)0/9 (0.0%)0/9 (0.0%)0/1 (0.0%)1/27 (3.7%)
Note: Resistance breakpoints applied according to the EUCAST Clinical Breakpoint Tables [57] for C. jejuni, C. coli and Enterobacterales: ampicillin < 14 mm; ciprofloxacin < 26 mm; erythromycin < 24 mm (C. jejuni) and <20 mm (C. coli); gentamicin < 17 mm and tetracycline < 30 mm.
Table 3. Prevalence of Arcobacter species among water sources, and species distribution.
Table 3. Prevalence of Arcobacter species among water sources, and species distribution.
Sample SourceN. of Positive Samples/N. Total of Samples (%)N. of Species/N. of Positive Samples (%)
A. butzleriA. cryaerophilusA. skirrowiiA. butzleri + A. cryaerophilusA. butzleri + A. skirrowii
River25/25 (100.0%)25/25 (100.0%)4/25 (16.0%)0/25 (0.0%)4/25 (16.0%)0/25 (0.0%)
River tributary37/50 (74.0%)37/37 (100.0%)5/37 (13.5%)1/37 (2.7%)5/37 (13.5%)1/37 (2.7%)
Spring water10/50 (20.0%)9/10 (90.0%)2/10 (20.0%)0/10 (0.0%)1/10 (10.0%)0/10 (0.0%)
Total72/125 (57.6%)71/72 (98.6%)11/72 (15.3%)1/72 (1.4%)10/72 (13.9%)1/72 (1.4%)
Table 4. Distribution of antibiotic resistance of Arcobacter species isolated from water bodies according to species.
Table 4. Distribution of antibiotic resistance of Arcobacter species isolated from water bodies according to species.
AntibioticN. of Resistant Strains/N. of Total Strains (% Resistance)MIC50MIC90
A. butzleriA. cryaerophilusA. skirrowiiTotal
n = 165n = 11n = 1n = 177
Ampicillin152/165 (92.1%)11/11 (100.0%)1/1 (100.0%)164/177 (92.7%)64128
Ciprofloxacin18/165 (10.9%)3/11 (27.3%)0/1 (0.0%)21/177 (11.9%)≤0.064
Erythromycin17/165 (10.3%)2/11 (18.2%)1/1 (100.0%)20/177 (11.3%)216
Gentamicin5/165 (3.0%)3/11 (27.3%)1/1 (100.0%)9/177 (5.1%)0.252
Tetracycline146/165 (88.5%)2/11 (18.2%)0/1 (0.0%)148/177 (83.6%)816
MDR profile24/165 (14.5%)3/11 (27.3%)1/1 (100.0%)28/177 (15.8%)
Note: Resistance breakpoints applied accordingly with the EUCAST Clinical Breakpoint Tables [57] and molecular cutoff [58]: ampicillin > 8 mg/L; ciprofloxacin > 0.5 mg/L; erythromycin > 8 mg/L; gentamicin > 2 mg/L and tetracycline > 2 mg/L. MIC50 and MIC90 indicate the concentration (mg/L) at which 50 and 90% of the isolates were inhibited by the antibiotic, respectively.
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Venâncio, I.; Martins, I.; Martins, R.M.; Oleastro, M.; Ferreira, S. Prevalence and Resistance Patterns of Campylobacter spp. and Arcobacter spp. in Portuguese Water Bodies. Water 2025, 17, 2767. https://doi.org/10.3390/w17182767

AMA Style

Venâncio I, Martins I, Martins RM, Oleastro M, Ferreira S. Prevalence and Resistance Patterns of Campylobacter spp. and Arcobacter spp. in Portuguese Water Bodies. Water. 2025; 17(18):2767. https://doi.org/10.3390/w17182767

Chicago/Turabian Style

Venâncio, Igor, Inês Martins, Rodrigo M. Martins, Mónica Oleastro, and Susana Ferreira. 2025. "Prevalence and Resistance Patterns of Campylobacter spp. and Arcobacter spp. in Portuguese Water Bodies" Water 17, no. 18: 2767. https://doi.org/10.3390/w17182767

APA Style

Venâncio, I., Martins, I., Martins, R. M., Oleastro, M., & Ferreira, S. (2025). Prevalence and Resistance Patterns of Campylobacter spp. and Arcobacter spp. in Portuguese Water Bodies. Water, 17(18), 2767. https://doi.org/10.3390/w17182767

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