This study aimed to investigate AMR profiles of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from Nile tilapia (Oreochromis spp.) (n = 276) purchased from fresh markets and supermarkets in Bangkok, Thailand. A sample of tilapia was divided into three parts: fish intestine (n = 276), fish meat (n = 276), and liver and kidney (n = 276). The occurrence of A. hydrophila, Salmonella, and V. cholerae was 3.1%, 7.4%, and 8.5%, respectively. A high prevalence of these pathogenic bacteria was observed in fresh market tilapia compared to those from supermarkets (p < 0.05). The predominant Salmonella serovars were Paratyphi B (6.4%), followed by Escanaba (5.7%), and Saintpaul (5.7%). All isolates tested positive for the virulence genes of A. hydrophila (aero and hly), Salmonella (invA), and V. cholerae (hlyA). A. hydrophila (65.4%), Salmonella (31.2%), and V. cholerae (2.9%) showed multidrug resistant isolates. All A. hydrophila isolates (n = 26) exhibited resistant to ampicillin (100.0%) and florfenicol (100.0%), and often carried sul1 (53.8%) and tetA (50.0%). Salmonella isolates were primarily resistant to ampicillin (36.9%), with a high incidence of blaTEM (26.2%) and qnrS (25.5%). For V. cholerae isolates, resistance was observed against ampicillin (48.6%), and they commonly carried qnrS (24.3%) and tetA (22.9%). To identify mutations in the quinolone resistance determining regions (QRDRs), a single C248A point mutation of C248A (Ser-83-Tyr) in the gyrA region was identified in six out of seven isolates of Salmonella isolates. This study highlighted the presence of antimicrobial-resistant pathogenic bacteria in Nile tilapia at a selling point. It is important to rigorously implement strategies for AMR control and prevention.

Background

Tilapia is a primary aquaculture fish in Thailand, but little is known about the occurrence of antimicrobial resistance (AMR) in Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae colonizing healthy tilapia intended for human consumption and the co-occurrence of these AMR bacteria in the cultivation water.

Methods

This study determined the phenotype and genotype of AMR, extended-spectrum β-lactamase (ESBL) production, and virulence factors of A. hydrophila, Salmonella spp., and V. cholerae isolated from hybrid red tilapia and cultivation water in Thailand. Standard culture methods such as USFDA's BAM or ISO procedures were used for the original isolation, with all isolates confirmed by biochemical tests, serotyping, and species-specific gene detection based on PCR.

Results

A total of 278 isolates consisting of 15 A. hydrophila, 188 Salmonella spp., and 75 V. cholerae isolates were retrieved from a previous study. All isolates of A. hydrophila and Salmonella isolates were resistance to at least one antimicrobial, with 26.7% and 72.3% of the isolates being multidrug resistant (MDR), respectively. All A. hydrophila isolates were resistant to ampicillin (100%), followed by oxytetracycline (26.7%), tetracycline (26.7%), trimethoprim (26.7%), and oxolinic acid (20.0%). The predominant resistance genes in A. hydrophila were mcr-3 (20.0%), followed by 13.3% of isolates having floR, qnrS, sul1, sul2, and dfrA1. Salmonella isolates also exhibited a high prevalence of resistance to ampicillin (79.3%), oxolinic acid (75.5%), oxytetracycline (71.8%), chloramphenicol (62.8%), and florfenicol (55.3%). The most common resistance genes in these Salmonella isolates were qnrS (65.4%), tetA (64.9%), bla _TEM (63.8%), and floR (55.9%). All V. cholerae isolates were susceptible to all antimicrobials tested, while the most common resistance gene was sul1 (12.0%). One isolate of A. hydrophila was positive for int1, while all isolates of Salmonella and V. cholerae isolates were negative for integrons and int _SXT. None of the bacterial isolates in this study were producing ESBL. The occurrence of mcr-3 (20.0%) in these isolates from tilapia aquaculture may signify a serious occupational and consumer health risk given that colistin is a last resort antimicrobial for treatment of Gram-negative bacteria infections.

Conclusions

Findings from this study on AMR bacteria in hybrid red tilapia suggest that aquaculture as practiced in Thailand can select for ubiquitous AMR pathogens, mobile genetic elements, and an emerging reservoir of mcr and colistin-resistant bacteria. Resistant and pathogenic bacteria, such as resistance to ampicillin and tetracycline, or MDR Salmonella circulating in aquaculture, together highlight the public health concerns and foodborne risks of zoonotic pathogens in humans from cultured freshwater fish.

Microbial food safety in cultured tilapia remains a challenge to public health worldwide, due in part to intensive aquaculture leading to poor water quality and high organic matter deposition. This study aimed to determine the prevalence of indicator and potential pathogenic bacteria in hybrid red tilapia (Oreochromis spp.) and their cultivation water and to identify environmental parameters and other bacterial contaminants associated with Salmonella contamination. A total of 120 fish were sampled, which were partitioned into fish carcasses (n=120), muscle (n=120), intestine (n=120), liver and kidney (n=120), and cultivation water (n=120) from three commercial farms in western Thailand from October 2019 to November 2020. The prevalence of fecal coliforms and Escherichia coli (E. coli) in these 600 samples was 74.8% and 56.7%, respectively. The prevalence of Salmonella, Vibrio cholerae (V. cholerae), Aeromonas hydrophila, and Vibrio vulnificus (V. vulnificus) was 23.0%, 17.5%, 2.5%, and 1.7%, respectively. None of the samples tested positive for Streptococcus agalactiae. Cultivation water exhibited a high prevalence for Salmonella (58.3%). Among fish samples, Salmonella had the highest prevalence at 14.1%, which was mainly from fish intestine. There was a significant association of Salmonella with the presence of fecal coliforms, E. coli, V. cholerae, and V. vulnificus. The predominant serovars of Salmonella included Saintpaul, Neukoelln, Escanaba, and Papuana. Grazing ducks that were raised in proximity to these cultured tilapia shared the same isolates of Salmonella based on the similarity of their rep-PCR DNA fingerprints, suggesting that ducks may function as either a biological reservoir for tilapia or at minimum participate in the environmental replication of this strain of Salmonella. Taken together, the results suggest that the environment used for tilapia aquaculture may be contaminated with pathogenic bacteria; therefore, food safety precautions are needed during processing, transportation, cooking, and consumption.

The occurrence of waterborne antimicrobial-resistant (AMR) bacteria in areas of high-density oyster cultivation is an ongoing environmental and public health threat given the popularity of shellfish consumption, water-related human recreation throughout coastal Thailand, and the geographical expansion of Thailand's shellfish industry. This study characterized the association of phenotypic and genotypic AMR, including extended-spectrum β-lactamase (ESBL) production, and virulence genes isolated from waterborne Escherichia coli (E. coli) (n = 84), Salmonella enterica (S. enterica) subsp. enterica (n = 12), Vibrio parahaemolyticus (V. parahaemolyticus) (n = 249), and Vibrio cholerae (V. cholerae) (n = 39) from Thailand's coastal aquaculture regions. All Salmonella (100.0%) and half of V. cholerae (51.3%) isolates harbored their unique virulence gene, invA and ompW, respectively. The majority of isolates of V. parahaemolyticus and E. coli, ~25% of S. enterica subsp. enterica, and ~12% of V. cholerae, exhibited phenotypic AMR to multiple antimicrobials, with 8.9% of all coastal water isolates exhibiting multidrug resistance (MDR). Taken together, we recommend that coastal water quality surveillance programs include monitoring for bacterial AMR for food safety and recreational water exposure to water for Thailand's coastal water resources.

Coastal pollution is of public health concern due to the possibility of bacterial contamination in aquaculture affecting health risk and seafood safety. This study determined the concentrations of total coliforms, fecal coliforms, E. coli, and V. parahaemolyticus and the presence of V. cholerae and Salmonella in water and measured water quality and meteorological factors from the major oyster cultivation areas in Thailand. The water samples (n = 363) were collected from Surat Thani (n = 60), Chanthaburi (n = 60), Trat (n = 60), Phetchaburi (n = 60), Chonburi (n = 63), and Phang Nga (n = 60) from February 2021 to January 2022. The prevalence of total coliforms (96.7%), fecal coliforms (60.6%), E. coli (22.9%), along with the prevalence of Salmonella (2.5%), Vibrio parahaemolyticus (74.9%), and V. cholerae (11.3%) were determined. Common Salmonella serovars were Othmarschen and Lamberhurst. The concentration of E. coli was significantly associated with dissolved oxygen and precipitation (p < 0.0001). Therefore, continuing microbiological monitoring and surveillance of water for coastal aquaculture is important to produce safe aquatic products. Furthermore, raising awareness of coastal pollution and seafood safety will help enhance sustainable coastal fisheries in Thailand.

This study investigated the prevalence and antimicrobial resistance (AMR) of Escherichia coli (E. coli) in Nile tilapia from fresh markets and supermarkets. A total of samples (n = 828) were collected from Nile tilapia including fish flesh (n = 276), liver and kidney (n = 276), and intestine (n = 276). Overall prevalence of fecal coliforms (61.6%) and E. coli (53.0%) were observed. High prevalence of E. coli was found in the intestine (71.4%), followed by the liver and kidney (45.7%). The highest prevalence of resistance was commonly found against tetracycline (78.5%), ampicillin (72.8%), and sulfamethoxazole (45.6%) with resistance to only tetracycline (15.2%) as the most common antibiogram. The prevalence of multidrug resistance (MDR) (54.4%) and Extended-spectrum beta-lactamases (ESBLs) (5.7%) were examined. The predominant virulence genes (n = 158) were st (14.6%), followed by eaeA (0.6%). The blaTEM (73.4%), tetA (65.2%), and qnrS (57.6%). There is statistical significance between Nile tilapia from fresh markets and supermarkets. Based on logistic regression analysis, ampicillin-resistant E. coli was statistically associated with the phenotypic resistance to tetracycline and trimethoprim, and the presence of blaTEM and tetA (p < 0.05). Further investigation of AMR transference and their mechanisms is needed for AMR control.

A Correction to this paper has been published: https://doi.org/10.1007/s10764-021-00222-6.