Detection and Characterization of Thermotolerant Campylobacter Resistant to Antibiotics of Priority Use in Humans Present in Broiler Slaughterhouses and Retail Markets

TL;DR

The study found antibiotic-resistant thermotolerant Campylobacter in poultry slaughterhouses and retail markets, highlighting a risk to public health due to multidrug resistance and virulence.

Contribution

This study characterizes multidrug-resistant thermotolerant Campylobacter in poultry supply chains, identifying genetic mutations and virulence factors.

Findings

Thermotolerant Campylobacter resistant to ciprofloxacin and erythromycin was detected in 48.55% of slaughterhouse samples.

Most isolates were multidrug resistant and carried virulence genes like flaA, flhA, and cadF.

Abstract

Background: This study aimed to assess the presence of thermotolerant Campylobacter resistant to ciprofloxacin and erythromycin in poultry slaughterhouses and retail markets, as well as to characterize their multidrug resistance profiles, genetic determinants, and clonal relationships. Methods: Samples were collected at slaughterhouses from cecal content (n = 270), neck skin (n = 270), and wastewater (n = 9), and at retail markets from breast skin (n = 241). Isolates were obtained from mCCDA agar supplemented with ciprofloxacin (2 μg/mL) and identified as C. jejuni or C. coli by PCR. The agar microdilution test was used to determine the minimum inhibitory concentration for ciprofloxacin and erythromycin, and other critical antibiotics. Point mutations in gyrA (Thr86Ile) and 23S rRNA (A2075G), virulence genes (flaA, flhA, cadF, and cdt), and clonal relationships were assessed by PCR and PFGE. Results: At the slaughterhouses, thermotolerant Campylobacter spp. resistant to erythromycin and ciprofloxacin were detected in 48.55% (107/549) of the samples, whereas 4.56% (11/241) of retail samples were positive. The Thr86Ile substitution in gyrA and the A2075G mutation in the 23S rRNA gene were detected in 92.97% and 89.84% of the isolates, respectively. Most isolates (>80%) were multidrug resistant and harbored key virulence genes (flaA, flhA, and cadF). C. jejuni exhibited the highest prevalence of cdt genes (76.19%). There was substantial genotypic diversity among isolates, with broad distribution across the sampled matrices and sites. Conclusions: These findings highlight the circulation of multidrug-resistant and potentially virulent thermotolerant Campylobacter spp. in the later stages of the poultry meat supply chain.

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1. Introduction

Antimicrobial-resistant (AMR) Campylobacter spp. is a globally important pathogen that represents a serious public health challenge [1,2]. Thermotolerant Campylobacter (TC), including C. jejuni and C. coli, is one of the most common causes of foodborne diarrheal disease worldwide [3]. In addition, these TC species are linked to serious long-term sequelae, including peripheral neuropathies, Guillain–Barré syndrome and Miller Fisher syndrome, and functional bowel diseases, such as irritable bowel syndrome [1,4]. In this context, several virulence-associated genes have been described in TC, and most of them are associated with pathogenicity [5]. Motility, bacterial adhesion, invasion of the intestinal epithelium, and toxin production appear to be the main virulence factors [6]. In addition, a significant association exists between virulence genes and antimicrobial resistance in bacterial pathogens, suggesting a link between antibiotic resistance and the potential colonization or invasion capacities of these bacteria [7,8].

Fluoroquinolones and macrolides, particularly ciprofloxacin and erythromycin, are the primary therapeutic options for severe campylobacteriosis [9]. Nevertheless, fluoroquinolone resistance has risen markedly across multiple reservoirs, including humans, food-producing animals, foods, and environmental sources [10,11,12,13]. Although macrolides represent the last effective option in many settings, they are not always accessible or affordable, and macrolide resistance, although less frequent, has also been described [13,14].

With regard to resistance mechanisms in Campylobacter spp., point mutations enable these bacteria to resist commonly used antibiotics. Fluoroquinolones target the DNA gyrase of bacteria, inhibiting DNA replication and transcription [15]. In TC, mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene, particularly the Thr86Ile (T86I) substitution, are the primary resistance mechanisms reported, which are strongly associated with high-level fluoroquinolone resistance [16,17]. On the other hand, macrolide resistance is mainly mediated by mutations in the 23S rRNA gene that alter the antibiotic binding site, particularly substitutions in the V domain, with A2075G being the most prevalent mutation described in Campylobacter spp. [13,18,19]. In addition, other point mutations, plasmid-mediated mechanisms, and the activity of multidrug efflux systems also play a role in resistance [20,21,22].

The development of antimicrobial resistance in Campylobacter spp. reflects the ongoing evolutionary adaptation of bacteria to environmental and therapeutic pressures [15]. However, the increase in AMR-TC worldwide is not only attributed to the overuse of antibiotics in human medicine. The use of antibiotics in animal farming as growth promoters (in the past) and to treat and prevent infections, particularly in poultry production, is mentioned as another cause for increased antimicrobial resistance [23].

Avian hosts constitute a natural reservoir for the TC species, and broiler flocks are frequently colonized in the intestinal tract with high numbers of these organisms [24]. At the slaughterhouse, the evisceration process can expose intestinal contents, facilitating cross-contamination of broiler carcasses and thereby contributing to the dissemination of resistant Campylobacter spp. along the food chain [25,26]. Broiler meat is a well-recognized source of human campylobacteriosis, and consumers may be exposed to AMR-TC or their resistance determinants through improperly cooked meat or via cross-contamination during food handling [25,26]. Consistently, numerous molecular typing studies have demonstrated that poultry-associated strains can be transmitted to humans, supporting the role of the broiler reservoir in the epidemiology of this zoonotic pathogen [26,27].

Although antimicrobial resistance, virulence, genetic mechanisms, and relationships of TC have been studied, these components have not been examined together, nor has there been a specific focus on the two critical drugs used for human campylobacteriosis. Therefore, from a One Health perspective and in alignment with the objectives of the WHO Global Action Plan for antimicrobial resistance [28], the present study aimed to assess the presence of ciprofloxacin- and erythromycin-non-susceptible TC obtained from samples collected at poultry slaughterhouses and retail markets. Additionally, the presence of target modifications potentially responsible for this resistance, multidrug resistance (MDR) profiles, the presence of selected virulence genes, and the clonal relationships among these isolates throughout the poultry production chain were evaluated.

2. Results

2.1. Presence of CIP/ERY-Non-Susceptible TC at Poultry Slaughterhouses and Retail Markets: Distribution and Minimum Inhibitory Concentrations

A total of 270 samples from cecal content, 270 samples from neck skin, and 9 samples from slaughterhouse wastewater from 9 visits to Argentinian slaughterhouses were collected (549 total samples). From these, a total of 272 isolates were obtained from mCCDA supplemented with ciprofloxacin (2 μg/mL), and 241 (88.60%; 241/272) were confirmed at the species level by PCR. In this sense, 165 isolates were confirmed as C. coli (68.46%; 165/241) and 76 as C. jejuni (31.54%; 76/241). A total of 31 isolates were not confirmed at the species level.

Of the 241 isolates, only 117 from slaughterhouses (48.55%; 95% CI: 42.08–55.05; 117/241) were categorized as CIP/ERY-non-susceptible TC by agar dilution. Of those, 106 (90.60%; 95% CI: 83.80–95.21; 106/117) were C. coli and 11 (9.40%; 95% CI: 4.79–16.20; 11/117) C. jejuni. The MIC values detected for ciprofloxacin from these 117 isolates ranged from 8 to ≥256 μg/mL (Table 1). On the other hand, the MIC values for erythromycin ranged from 16 to ≥ 1024 μg/mL and, in particular, C. coli showed high levels of erythromycin resistance (≥1024 µg/mL) (Table 1). In addition, some C. coli (3.77%; 95% CI: 1.04–9.38; 4/106) and, in particular, C. jejuni (54.55%; 95% CI: 23.38–83.25; 6/11) were classified as “intermediate resistant” (MIC = 16 μg/mL). Finally, 13 isolates (10 C. coli and 3 C. jejuni) did not grow during the antimicrobial susceptibility testing, so their data were not available.

Notably, the highest proportion of samples having CIP/ERY-non-susceptible TC was detected in wastewater (44.44%; 95% CI: 13.70–78.80; 4/9), followed by cecal content (33.33; 95% CI: 27.74–39.30; 90/270) and neck skin (8.52%; 95% CI: 5.48–12.51; 23/270) (p < 0.001) (Table 2; Figure 1). The distribution of isolates by species, sample type, and province is shown in Table 2. On the other hand, while cecal content and neck skin samples showed a higher proportion of CIP/ERY-non-susceptible C. coli, the slaughterhouse wastewater samples exhibited a higher proportion of CIP/ERY-non-susceptible C. jejuni (p < 0.001) (Table 2; Figure 1).

With respect to the poultry retail market, 241 breast skin samples were analyzed. A total of 49 isolates were obtained from mCCDA supplemented with ciprofloxacin (2 μg/mL), with 44 (89.79%; 44/49) being confirmed at the species level. In contrast with the slaughterhouses, most of them were classified as C. jejuni (86.63%; 38/44) and the rest as C. coli (16.64%; 6/44). A total of 5 isolates were not confirmed at the species level.

A total of 11 TC isolates from 241 breast skin samples (4.56%; 95% CI: 2.30–8.02; 11/241) were categorized as CIP/ERY-non-susceptible TC by agar dilution. Of those, 1 (9.09%; 95% CI: 0.23–41.28; 1/11) was C. coli and 10 (90.91%; 95% CI: 58.72–99.77; 10/11) C. jejuni. The MIC values for ciprofloxacin from these 11 isolates ranged from 64 to 256 μg/mL, with a predominance of 128 μg/mL. In addition, most of the C. jejuni (70%; 95% CI: 34.75–93.33; 7/10) showed a MIC level corresponding with intermediate resistance to erythromycin (Table 1). Two C. jejuni did not grow during the antimicrobial susceptibility testing, so their data were not available in this study.

The distribution of CIP/ERY-non-susceptible TC by species and province is shown in Table 3. Notably, breast skin samples from retail markets showed a higher proportion of CIP/ERY-non-susceptible C. jejuni with respect to cecal content and neck skin samples from slaughterhouses (p < 0.001) (Table 3, Figure 1).

2.2. Presence of Antimicrobial Resistance Determinants: Target Modifications in the gyrA and 23S rRNA Genes

Among the 128 CIP/ERY-non-susceptible TC recovered (117 from slaughterhouses and 11 from retail markets), 92.97% (95% CI: 87.07–93.73; 119/128) were shown to have the mutant type T86I region of the quinolone resistance-determining region (QRDR) of the gyrA gene by MAMA-PCR. Accordingly, 90.48% (95% CI: 69.62–98.83; 19/21) of the ciprofloxacin-resistant C. jejuni and 92.52% (95% CI: 85.80–96.72; 99/107) of C. coli presented the mutation (p = 0.79). The distribution of CIP/ERY-non-susceptible TC having this mutation by species and sampling point is shown in Table 1.

On the other hand, of the 128 CIP/ERY-non-susceptible TC, 89.84% (95% CI: 83.26–94.48; 115/128) were shown to have the A2075G mutation in the 23S rRNA gene by MAMA-PCR. Accordingly, 75% (95% CI: 34.91–96.81; 6/8) of C. jejuni and 90.29% (95% CI: 82.87–95.24; 93/103) of C. coli classified as resistant to erythromycin (MIC ≥ 32 μg/mL) presented the A2075G mutation in the 23S rRNA gene (p = 0.52). Moreover, 92.31% (95% CI: 63.97–99.81;12/13) of C. jejuni and 100% (95% CI: 39.76–100; 4/4) of C. coli classified as intermediate resistant to erythromycin (MIC = 16 μg/mL) presented this mutation. The distribution of CIP/ERY-non-susceptible harboring this mutation by species and sampling point is shown in Table 1

2.3. Antimicrobial Resistance Profile of CIP/ERY-Non-Susceptible TC Isolates

Several profiles of antimicrobial resistance were observed among the evaluated isolates. The proportions of antimicrobial resistance, intermediate resistance, and susceptibility of C. coli and C. jejuni and their MIC distributions are shown in Table 4.

The majority of the isolates were resistant to most of the antibiotics tested. The high proportion of resistance to enrofloxacin (C. coli: 100%, 95% CI: 96.61–100; C. jejuni: 100%, 95% CI: 83.89–100) and the elevated MIC values detected in both species were consistent with the observed ciprofloxacin resistance. In addition, high levels of tetracycline resistance were observed in both C. coli (98.13%, 95% CI:93.41–99.77) and C. jejuni (80.95%, 95% CI: 58.09–94.55), with MICs between 64 and 256 µg/mL and no significant differences between species (p = 0.16). Ampicillin resistance was 100% (95% CI: 83.89–100) in C. jejuni and 86.92% (95% CI: 79.02–92.66) in C. coli; however, no statistically significant differences were detected between species (p = 0.97). With respect to chloramphenicol and gentamicin, both species showed high levels of susceptibility. Only 7.48% (95% CI: 3.28–14.20) of C. coli and 4.76% (95% CI: 0.12–23.82) of C. jejuni were resistant to gentamicin (p = 0.59), and no chloramphenicol-resistant isolates were detected. Furthermore, no relevant resistance or intermediate resistance profiles were observed for either antibiotic.

Considering the initial ciprofloxacin and erythromycin resistance evaluated and the other five antimicrobial classes tested in this study (fluoroquinolones, aminoglycosides, penicillins, tetracyclines, and amphenicols), 99.07% (95% CI: 94.90–99.98; 106/107) of C. coli and 85.71% (95% CI: 63.66–96.95; 18/21) of C. jejuni were categorized as MDR (p = 0.059) (Table 5). All the isolates in which the MDR profile was not detected were recovered from Entre Rios province: 3 C. jejuni from a retail market and 1 C. coli from a slaughterhouse.

The predominant MDR pattern in C. coli combined resistance to ciprofloxacin and enrofloxacin fluoroquinolones, erythromycin, tetracycline, and ampicillin (77.36%; 95% CI: 68.21–84.92; 82/106). The most prevalent MDR profile for C. jejuni was fluoroquinolone (ciprofloxacin and enrofloxacin), tetracycline, and ampicillin (55.56%; 95% CI: 30.76–78.47; 10/18). The MDR resistance profiles according to the classes and types of antimicrobials tested are shown in Table 5.

2.4. Detection of Virulence Gene

Overall, two of the six virulence genes were detected in all the CIP/ERY-non-susceptible TC evaluated. In this sense, 100% (95% CI: 97.16–100; 128/128) of the isolates were found to have the flaA and flhA genes. The third most prevalent gene was cadF, present in 85.16% of the isolates (95% CI: 77.79–90.82;109/128).

Regarding the cdt genes, cdtC was detected in 68.75% of isolates (95% CI: 59.96–76.65; 88/128), while cdtA and cdtB were detected in 30.47% (95% CI: 22.65–39.22; 39/128) and 42.19% (95% CI: 33.51–51.23; 54/128), respectively. A total of 27 isolates (21.09%; 95% CI: 14.38–29.19; 27/128) simultaneously harbored cdtA, cdtB, and cdtC. Notably, the detection of all three genes was substantially higher in C. jejuni (76.19%; 95% CI:52.83–91.78; 16/21) than in C. coli (10.28%; 95% CI: 5.24–17.65; 11/107) (p < 0.001). The distribution of the virulence genes evaluated according to TC species is shown in Figure 2.

2.5. Clonal Relationship Among CIP/ERY-Non-Susceptible CT

A high level of genotype diversity was found for CIP/ERY-non-susceptible TC. For C. coli isolates, 72 strains were found clustered in 15 profiles with 3 predominant genotypes (profiles A, B, C, D, E, F) shared by 8, 7, 6, 5, and 4 isolates, respectively (Table 6). Many small profiles with two, three, or four isolates were found (profiles F to O). A high number of isolates presented unique patterns, and several strains did not show any restriction pattern by PFGE.

Similarly, C. jejuni were clustered in 2 groups, with many isolates presenting unique profiles and several isolates not showing any restriction pattern by PFGE (Table 7). As can be observed, profiles A and B were predominant, and each of them had 3 and 2 isolates, respectively.

At the slaughterhouse level, clonal relationships were observed among isolates obtained from different sample types. In this sense, the same PFGE profiles were detected among isolates from the cecum, neck skin, and wastewater. For C. coli, several profiles grouped isolates obtained from different samples from slaughterhouses in different provinces (A, B, E, F, G, I, K, O). Notably, profile B included isolates obtained from different samples of multiple slaughterhouses in different provinces and from a retail market (Table 6). In addition, three gentamicin-resistant isolates exhibiting distinct profiles (B, F, and O) were identified, all having been isolated from the same province (ER).

As regards C. jejuni, all isolates of profile A were obtained from breast skin samples collected during the same sampling event (Table 7).

3. Discussion

For food safety and public health reasons, the emergence and spread of TC resistant to the main antimicrobial agents for human use in food-producing animals, especially broilers, is worrisome. The findings of this study contribute to providing new and integrated epidemiological evidence on the presence and characteristics of AMR-TC in the poultry production chain.

At the slaughterhouse level, CIP/ERY-non-susceptible TC were detected in almost half of the samples (48.55%). In this context, the cecal content was identified as a relevant reservoir of these bacteria, with most isolates identified as C. coli (95.56%). This sample type is widely recognized as a marker of antimicrobial exposure at the farm level [30]. Accordingly, the predominance of C. coli in cecal content is consistent with the selective pressures of the intestinal environment, where previous antimicrobial exposure and microbial competition may favor its persistence and resistance. Consistent with these findings, several studies have reported higher resistance to multiple antimicrobials, particularly erythromycin, in C. coli compared to C. jejuni, in poultry [11,12,31]. For instance, Drame et al. [31] and Kovalenko et al. [32] observed that co-resistance to macrolides and fluoroquinolones in cecal samples was almost exclusively associated with C. coli. This species appears to be better adapted to survive under antimicrobial selection pressure, which allows it to develop resistance to these antimicrobials [33,34]. Furthermore, the reported reduced ability of erythromycin-resistant C. jejuni to colonize chickens may explain the overall low prevalence (4.44%) of these isolates detected in this study [35].

In post-chiller neck skin samples, despite fewer positive samples being detected (8.51%), a similar trend to cecal content was observed with a relative predominance of CIP/ERY-non-susceptible C. coli (82.61%). However, C. jejuni began to appear more frequently (17.39%), suggesting a shift in species distribution as the product moves through processing [35].

During the evisceration process, intestinal contents may be inadvertently exposed, leading to cross-contamination between carcasses, neck skins, and equipment [25,35,36]. This could explain the presence of CIP/ERY-non-susceptible TC in post-chiller neck skins from the slaughterhouses. Supporting these findings, the PFGE analysis revealed the presence of related pulse-types recovered from both cecal content and neck skin samples from the same slaughterhouse (Profiles B and L), reinforcing the hypothesis of cross-contamination occurring during slaughter and processing. These results underscore the need to control slaughterhouse processing to reduce the spread of antimicrobial-resistant bacteria.

From a One Health perspective, assessing the potential implications of the presence of AMR-TC on human, animal, and environmental health is essential [37]. In this study, the highest proportion of CIP/ERY-non-susceptible TC positive samples was detected in slaughterhouse wastewater (44.44%), with C. jejuni being the most frequently identified species (75%). This result indicates that slaughterhouse wastewater can act as an important reservoir and dissemination route for AMR-TC, underscoring the need to monitor effluents as a sentinel for antimicrobial resistance spread. In this context, the detection of C. jejuni isolates sharing the same PFGE pulse-type in slaughterhouse wastewater and in retail chicken meat (Profile B) is interesting since it suggests diffusion in the broiler meat supply chain. Taken together, these findings support the idea that wastewater from slaughterhouses could constitute a potential risk both to public and environmental health.

At the retail level, fewer CIP/ERY-non-susceptible TC isolates were recovered (4.56% positive samples), likely reflecting decreased viability during chilling, storage, and distribution [38,39]. Interestingly, most resistant isolates were C. jejuni (90.91%), which is consistent with Zbrun et al. [40]. Given that C. jejuni is one of the leading causes of enteritis worldwide [4,41], these results at the consumer interface warrant attention from a public health and food safety perspective.

The species distribution observed at the retail market contrasts with that detected at the slaughterhouse, which may reflect species-specific stress tolerance as downstream stages impose distinct selective pressures. In this sense, C. coli is generally considered less robust and more sensitive to adverse conditions, whereas the ability of C. jejuni to form biofilms may contribute to its persistence under stress conditions encountered during processing, storage, and retail [42,43]. Therefore, the high genetic diversity of C. jejuni detected at the retail market may be determined by the ability of the different strains to survive under stress, even after cleaning and disinfection [42]. Furthermore, the possibility of cross-contamination between broiler meats on the retail shelf can also be strongly suggested since all the isolates of profile A were obtained during the same sampling event. Finally, results suggest that certain C. coli clones may persist under diverse stress conditions throughout the poultry production chain, as previously described [35,44,45]. Accordingly, the detection of C. coli profile B in slaughterhouse samples (cecal content and neck skin), as well as in retail market samples (breast skin), suggests the persistence and dissemination of specific resistant clones across different stages of the poultry supply chain.

Another remarkable finding of the present study was the high frequency of MDR-TC isolates (>88%), comparable to global reports [11,46]. In Campylobacter spp., the resistance to several antimicrobial drugs has been attributed to point mutations in specific target genes or the acquisition of resistance determinants [1]. Accordingly, the co-occurrence of multiple resistance genes within the same isolates has been previously reported [11,47,48]. However, gaining a specific resistance determinant only allows bacteria to resist a particular antimicrobial, while the presence of multidrug efflux systems empowers bacteria with simultaneous resistance to multiple classes of antibiotics. In Campylobacter spp., the tripartite efflux pump CmeABC represents the predominant efflux system and plays a central role in mediating resistance to a broad range of structurally diverse antimicrobials [49]. Furthermore, the resistance-enhanced CmeABC (RE-CmeABC) efflux pump has been recently described, which confers significantly enhanced resistance to multiple antibiotics and shifts antibiotic MIC distributions to a much higher range [48]. These mechanisms likely contribute substantially to the high MDR rates and the high MICs for several antibiotics detected among the isolates analyzed in the present study.

This scenario may reflect the overuse of different antimicrobial agents in poultry production. For instance, enrofloxacin resistance was common in both species (100%), with MIC values clustering at the highest concentrations tested (8–32 µg/mL). This is consistent with previous reports at the global and regional levels and may be attributed to the extensive use of fluoroquinolones in poultry production [13,50]. It is important to note that fluoroquinolone-resistant Campylobacter spp. does not exhibit an evident fitness cost, allowing resistant strains to persist and compete effectively even in the absence of antimicrobial pressure [17,51].

On the other hand, the detection of high levels of erythromycin resistance, particularly among C. coli isolates and notably also in a subset of C. jejuni, represents a critical finding of the present study. From a public health perspective, the presence of isolates exhibiting erythromycin MICs ≥ 1024 µg/mL is particularly concerning, as macrolides remain the first-line treatment for severe campylobacteriosis in humans. Moreover, these results are consistent with recent findings by Quino et al. [52] and Cáceres Bautista et al. [53], who also reported elevated erythromycin MICs in strains isolated from chicken and human clinical samples, reinforcing concerns regarding the persistence and dissemination of highly resistant Campylobacter spp. along the poultry supply chain. Additionally, erythromycin resistance in C. jejuni from retail market samples was predominantly classified as intermediate, which remains a relevant public health concern given that this category includes isolates with reduced susceptibility compared to fully susceptible strains [54].

Finally, while tetracycline and ampicillin also exhibited a high resistance rate, gentamicin and chloramphenicol showed mostly susceptible or intermediate profiles, in line with previous reports [11,50,55]. This finding suggests that different antimicrobial classes may exert distinct selection pressures in poultry production systems, potentially reflecting differences in their use or regulatory status.

The investigation of genotypic fluoroquinolone and erythromycin resistance revealed a high prevalence of well-established genetic determinants. In the present study, T86I substitution in the gyrA gene was the most prevalent mutation (>90%) detected, in accordance with previous reports [2,11]. This single-step mutation in gyrA was associated with high-level fluoroquinolone resistance in Campylobacter spp., typically resulting in ciprofloxacin MICs > 16 µg/mL, which supports the phenotypic resistance levels observed in this study [17]. Nevertheless, additional mechanisms not evaluated in the present study may contribute to the extremely high MIC values detected. In this regard, in the presence of RE-CmeABC and a gyrA mutation, Campylobacter spp. expresses an exceedingly high resistance level to ciprofloxacin (MICs ≥ 256 µg/mL) [35].

On the other hand, the A2075G mutation in the 23S rRNA gene was frequently detected among erythromycin-resistant isolates in agreement with previous studies [11,18,56]. High macrolide MICs in Campylobacter spp. such as those detected in the present study, have been associated with mutations affecting multiple copies of the 23S rRNA gene, often acting in concert with intact multidrug efflux systems such as CmeABC or enhanced variants like RE-CmeABC. Together, these mechanisms can markedly reduce intracellular antimicrobial concentrations and contribute to high-level macrolide resistance [35].

However, neither mutation was universally present in resistant isolates, a discrepancy that has also been reported for both fluoroquinolones and macrolides, supporting the involvement of additional resistance mechanisms (e.g., other mutations, plasmid-borne determinants, and efflux systems) [18,56,57]. In this context, further genomic investigations are needed to clarify the contribution of these mechanisms.

Colonization of the gastrointestinal mucosa represents the initial step in Campylobacter spp. infection, and flagellar-associated determinants such as flaA and flhA are central to motility and host interaction. In the present study, all CIP/ERY-non-susceptible TC isolates harbored flaA and flhA, irrespective of species or sample origin, reinforcing the highly conserved nature of these genes, as previously reported [35,58,59]. Likewise, a high proportion of CIP/ERY-non-susceptible TC carried the cadF gene (>80%), which is consistent with its widespread prevalence among poultry-associated Campylobacter spp. isolates and its recognized role in intestinal colonization [60,61]. Regarding toxin production, the cdt cluster was more frequently detected in C. jejuni than in C. coli, suggesting a comparatively higher virulence potential of this species. Similar patterns have been widely reported in poultry and human isolates [35,59,62]. Notably, as most C. jejuni isolates in this study originated from retail samples, these findings raise public health concerns due to potential consumer exposure to strains combining antimicrobial resistance and virulence traits.

Finally, the use of mCCDA agar supplemented with ciprofloxacin for initial isolation, followed by MIC determination for ciprofloxacin and erythromycin to select non-susceptible isolates, limited the estimation of the overall prevalence of Campylobacter spp. and its distribution by species. Nevertheless, this approach allowed a focused analysis of clinically relevant resistant strains in a context where national data are scarce.

Although the analyzed region is limited and may be considered a study limitation, this region accounts for the majority of Argentina’s poultry meat production (85–90% of poultry activity; Figure S1) [63]. In addition, a single sample from the effluent treatment pool was collected during each slaughterhouse visit, which may also be considered a limitation. Further studies should be conducted to evaluate Campylobacter spp. presence and resistome in slaughterhouse effluents to assess their environmental and public health impact.

As mentioned above, from a One Health perspective, evaluating the implications of AMR-TC presence across human, animal, and environmental compartments is essential. A key limitation of this study is the lack of data on AMR-TC isolates from humans; future research should be conducted to elucidate epidemiological links between isolates from different sources. Overall, these findings provide novel evidence and highlight the need for larger-scale studies incorporating genomic characterization to further elucidate resistance mechanisms, transmission routes, and effective prevention and control strategies.

4. Materials and Methods

4.1. Sample Collection

This study was conducted in Argentina between August 2022 and December 2023. A total of 790 samples were collected from nine broiler meat supply chains in the central region of Argentina (Santa Fe, Entre Rios and Buenos Aires provinces) (Figure S1). The samples of each broiler meat chain were obtained first at the slaughterhouse (n = 549 total samples) and then at the retail market, where the slaughterhouse sells its carcasses (n = 241 total samples).

At the slaughterhouses, samples were taken from three areas: a—evisceration zone: broiler cecum (n = 30); b—chiller zone: neck skin (n = 30); c—wastewater treatment facility area: wastewater (n = 1). Cecum samples in the slaughterhouse were randomly collected from the evisceration line and placed into sterile plastic bags. Neck skins were taken from the processing line after chilling and placed into a sterile bag with 400 mL of 0.1% buffered peptone water (Oxoid Ltd., Basingstoke, UK). Neck skins were rinsed, and the solution was recovered in sterile plastic tubes. The wastewater sample (100 mL) was obtained from a tank in the effluent treatment and placed into a sterile bottle. Chicken breast skins from approximately 30 carcasses were taken at each retail market and rinsed as mentioned above with 400 mL of 0.1% buffered peptone water (Oxoid Ltd., Basingstoke, UK). All samples were transported to the laboratory under refrigeration conditions within 4 h.

4.2. Campylobacter spp. Isolation in mCCDA Agar Plate with Ciprofloxacin (2 μg/mL)

In the laboratory, Campylobacter spp. was isolated using selective media Bolton broth (Oxoid Ltd., Basingstoke, UK)and Modified Charcoal Cefoperazone Deoxycholate (mCCDA) agar plates (Oxoid Ltd., Basingstoke, UK) (ISO 10272–1 [64]) with modifications. The isolation was performed using a passive filtration method [65]. Sterile membrane filters, with a 47 mm diameter and a 0.45 μm pore size (Sartorius AG, Goettingen, Germany), were placed on the surface of mCCDA agar plates with 2 μg/mL of ciprofloxacin (Sigma-Aldrich, St. Louis, MO, USA). Briefly, one gram of broiler cecal content or pellet obtained from Bolton broth from rinsed skin and wastewater was inoculated on top of the sterile membrane filters [35]. Plates with filters were incubated at 37 °C for 10 min to allow motile cells to cross the membrane. After filtration, the filters were carefully removed and discarded, and all plates were incubated at 42 °C for 48 h under microaerophilic conditions (5% O_2_, 10% CO_2_, and 85% H_2_) in anaerobic jars (Oxoid Ltd., Basingstoke, UK).

4.3. Identification of Thermotolerant Campylobacter spp.

Following incubation, suspect colonies (grey, smooth, glossy, convex, and well-defined edges) were selected from the plates. Initial Campylobacter identification was based on colony morphology, Gram staining, and microscopy (revealing motile, curved rods). All suspect TC strains were identified at the species level (C. jejuni and C. coli) via multiplex PCR using a Heal Force T960 Thermocycler (Biometra, Shanghai, China), following the method of Vandamme et al. [66]. For PCR analysis, genomic DNA templates were extracted from the isolates using a Wizard Genomic DNA Purification Kit (Promega^®^, Madison, WI, USA) according to the manufacturer’s instructions. Confirmed positive isolates were subcultured onto mCCDA agar and preserved in glycerol broth at −80 °C [67].

4.4. Antimicrobial Susceptibility Testing of Ciprofloxacin and Erythromycin and Determination of Minimal Inhibitory Concentrations

The antimicrobial sensitivity of TC isolates was tested against ciprofloxacin and erythromycin (Sigma-Aldrich, St. Louis, MO, USA) by the agar dilution assay as recommended by the Clinical and Laboratory Standards Institute in the standard [29,54]. Isolates were recovered from freezer storage, streaked onto Columbia blood agar (Oxoid Ltd., Basingstoke, UK), and incubated for 48 h at 42 °C under microaerobic conditions. Multiple colonies were then transferred to a tube with 5 mL of Mueller–Hinton broth (Britania, Buenos Aires, Argentina) to achieve a standard inoculum (0.5 McFarland), followed by a 1:10 dilution in sterile saline to obtain a concentration of 10^7^ CFU/mL. Suspensions (~10^4^ CFU/mL) were inoculated using a multipoint inoculator (Steers replicator, CMI-Promex, Inc., Auburn, NJ, USA) onto Mueller–Hinton agar plates (Britania, Buenos Aires, Argentina) containing twofold antimicrobial dilutions and 5% defibrinated sheep blood. Plates were incubated for 24 h at 42 °C under microaerobic conditions. C. jejuni ATCC 33560 was used as a reference strain. The inhibition was evaluated according to the standards of the Clinical and Laboratory Standards Institute [54]. Isolates that showed a minimal inhibitory concentration (MIC) value within the intermediate or resistant category for both ciprofloxacin (MIC ≥ 2 μg/mL) and erythromycin (MIC ≥ 16 μg/mL) were classified as CIP/ERY-non-susceptible TC. Only these isolates were subjected to the subsequent analyses described below.

4.5. Detection of Antimicrobial Resistance Determinants

There are several mechanisms associated with resistance to ciprofloxacin and erythromycin; this study addressed the main point mutations involved in such resistance. Mutations in the gyrA quinolone resistance-determining region (T86I), associated with quinolone resistance, were detected by MAMA-PCR as described by Zirnstein et al. [68] and Zirnstein et al. [69]. Mutations at position 2075 in domain V of the 23S rRNA gene, associated with high-level erythromycin resistance, were detected by the mismatch amplification mutation assay PCR (MAMA-PCR) [70]. All reactions were carried out using a Heal Force T960 Thermocycler (Biometra, Shanghai, China). PCR products were visualized by electrophoresis in 1.5% agarose gels, stained with GelRed^®^ (Biotium, Fremont, CA, USA), and viewed under UV light. The size of the PCR amplicons was compared to the 100 bp DNA marker (PB-L^®^, Buenos Aires, Argentina).

4.6. Antimicrobial Susceptibility Testing Against Other Antimicrobials: Determination of Minimum Inhibitory Concentrations and Multidrug-Resistance Profiles

The antimicrobial sensitivity of CIP/ERY-non-susceptible TC isolates was tested by an agar dilution assay as recommended by the CLSI [29,54], as mentioned above. The MICs of gentamicin, tetracycline, enrofloxacin, chloramphenicol, and ampicillin (Sigma-Aldrich, St. Louis, MO, USA) were determined to assess phenotypic resistance to these antibiotics. C. jejuni ATCC 33560 was used as the quality control strain. Isolates resistant to three or more antimicrobial classes were classified as MDR [71].

4.7. Identification of Virulence Genes

The presence of six pathogenic genes responsible for the expression of adherence, colonization, and cytotoxin production was examined. Sets of primers were described by Konkel et al. [72] cadF, Hickey et al. [73] cdtA, and Müller and Hänel [74] flhA. Three further sets of primers were described by Datta et al. [75] flaA, cdtB, and cdtC. The PCRs were carried out as described by Rossler et al. [35] using a Heal Force T960 Thermocycler (Biometra, Shanghai, China). DNA bands were visualized by staining with GelRed^®^ (Biotium, Fremont, CA, USA) and viewed under UV light. The size of the PCR amplicons was compared to the 100 bp DNA marker (PB-L^®^, Buenos Aires, Argentina). To carry out the analysis, the cdtA, cdtB, and cdtC genes were grouped in a cdt cluster.

4.8. Pulsed-Field Gel Electrophoresis (PFGE)

CIP/ERY-non-susceptible TC were subtyped by PFGE following the PulseNet protocol (https://www.pulsenetinternational.org/protocols?url=%2Findex.php (accessed on 10 November 2024)). Briefly, isolates were incubated on blood agar base plates (Oxoid Ltd., Basingstoke, UK) at 42 °C for 48 h under microaerophilic conditions for the preparation of genomic DNA in agarose plug (Pulsed Field Certified Agarose, Bio-Rad Laboratories, Hercules, CA, USA) as mentioned in the “Standard Operating Procedure for PulseNet PFGE of Campylobacter jejuni” (https://www.pulsenetinternational.org/assets/PulseNet/uploads/pfge/PNL03_CampyPFGEprotocol.pdf (accessed on 10 November 2024)). Bacterial genomic DNA in agarose plugs was digested with 40 U of SmaI (Thermo Fisher Scientific, Waltham, MA, USA) for 4 h at 25 °C. Digested fragments were separated by pulsed-field gel electrophoresis (PFGE) in 1.0% agarose gels (0.5× TBE buffer) using a CHEF-DR^®^ III system (Bio-Rad, Hercules, CA, USA). Electrophoresis was performed at 14 °C for 18 h. After staining with ethidium bromide, DNA bands were obtained by UV transillumination. PFGE banding patterns were analyzed using BioNumerics Software V6.6. Clonal relationship among strains was assessed using the Dice coefficient, with a dendrogram constructed via the unweighted pair-group method with arithmetic mean (UPGMA; 2% optimization, 4% tolerance). The Salmonella enterica subsp. enterica serovar Braenderup strain H9812 (ATCC BAA-664), restricted with XbaI (Thermo Fisher Scientific, Waltham, MA, USA), was used as a DNA size marker [76].

4.9. Statistical Analysis

Data were compiled in a Microsoft Excel spreadsheet (Microsoft, Redmond, WA, USA). Proportions of CIP/ERY-non-susceptible TC by sample type, as well as the presence of resistance-associated point mutations, resistance to other antimicrobials, MDR, and virulence genes, were estimated and presented with 95% confidence intervals (CI).

Differences in the proportions of CIP/ERY-non-susceptible TC and species distribution among sample types, and differences in antimicrobial resistance profiles, MDR rates, and virulence gene prevalence between C. coli and C. jejuni, were assessed using the Generalized Mixed Linear Model (GzMLM) with a binomial distribution with logistic link function and the sampling point as a random variable. A p-value < 0.05 was considered statistically significant. All of the analyses previously described were performed using the Infostat software version 2017 (Universidad Nacional de Córdoba, Córdoba, Argentina).

5. Conclusions

This study provides an integrated characterization of CIP/ERY-non-susceptible TC circulating along the broiler meat supply chain. The distribution of C. jejuni and C. coli varied across sample types, with slaughterhouses showing the highest proportion of resistant C. coli. Although detected at lower frequencies, the presence of CIP/ERY-non-susceptible C. jejuni at the retail level remains epidemiologically relevant from a public health and food safety perspective.

A high proportion of isolates were MDR, which includes key drugs for the treatment of human campylobacteriosis, and most of them carried virulence genes, potentially exacerbating risk to human health. While point mutations partially explain the observed resistance to ciprofloxacin and erythromycin, other mechanisms should be evaluated as contributing to the detected phenotypes.

Furthermore, the identification of clonal relationships among isolates recovered from different sample types supports the dissemination of resistant strains throughout interconnected compartments of the poultry production chain at a regional level.

These results highlight the need for coordinated strategies, including prudent antimicrobial stewardship, strengthened slaughterhouse biosecurity, and improved management and treatment of slaughterhouse wastewater in order to mitigate the spread of MDR Campylobacter spp. and reduce associated public health risks.