Wild Fishes as Reservoirs of Gut Bacteria Carrying Antimicrobial Resistance Encoding Genes in Chilean Bays

TL;DR

Wild fish in Chilean bays carry bacteria with antibiotic resistance genes, suggesting they play a role in spreading resistance in marine environments.

Contribution

This study identifies wild fish as reservoirs of antimicrobial resistance genes in anthropogenically impacted marine areas in Chile.

Findings

Pseudomonas, Vibrio, and Shewanella were the most common bacteria carrying resistance genes.

High resistance to streptomycin, amoxicillin, and furazolidone was observed, with 76.9% of isolates showing multi-drug resistance.

blaCTX-M1 and carbapenemase genes were detected, indicating potential for extended-spectrum β-lactamase and carbapenem resistance.

Abstract

Objective: The main aim of the study was to evaluate the role of wild fishes inhabiting in three anthropogenic-impacted Bays in Chile as reservoirs of antimicrobial resistance genes (ARGs). Methods: A total of 245 antimicrobial-resistant isolates were isolated from fish captured in the Coquimbo (142 isolates), Concepción (44 isolates), and Puerto Montt (59 isolates) Bays, and were identified by 16S rRNA gene sequence analysis, Antimicrobial-resistant isolates were tested for susceptibility to 12 antimicrobials by an agar disk diffusion method, and the carriage of genes encoding for resistance to main antimicrobial classes, such as β-lactams, amphenicols, tetracyclines, and sulfonamides by PCR (Polymerase Chain Reaction). Results: A predominance of the Pseudomonas (37.04%), Vibrio (14.40%), and Shewanella (13.99%) genera. Antimicrobial-resistant isolates were tested for susceptibility to 12 antimicrobials by an agar disk diffusion method, showing highest resistance to streptomycin (82.4%), amoxicillin (67.4%), and furazolidone (63.3%), and lowest resistance to ciprofloxacin (3.7%), meropenem (22.5%), and oxytetracycline (29.8%) and exhibiting a high occurrence of the multi-drug resistance phenotype (76.9%). Furthermore, an important number of isolates recovered from sampled fish species carried plasmids (53.5%), floR gene (36.7%), and tet genes (19.2%), whereas the detection of sul genes and class 1-integron was rare. As an overall result, 10.6% of isolates carried at least one bla gene, encoding an extended-spectrum-β-lactamase, with a high predominance of the blaCTX-M1 gene (23 isolates), whereas 14 out of 245 isolates (5.7%) were positive for the carriage of carbapenemases encoding genes, which both groups exhibited the β-lactam resistance phenotype. Conclusions: The wide distribution of ARG-carrying bacteria in wild fishes from all sampled Bays provides evidence that wild fish are important reservoirs and drivers of spread of ARGs in the marine environment, prompting the need of a continuous surveillance of these genes in wild fishes inhabiting anthropic impacted coastal marine environments in Chile.

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1. Introduction

The rapid global spread of antimicrobial resistant bacteria represents one of the most serious threats to human health today, resulting in approximately 700,000 deaths worldwide each year due to drug-resistant infections [1,2].

As noted, antimicrobial resistance is a growing public health concern worldwide, and it is now regarded as a critical One Health issue [3,4]. One Health is an interdisciplinary concept, which recognizes that interdependent human, animal, and environmental domains contribute to the emergence, evolution, and spread of antibiotic-resistant microorganisms, and their related antimicrobial-resistance genes (ARGs) on a local and global scale [3,4], which demands knowledge and understanding of their role in the dissemination of ARGs under a One-Health perspective. Thus, under a One Health approach, it is critical to understand the role of marine fishes residing in anthropic-impacted coastal waters in the spread of molecular elements involved in antimicrobial resistance and transfer [5,6,7]. Carriage of ARGs by resistant bacteria residing in wild fishes raises ecological and public health concerns, emphasizing the need for further studies, especially in relation to the ARGs of clinical importance [8,9].

Most of studies on the occurrence of ARGs in fish gut microbiota are related with aquaculture settings [10,11,12,13]. Otherwise, studies on the ARGs carried by bacteria composing the microbiota of wild fishes from non-aquaculture settings are scarce, and mostly related to freshwater and estuarine environments [8,9,14,15]. In this trend, Ballash et al. [16] concluded that fishes are effective bioindicators of freshwater environments contaminated with antimicrobial-resistant bacteria, and antimicrobial-resistance encoding genes. In another study, Sellera et al. [17] reported for the first time the occurrence of Escherichia coli carrying β-lactamase-encoding genes among gut bacteria from wild fishes from Brazilian polluted estuarine waters. Furthermore, Mills et al. [18] demonstrated that fish gut bacteria carry higher levels of genes encoding for carbapenem resistance than do the surrounding water and sediment of a human-impacted river.

Despite significant anthropogenic impacts in many coastal areas in Chile, which are usually used for human recreational activities and by artisanal fishing for capturing seafood, no studies of the resistome of gut bacteria inhabiting wild fishes have been previously performed. In Chile, only two studies, conducted more than two decades apart, on the occurrence of antimicrobial resistant bacteria by marine wild fishes have been performed, confirming the role of these species as reservoirs of antimicrobial resistant bacteria [19,20]. We reported a high incidence of multi-drug-resistant bacteria among gut microbiota of wild fishes captured in Southern [19] and Northern [20] Bays in Chile; however, to date no studies have been conducted in this country, to evaluate the occurrence of antimicrobial-resistance encoding genes carried by gut bacteria recovered from marine wild fishes.

The main aim of the study was to evaluate the role of marine wild fishes residing in three main human-impacted Chilean Bays as reservoirs and routes for the spread of genes encoding antimicrobial resistance against antimicrobials important in human and animal therapy, posing a significant public health risk.

It is expected that marine wild fishes residing in different bays impacted with anthropogenic contaminants, will carry a high taxonomic diversity of antimicrobial resistant bacteria carrying diverse ARGs, as well as plasmids, confirming the role of wild fishes as highly important contributors of maintenance and spread of main antimicrobial resistant genes of clinical importance in marine environments. Thus, this study will address a critical knowledge gap and emphasizes the public health risks posed by anthropogenic contamination in coastal ecosystems.

2. Results

2.1. Bacterial Identification

The identification of 245 antimicrobial-resistant isolates recovered from the Coquimbo, Concepción, and Puerto Montt Bays showed a high predominance of Pseudomonas genus (36.7%), and a lesser proportion the Vibrio and Shewanella genus (14.3 and 13.9%, respectively), as is described in Table 1. All Bays exhibited a dominance of Pseudomonas representatives, comprising the 38.7%, 43.2%, and 27.1%, of antimicrobial resistant isolates recovered from intestinal content of wild fishes from Coquimbo, Concepción, and Puerto Montt Bays, respectively, whereas Vibrio (21.1%), Psychrobacter (13.6%), and Shewanella (18.6%) were the second largest predominant genus in wild fishes recovered from these Bays (Table 1). Antimicrobial-resistant isolates from wild fishes belonged from ten to fourteen different genera, and from these, only Pseudomonas, Psychrobacter, Shewanella and Photobacterium, Vibrio and Brochothrix genera were detected in fishes from all sampled Bays (Supplementary Tables S1–S3).

2.2. Antimicrobial Resistance Patterns

Antimicrobial resistance patterns of the recovered strains were not related to specific bacterial species or Bay sampled. Overall, highest percentages of antimicrobial resistance among Chilean isolates were observed against streptomicin (82.4%), amoxicillin (67.4%), and furazolidone (63.3%), whereas the lowest incidence of resistance to ciprofloxacin (3.7%), meropenem (22.5%), and oxytetracycline (29.8%), was detected among the studied isolates (Figure 1a). When sampled Bays were compared, isolates recovered from Concepción and Puerto Montt Bays exhibited the lowest proportions of resistance to the antimicrobials, meropenem (11.4% and 3.6%), ciprofloxacin (4.5% and 12.5%), and kanamycin (22.7% and 30.4%), whereas isolates from Coquimbo Bay evidenced lowest resistance proportions against the antimicrobials ciprofloxacin (0.7%), oxytetracycline (23.2%), and trimethoprim/sulfamethoxazole (28.9%) (Figure 1a). Conversely, isolates recovered from fishes captured at Coquimbo, Concepción, and Puerto Montt Bays, exhibited highest proportions of resistance to the antimicrobials streptomycin (85.2%, 63.6% and 83.9%), amoxicillin (64.8%, 70.4% and 73.2%), and furazolidone (56.6%, 85.5% and 73.2%), as shown in Figure 1a.

High levels of multiresistance among antimicrobial-resistant isolates from sampled Bays were detected, observing that 29.6%, 54.8% and 33.9% of isolates from Coquimbo Bay, Concepción Bay, and Puerto Montt Bay, respectively, showed simultaneous resistance to 7–11 antimicrobials (Figure 1b). In this trend, similar antimicrobial resistance indexes (ARI) were observed among the antimicrobial-resistant bacteria isolated from the studied Bays, exhibiting ARI values of 0.37, 0.47, and 0.44 for isolates recovered from Coquimbo, Concepción, and Puerto Montt Bays, respectively. Otherwise, a high proportion of isolates from all Bays showed high levels of simultaneous resistance to at least three antimicrobial classes (71.1%, 81.8%, and 89.3% of isolates recovered from Coquimbo, Concepción, and Puerto Montt Bays, respectively), thus exhibiting a multi-drug resistance phenotype. Reference strain Escherichia coli ATCC 25922 was used for quality control in all susceptibility assays exhibited. Antimicrobial inhibition values within the ranges were considered acceptable by CLSI [21].

2.3. Carriage of Antimicrobial Resistance Genes (ARGs)

From a comprehensive perspective, isolates recovered from wild fishes residing in Chilean anthropogenically impacted waters evidenced the carriage of a wide variety of ARGs encoding for resistance to antimicrobial classes of critical importance for human and animal health, confirming the role of wild fishes as reservoirs of these elements. Geographically, no significant differences in the distribution of studied ARGs among the studied Chilean Bays were observed.

As shown in Table 2, a considerable number of gut isolates from wild fishes carried at least one bla gene (37 isolates), encoding for resistance against cephalosporins (10.6%), exhibiting a high predominance of representatives of the Pseudomonas genus (28 isolates). Among the bla-carrying isolates, a high predominance of the blaCTX-M1 gene (23 isolates), followed by the carriage of the blaCTX-M4 gene (four isolates), was observed. All of these bla-carrying isolates exhibited a positive resistance phenotype.

As an overall result, 15 out of 245 isolates (6.1%) were positive for the carriage of a gene, encoding for the synthesis of a metallo-β-lactamase, observing the highest carriage of the blaSPM gene (eight isolates). Among the five assayed carbapenemase-producing genes, four of them were detected among some isolates from wild fishes, and only the blaGYM gene was not detected in any isolate (Table 2). It is important to note that all bla-carrying isolates encoding for cephalosporinases and carbapenemases exhibited the resistant phenotype against the third-generation cephalosporin cefotaxime, and the carbapenem meropenem, respectively. In addition, the phenotypic detection of Extended-Spectrum-β-Lactamase (ESBL) and carbapenemases activity were demonstrated in all of these isolates.

When the carriage of chromosomal Ampc β-lactamase was investigated, only five isolates from wild fishes were captured at Coquimbo Bay (three isolates carrying blaCMY, and one isolate carrying blaFOX or blaDHA) and two resistant isolates recovered from fishes captured at Concepción (one isolate carrying blaFOX or blaACC) Bay were positive for this β-lactamase group, whereas no carriage of this β–lactamase enzyme was detected among isolates from Puerto Montt Bay (Table 3).

Additionally, resistance against phenicols was extensively detected among the studied bacterial isolates (Table 2). A high percentage of antimicrobial-resistant isolates from wild fishes from the studied Bays carried the floR gene (31.7% of isolates from Coquimbo Bay, 52.3% of isolates from Concepción Bay, and 37.3% of isolates from Puerto Montt Bay), whereas none of the isolates was positive for the fexA gene, encoding for another florfenicol specific exporter (Table 2).

A high diversity of tetracycline resistance (tet) genes was detected among the gut bacteria from wild fish species, with at least four different tet genes detected in isolates from each Bay, as shown in Table 2. Overall, 47 out of 245 isolates (19.2%) were positive for the carriage of a tet gene, and seven out of the nine assayed tet genes were detected in the resistant bacteria recovered from wild fishes inhabiting the sampled Bays (Table 2). Only the tet(34) and tet(35) genes were not detected among assayed isolates. A high occurrence of resistant isolates carrying a tet gene was observed among isolates from wild fish species captured at Puerto Montt Bay, with a remarkable predominance of the tet(E) gene (18.6% of isolates). In accordance, tet(E) gene was the predominant tet gene among isolates from wild fishes from Coquimbo and Concepción Bays, but at lesser percentages.

The occurrence of sulfonamide-resistance encoding genes was infrequent, and only two isolates from each Bay were carrying the sul1 gene, whereas only one isolate from Coquimbo and Puerto Montt Bays were positive for the sul2 gene, as shown in Table 2.

2.4. Plasmid Content

A considerable number of isolates from gut microbiota of wild fishes captured from Chilean Bays carried plasmids (114 out of 245), and from these, 34 isolates contained two or more plasmid bands (Figure 2). Thus, the percentage of plasmid carrying isolates from studied Bays varied between 31.8% and 53.5%. As previously reported, a high percentage of isolates from wild fishes captured at Coquimbo Bay harbored plasmid content (76 out of 142 isolates), observing a predominance of plasmid weighting 20, 50, and 100 kb (11, 22, and 16 isolates, respectively), and from these, 18 and four isolates contained two and three plasmids, respectively [20]. Plasmid DNA was detected in 14 out of 44 isolates from fishes captured at Concepción Bay, and from these, four and one isolates carried two and three plasmids, respectively. Among the plasmid-carrying isolates, a high diversity of plasmid bands with molecular weights in the range from 10 to 80 kb was observed, exhibiting plasmid bands with molecular weight of 10 kb (two isolates), 15 kb (three isolates), 50 kb (two isolates), 60 kb (two isolates), and 80 kb (two isolates) followed by plasmids with a lower molecular weight. Plasmid DNA was detected in 24 out of 59 resistant isolates recovered from wild fishes captured at Puerto Montt Bay, and of these, seven isolates contained two plasmid bands. Among these isolates, the most found plasmid band had a molecular weight of 50 kb (18 isolates), followed by plasmids of 100 kb (three isolates).

As is shown in Table 3, gut bacteria from Chilean wild fishes carrying at least one β-lactamase encoding gene were predominantly identified as belonging to the Pseudomonas genus, comprising the 75.7%, and Shewanella, in a lower percent (16.2%) of the isolates positive for a bla gene. Furthermore, an important percentage of bla-carrying isolates were also harboring plasmid elements (48.6%), suggesting the feasibility of their horizontal dissemination in the aquatic system (Table 3).

Additionally, wild fish species, where bla-carrying bacteria were isolated vary greatly, and eight out of nine studied fish species carried isolates, at least observing that these bacteria were recovered from eight different fish species, confirming the widespread of fish species acting as reservoirs of these genes (Table 3).

Moreover, among bla-carrying isolates, a high percentage (70.3%), carried simultaneously the florfenicol resistance gene, floR, whereas only three of these isolates carried simultaneously a tet gene (Table 3).

2.5. Carriage of Class-1 Integron

As was previously reported by Miranda et al. [20], only two out of 142 isolates from wild fishes captured at Coquimbo Bay carried a class 1 integron, which were identified as Pseudomonas sp. NCIA62 and Pseudomonas sp. NCIF20, recovered from fish species Trachurus murphyi and Menticirrhus ophicephalus, respectively. Isolate NCIF20 harbored two plasmids (1.5 and 50 kb), whereas no plasmid content was detected in isolate NCIA62. Among the isolates from wild fishes captured at Concepción Bay, only the isolates Pseudomonas CCIA1 from Merluccius gayi and Pseudomonas CCIA12 from Pinguipes chilensis were positive for carrying of a class 1 integron. Isolates CCIA1 and CCIA12 carried plasmids of 50 kb and 2 kb, respectively. Otherwise, only the isolate Vibrio CIF31 from T. murphyi captured at Puerto Montt Bay carried a class 1 integron, as well as carrying a plasmid element of 100 kb. It must be noted that none of the studied bacterial isolates carried a class 2 integron. Therefore, this study show that integron-carrying bacterial isolates from wild fishes inhabiting anthropogenically impacted Chilean coasts are very rare, because only five out of 245 isolates (2.0%) were positive for carriage of this mobilome element, evidencing a wide geographic distribution at very low levels.

3. Discussion

To our knowledge, this is the first report of the occurrence of ARGs carried by gut bacteria of marine wild fishes in Chile, despite the extensively impacted coastal waters in this country. As we previously noted [20], the high incidence of untreated urban effluents disposed into aquatic waters in Chile [22], demands performing studies to assess the spread of bacteria carrying genes encoding antimicrobial resistance which could be spread in these water bodies. Some aspects of the anthropic impact on each sampled Bay that must be considered to explain differences among them is that, in addition to disposal of urban effluents in all three Bays, Concepción Bay is subject to the intense impact of industrial discharges due to the intensive activity of fishing and fish processing companies producing fishmeal, while Coquimbo and Puerto Montt Bays mainly receives domestic and gastronomic wastes, because of their high importance as tourism sites.

In agreement with the present results, ARGs have been frequently reported in isolates belonging to the Pseudomonadaceae family [23]. However, the high predominance of antimicrobial resistant isolates belonging to the Pseudomonas genus contrasted with the study of Miranda and Zemelman [19], who found that among the antimicrobial resistant gut bacteria from wild fish inhabiting the Concepción Bay in Chile, Pseudomonas representatives were almost absent. However, that study was developed 20 years ago, when the consumption by Chilean population of antimicrobials was not regulated and not requiring a medical prescription, thus favoring self-medication and excessive consumption of these drugs, suggesting that higher levels of antimicrobial residues were entering into coastal waters by disposal of urban effluents, most probably influencing the structure and levels of antimicrobial resistant microbiota residing in the ecosystems.

As previously noted by Muziasari et al. [11], fish gut has been found to contain much more abundant ARGs than the other parts of the fish body, such as skin and gills, thus we investigated the carriage of ARGs by gut microbiota of wild fishes. ARGs encoding resistance to cephalosporins of second to fourth generation, carbapenems, amphenicols, tetracyclines, and sulfonamides were investigated. These antimicrobials have been classified as critically important (cephalosporins of third to fourth generation, quinolones, and carbapenems), or highly important (amphenicols, tetracyclines, and sulfonamides), for human health, according the WHO classification [24,25].

As a major concern, the most widespread mode of clinical resistance development to β–lactam antibiotics is the expression of β–lactamases that hydrolyze the β–lactam ring [26,27], which are increasingly being reported among environmental bacteria, and exhibiting a transmissible antimicrobial resistance to third and fourth-generation cephalosporins mostly mediated by Extended Spectrum Beta-Lactamases (ESBL).

In a recent study, using functional metagenomic analysis, Ren et al. [28] demonstrated that wildlife are natural reservoirs of novel β–lactamases declaring that ARGs develop subtly in wildlife, which may interplay with human resistome over time, thus concluding that wildlife and associated environments serve as natural reservoirs for novel β–lactamases. In another study, de Araujo et al. [29], found that operating wastewater treatment plants are not sufficient to eliminate the carbapenemase genes, finding the occurrence of various carbapenemase genes in two important aquatic environments in Brazil, highlighting the role of aquatic environments as gene pools.

The only few studies investigating ARGs in wild fishes, found a predominance of blaCTX-M and blaTEM genes among gut microbiota [30,31,32,33]. Baothman et al. [30] investigated the incidence of ESBL-expressing Enterobacteriaceae in the gut of wild fish species from the Red Sea coastal region of Saudi Arabia, concluding that the evolution of the blaCTX-M gene in Morganella morganii is a consequence of aquatic pollution. Furthermore, Brahmi et al. [31] studied the prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in wild fish from the Mediterranean Sea in Algeria, observing a predominance of the blaCTX-M gene.

In contrast, in disagreement with the results of this study, most of the studies describing ARGs among fish gut microbiota reported the carriage of blaTEM gene, encoding for a β–lactamase [28,30,31,32], but surprisingly none of the isolates from this study carried that gene. In this trend, Sellera et al. [17] reported for the first time the occurrence of Escherichia coli carrying β-lactamases genes among gut bacteria from wild fishes from Brazilian polluted waters, observing an important carriage of blaTEM and intI1-integrase genes. In this trend, Ryu et al. [34] suggested that commercial fish and seafood may act as the reservoir for multi-resistant bacteria and facilitate the dissemination of the resistance genes, mostly blaTEM genes [31].

Furthermore, Khurana et al. [35] found that extended-spectrum β-lactamase (ESBL) was predominantly associated with Klebsiella pneumoniae present in the gut of Tor putitora, whereas Tyagi et al. [36] found that genes encoding extended spectrum β-lactamase (blaTEM, blaCTX-M-1) were also found in the gut microbiome of freshwater Indian carp [36]. When studied common antibiotic resistance genes and integrons among antimicrobial-resistant bacteria isolated from eels and aquaculture ponds in China, Lin et al. [36] found that genes blaTEM, tet(C), sul1, aadA, floR, and qnrB were detected at high percentages, whereas class-1 integrons were present in 79.6% of the isolates [37].

This study demonstrated the occurrence and distribution of diverse β–lactamase-encoding genes, and among them, blaCTX-M1 gene was predominant in wild fishes from all Bays, followed by blaSPM and blaCTX-M4 genes.

This is the first report of the presence of gut bacteria carrying ARGs encoding the production of β-lactamases in wild fish in Chile. Compared to other studies investigating the carriage of ARGs by gut bacteria from wild fish recovered in other countries, the predominance of the gene blaCTX-M is in agreement with many of these studies [16,17,30,31,32,33,34], whereas the absence of the blaTEM gene in this study is contrary to other studies, usually reporting a predominance of this gene [34]. On the other hand, the diversity and low incidence of ARGs, encoding the synthesis of metallo-β-lactamases, which included the blaSPM, blaSIM, blaVIM, and blaIMP genes detected in Chilean wild fish, is contrasting with other studies in which a complete absence or predominance of a particular metallo-β-lactamase encoding-gene is commonly observed [29].

In agreement with the results of this study, which have shown that integron-carrying antimicrobial-resistant bacteria from wild fishes are very rare, previous studies have demonstrated that carriage of integrons is not usually detected among wild fish gut bacteria [38,39,40,41,42]. In this context, Jia et al. [38] found a low abundance of intI1–integrase gene in the gut of guppies (Poecilia reticulata), demonstrating that intI1 is not the main element in diffusing ARGs in this ecosystem.

Furthermore, Barraud et al. [39] did not detect integrons among bacteria from wild fish, in comparison with farmed fish, with a moderate prevalence of integrons; and diseased fish, in which integron prevalence was high. In another study, Vivant et al. [40] studied Enterobacterales resistant to antimicrobials, isolated from gut microbiota of wild fish inhabiting a highly urbanized river in France, not detecting ESBL-producing bacteria, and only few of them carried a class-1 integron, whereas Bollache et al. [41] observed a high spread of blaCTX-M carrying E. coli in freshwater fishes from French watershed. In another study, the abundances of ARGs were quantified in the gut of Chinese freshwater carp, observing that sul1, bla_TEM-1_, and tet(A) genes were the most prevalent ARG [42].

Additionally, floR gene, encoding a phenicol-specific exporter was detected among gut bacteria from wild fishes from all Bays, despite that only Puerto Montt Bay is located at south of Chile, near salmon farming settings. Apparently, this gene is ubiquitous in microbiota associated to marine wild fishes in Chile.

A high percentage of antimicrobial-resistant bacteria from wild fish from all sampled Bays are carrying plasmid elements, as was previously reported for gut bacteria from Coquimbo Bay [20], which could provide very favorable conditions for genetic exchange through conjugative plasmids among gut microbiota of wild fishes destined for human consumption. Thus, it can be concluded that wild fishes along Chilean coasts are highly relevant as reservoirs of these mobilome elements, although it should be mentioned as a limitation of this study the analysis of only three bays along the long Chilean coast.

As previously noted, bacterial pathogens are the main pathway for the spread of ARGs [43], thus it is highly expected that antimicrobial resistant fecal bacteria carrying ARGs encoded on mobile DNA elements, such as conjugative plasmids, integrons, or transposons [44], from domestic and wastewater treatment plant discharges might transfer their ARGs to indigenous bacteria of fish provoking their spread and prevalence in the marine environment, as well as their potential transfer to fish handlers and consumers.

Fu et al. [45] investigated the dissemination of ARGs in microbial communities of zebrafish guts after the introduction in the aquaria of bacterial donors carrying self-transferring plasmids that encode ARGs, observing that 15% of fecal bacteria obtained ARGs through conjugal transfer, concluding that aquatic animal guts contribute to the spread of ARGs in water environments. Thus, the high carriage of plasmid elements among the antimicrobial-resistant bacteria associated with wild fish guts prompts the need of further studies to elucidate which ARGs are carried by these plasmids, and their capability to be horizontally transferred, in order to confirm that carriage of ARGs by wild fishes have ecological and public health implications, emphasizing the need for further studies related to ARGs of human clinical importance.

Although the occurrence of antimicrobial resistant bacteria carrying ARGs has been attributed mainly to the extensive and indiscriminate use of antibiotics in human and animal therapies the role of other contaminants usually entering to the marine coastal waters, such as biocides and heavy metals as significant contributors to this selective pressure in water systems must be highlighted [46,47]. As previously reported, heavy-metals and biocides are introduced into aquatic environment via discharges from pharmaceutical industries, hospital effluents, direct excretion from humans and livestock, and runoff from farms [48], and creating environmental reservoirs of these contaminants [49]. These agents could drive the selection and proliferation of ARGs through cross-resistance and co-resistance mechanisms, even without direct antibiotic exposure, because metal and biocide resistance genes could be co-located with ARGs on mobile genetic elements, such as plasmids, facilitating their spread [46,47].

Antimicrobial resistant bacteria carrying ARGs in commercial fish intestines have ecological and public health implications, raising questions on the origin of these ARGs, their spread and prevalence in the marine environment as well as fish handlers and consumers, as well as the possibility of the returning of ARGs to human population through fish handling and consuming, considering as potential pathways of passing ARGs to transient zoonotic bacterial pathogens that cause disease in humans, an improperly cooking or otherwise mishandling, demanding further epidemiological and molecular investigations to evaluate their public health importance, especially in relation to the ARGs of clinical importance. In this context, we are currently studying, antimicrobial resistant bacteria carrying ARGs isolated from wild fish mucus.

4. Materials and Methods

4.1. Bacterial Isolates

A total of 245 antimicrobial-resistant isolates were obtained by selecting different-looking colonies grown on plates with Tryptic soy agar (BBL BD Becton Dickinson™, Sparks, MD, USA), added with 2% NaCl (TSA2) containing an antimicrobial agent and seeded with intestinal content samples, as described in Miranda et al. [20]. Antimicrobial resistant isolates were recovered from various marine fish species as shown in Supplementary Tables S1–S3, captured by artisanal fishing from three main Chilean bays located along the country (Figure 3). Antimicrobial resistant isolates were recovered from wild fishes captured at Coquimbo Bay (142 isolates), Concepción Bay (44 isolates), and Puerto Montt Bay (59 isolates) (Supplementary Tables S1–S3). Fish samples were obtained from the fishermen’s boats, transported on ice to the Aquatic Pathobiology Lab of the Universidad Católica del Norte, and processed as described [20]. Sampled fishes were aseptically eviscerated, collecting and processing intestinal content samples, as previously described [20]. Bacterial isolates were purified using plates with TSA2 medium and stored at −85 °C in CryoBank^TM^ vials (Mast Diagnostica, Reinfeld, Germany) before being grown in TSA2 at 20 °C for 24–48 h until use.

4.2. Bacterial Identification

Antimicrobial-resistant isolates were identified using bacterial 16S rRNA gene sequence analysis. Isolates were cultured in Tryptic soy broth (TSB2, BBL BD Becton Dickinson™, Sparks, MD, USA), with 2% NaCl at 22 °C for 12–24 h and pellets were obtained centrifuging bacterial cultures at 9000× g for 3 min using an Eppendorf 5415D (Eppendorf AG, Hamburg, Germany) microcentrifuge. DNA extraction was performed using the WizardR Genomic DNA Purification commercial kit (Promega, Madison, WI, USA) following the supplier’s instructions. The amplification of the 16S ribosomal genes of isolates was performed using PCR, using the procedure described by Opazo et al. [50]. The resulting amplified PCR products were sequenced by Macrogen (Rockville, MD, USA), using the ABI PRISM 373 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were edited and matched to the Silva database (https://www.arb-silva.de/aligner, accessed on 26 November 2025) to identify the bacterial isolates, and 16S rDNA gene sequences were deposited in the GenBank database (Supplementary Tables S1–S3).

4.3. Antimicrobial Susceptibility Patterns

The antimicrobial susceptibility of resistant isolates was determined using a disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guideline M02-A12 [51], using plates containing Cation-Adjusted Mueller–Hinton agar (CAMH, BBL BD Becton Dickinson™, Sparks, MD, USA), and disks (Oxoid Ltd., Basingstoke, Hampshire, UK) containing the antimicrobials amoxicillin (AML, 25 µg), cefotetan (CTT, 30 µg), cefotaxime (CTX, 30 µg), meropenem (MEM, 10 µg), streptomycin (S, 10 µg), kanamycin (K, 30 µg), chloramphenicol (CM, 30 µg), florfenicol (FFC, 30 µg), oxytetracycline (OTC, 30 µg), ciprofloxacin (CIP, 5 µg), furazolidone (FR, 100 µg), and sulfamethoxazole-trimethoprim (SXT, 25 µg), as described in Miranda et al. [20]. Plates were incubated at 22 °C for 24 h, and isolates were considered resistant according to the CLSI criteria [52]. As suggested by the CLSI guideline M02-A12, the quality control strain Escherichia coli ATCC 25922 was included in all antimicrobial susceptibility test groups, and results were accepted when results of E. coli ATCC 25922 were within the Disk diffusion QC ranges recommended by CLSI [53]. All isolates were re-examined to check the assay reproducibility. Isolates showing resistance to at least three groups of antimicrobials were considered exhibiting the Multi-Drug Resistance (MDR) phenotype. In addition, the antimicrobial resistance index (ARI) of sampled Bays were determined according to Hinton et al. [54].

4.4. Phenotypic Detection of Extended-Spectrum-β-Lactamase (ESBL) Activioty

The production of ESBL was investigated in isolates showing reduced susceptibility (≤23 mm), to cefotaxime [55], using two phenotypic methods, as described in Miranda et al. [20]. ESBL production was detected by the Combination Disc Diffusion Test (CDDT) method, according to CLSI guidelines [56,57], using disks containing cefotaxime, both alone (CTX, 30 µg) and in combination with clavulanic acid (CTL), placed onto agar plates 15 mm apart. Plates were incubated at 22 °C for 24 h and production of ESBL was confirmed in the isolates exhibiting a ≥5 mm increase in the inhibition zone diameter of the CTL, compared with the CTX disc [58]. Furthermore, ESBL production was phenotypically detected by using the Double Disc Synergy Test (DDST) method [59]. An amoxicillin–clavulanic acid disc (AMC, 20/10 µg) was placed at the center of the CAMHA plate inoculated with the test isolate, and a ceftazidime disc (CAZ, 30 µg), ceftriaxone disc (CRO, 30 µg), and cefotaxime disc (CTX, 30 µg) were each placed 25–30 mm apart from the center disk. Plates were incubated at 22 °C for 24 h, and an increase of inhibition zone towards the AMC disc was considered positive for ESBL production [60]. In all assays, Klebsiella pneumoniae ATCC 700603 (ESBL producing strain) and E. coli ATCC 25922 (susceptible strain) were used as positive and negative control strains for the ESBL production, respectively [61].

4.5. Phenotypic Detection of Carbapenemase Activity

Bacterial isolates showing inhibition zones of ≤23 mm to meropenem were considered likely to produce a Metallo-β-Lactamase (MBL) enzyme [62], and were screened for MBL production using two methods. The production of MBLs, was investigated employing a combined disc diffusion test (CDDT) method [52], using discs containing meropenem, both alone (MRP, 10 µg) and combined with the metal chelator, ethylene diamine tetra-acetic acid (EDTA) (MRP EDTA, 30 µg), placed 15 mm apart. Plates were incubated at 22 °C for 24 h [63], and a ≥7 mm increase in the inhibition zones of discs of MRP EDTA compared with those from MRP discs phenotypically confirmed the production of MBLs [64,65,66]. Furthermore, MBL production was assayed using the modified Hodges test [67,68], aseptically swabbing CAMHA plates with the E. coli ATCC 25922 strain, and then meropenem (MRP, 10 µg) discs were aseptically placed at the center of the plates, and finally assayed isolates were heavily streaked on a straight line from the edge of the meropenem disc to the edge of the plate. Plates were incubated at 22 °C for 24 h and a clover-leaf-type indentation or flattening at the intersection of the tested isolate and the E. coli ATCC 25922 within the zone of inhibition of the meropenem disc was considered positive for MBL production [66].

4.6. Detection of Antimicrobial Resistance Genes

The carriage of genes encoding for resistance to main antimicrobial classes, such as β-lactams, amphenicols, tetracyclines, and sulfonamides was investigated, using primers previously described, as shown in Supplementary Table S4.

4.6.1. Genes Encoding for β-Lactam Resistance

The occurrence of various bla genes, encoding resistance against β-lactams, was detected using the procedure described by Dey et al. [57]. The amplification conditions for blaTEM and blaSHV genes were as described in Kojima et al. [69], for the blaCTX-M1, blaCTX-M2, blaCTX-M3, and blaCTX-M4 genes were as described in Pitout et al. [70], and for the blaFOX, blaMOX, blaEBC, blaACC, blaDHA, and blaCMY genes were as described in Pérez-Pérez and Hanson [71], whereas for the carbapenemase-encoding genes blaIMP, blaVIM, blaGIM, blaSIM, and blaSPM, were as described in Mendes et al. [72]. The amplification conditions were as follows: denaturation at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 60 s, annealing at 60 °C (blaTEM, blaSHV, blaCTX-M1, blaCTX-M2, blaCTX-M3, and blaCTX-M4), 53 °C (blaIMP, blaVIM, blaGIM, blaSIM, and blaSPM), or 64 °C (blaFOX, blaMOX, blaEBC, blaACC, blaDHA, and blaCMY) for 40 s, and elongation at 72 °C for 60–65 s; and finally, extension at 72 °C for 7 min using the GE-96G thermocycler (BIOER Technology, Hangzhou, China) using the primers described in Supplementary Table S4.

4.6.2. Genes Encoding for Amphenicol Resistance

The carriage of floR and fexA genes conferring resistance to amphenicols, was investigated using PCR assays according to Hurtado et al. [73]. The amplification conditions were as described in Hurtado et al. [73], for the floR gene, and Higuera-Llantén et al. [10] for the fexA gene, using the primers shown in Supplementary Table S4. Briefly, the amplification conditions were denaturation at 95 °C for 4 min; 30 cycles of denaturation at 95 °C for 40 s, annealing at 58 °C for 30 s, elongation at 72 °C for 60 s; and finally, extension at 72 °C for 5 min using the GE-96G thermocycler (BIOER Technology, China). The positive controls Citrobacter freundii FB98 for floR gene, and Vibrio tasmaniensis AVF09 for fexA gene [73], were included in each gel run.

4.6.3. Genes Encoding for Tetracycline Resistance

The occurrence of the tet genes, encoding for tetracycline resistance was investigated using PCR using the procedure described by Ng et al. [74] for tet(A), tet(C), tet(G), and tet(E) genes, whereas tet(D), tet(34), and tet(35) genes were investigated using the methodology described by Miranda et al. [75]. The presence of the tet(B) gene was investigated following the methodology described by Higuera-Llantén et al. [10], whereas tet(39) gene was detected using the methodology described by Hamidian et al. [76]. Primers used are shown in Supplementary Table S4. Briefly, the amplification conditions for tet(A), tet(C), tet(G), and tet(E) genes were denaturation at 95 °C for 5 min; 30 cycles of denaturation at 94 °C for 60 s, annealing at 55 °C for 40 s, elongation at 72 °C for 60 s; and finally, extension at 72 °C for 7 min using the GE-96G thermocycler (BIOER Technology, China), whereas amplification conditions for tet(B), tet(D), tet(34), tet(35), and tet(39) were as previously described [10,75,76].

4.6.4. Genes Encoding for Sulfonamide Resistance

The occurrence of sul1 and sul2 genes, encoding for resistance to sulfonamides, was investigated using PCR according to procedures described by Domínguez et al. [77], using the primers previously described [77], as shown in Supplementary Table S4. Briefly, amplifications were performed in a GE-96G thermocycler (BIOER Technology, China). with one cycle of 95 °C for 10 min, 30 cycles of 94 °C for 45 s, annealing at 64 °C for 30 s, elongation at 51 °C for 45 s and 72 °C for 2 min, with a final extension at 72 °C for 10 min. The control strain Citrobacter gillenii FP75, positive for both genes was included in each assay [77].

4.7. Detection of Class-1 Integron

The carriage of intl1 gene (class 1 integron integrase) was investigated using the methodology and primers described in Domínguez et al. [77]. Briefly, the amplification of the intI1-integrase gene was performed using a BIOER Technology^TM^ model GE-96G thermal cycler using the cycle conditions, 95 °C for 30 min, followed by 30 cycles of 95 °C for 30 s, 58 °C for 30 s (annealing temperature), 72 °C for 1 min, and a final extension at 72 °C for 5 min. The production of two fragments (393 and 499 bp) from the intl1 amplicon treated with the restriction enzyme, SphI confirmed the presence of the intl1 gene [20]. The strain Citrobacter gillenii FP75 was used as positive control for intl1 gene [77].

4.8. Plasmid Content

Isolates were cultured in TSB2, for 12 h at 22 °C and pellet was obtained by centrifugation at 9000× g for 5 min using an Eppendorf 5415D microcentrifuge to obtain a pellet. Plasmid DNA was extracted by using the Wizard^®^ Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA) following the supplier’s instructions. Plasmid DNA was ran on 1.5% agarose gel electrophoresis for plasmids less than 20 kb and 0.8% agarose gel for plasmid greater than 20 kb, as described by Domínguez et al. [77]. Gels were stained with GelRed^TM^ (Biotium, Fremont, CA, USA), viewed by UV transillumination, and plasmid size was estimated by comparing with standard molecular weight markers Quick-Load^®^ 1 kb Extend DNA Ladder and known plasmid weight standards [73].

5. Conclusions

The impact of anthropogenic activities in marine coastal waters in Chile is undeniable but to date there are no studies of the resistome associated with wild fishes residing in these systems. This study demonstrated that intestinal contents of wild fishes inhabiting Chilean Bays impacted by diverse anthropogenic activities are acting as reservoirs of antimicrobial-resistant bacteria carrying a plethora of antimicrobial-resistant genes (ARGs) encoding for resistance against various classes of antimicrobials of human and animal importance, as well as plasmids, becoming important drivers of spread of resistome and mobilome components, even in the absence of antimicrobial use, threatening human health safety.