Exploring Plasmid-Mediated Multidrug Resistance, Virulence, and Metal/Biocide Resistance in Vibrio Spp. Isolated from Influent and Effluent Water Samples of Fish Farms in Jeju, South Korea

Abstract

The objective of this study was to investigate the plasmid profiling, virulence genes, metal/biocide resistance genes and plasmid sequencing of multi-drug resistant (MDR) Vibrio in influent (inflow) and effluent (discharged) water samples of fish farms in Jeju, South Korea. MDR isolates identified through disc diffusion susceptibility tests were subjected to plasmid profiling. One hundred fifty Vibrio species isolates were obtained from each influent and effluent water sample. All MDR isolates were subjected for plasmid profiling. The greater number of bacteria were enumerated from effluents (61%) comparing to influents (39%). High incidence of neomycin, sulfamethoxazole, amoxicillin and oxytetracycline resistance was observed among the isolates, particularly more resistance rate in effluent Vibrio. In contrast, Vibrio isolates were more susceptible to florfenicol, chloramphenicol, ciprofloxacin, and nalidixic acid. Among 99 (influent 39 and effluent 60) MDR isolates, a total of 58 plasmid harboring isolates (influent 38 and effluent 20) were identified and showed Sixteen different resistance antibiograms. Influent MDR isolates showed six distinct plasmid profiles with size ranging from 2 kb to >10 kb, whereas effluent MDR showed four plasmid profiles with a molecular weight ranging from 1.7 kb to >10 kb. All three hundred isolates were also screened for the presence of Vibrio virulence genes, thermostable direct hemolysin (tdh), cholera toxin (vct), and cytotoxin-hemolysin (vvh). There were 17 (12 influent and 5 effluent) and 27 (8 influent and 19 effluent) Vibrio isolates found to harbor thermostable direct hemolysin gene (tdh) and cytotoxin-hemolysin gene (vvh), respectively. In addition, two isolates (02 influent) were found to harbor cholera toxin gene (vct). Detection of 19 Vibrio vulnificus, vvh genes, and 8 Vibrio parahaemolyticus, the genes in the fish farm discharged water may be responsible for their potential pathogenicity. Largely virulence genes were confined to chromosomes, only 9 (3 influent, 6 effluents) detected in plasmid bearing MDR Vibrio. Twenty four (12 influent, 12 effluent) plasmid bearing MDR Vibrio isolates selected for sequencing at Illumina Miseq platform predicted 14 influent putative plasmid sequences and 17 effluent putative plasmid sequences. Furthermore, 17 antibiotic resistance genes (ARGs) in influent plasmids and 21 ARGs in effluent plasmids were identified. Influent and effluent putative plasmid sequences encoding ARGs were resistant to 12 classes of antibiotics, among which beta-lactams (BET), glycopeptides (GLY) and peptides (PEP) were only confined to effluents whereas macrolidelincosamide- streptogramin (MLS) and rifampin (RIF). Moreover, it was observed that chromosome-mediated antibiotic resistance contributed to the overall antibiotic resistance showed by plasmid bearing MDR Vibrio isolates. Two influent and three effluent putative MDR plasmids were carrying metal/biocide resistance genes. In addition, Illumina high throughput sequencing of metagenome plasmids from influents and effluents samples identified a higher abundance of ARGs encoding beta-lactams in effluents. Beta-lactam ARGs carrying bacterial genomes were studied to be involved in the foodborne diseases. Our results showed that more diverse plasmid profiles and antibiograms, higher abundance of plasmid carrying MDR Vibrio, virulence and metal/biocide resistance genes present in effluent samples, suggesting the accumulation of uneaten feed, fish excretion, residual antibiotics, metal/biocide contaminants and antibiotic resistance genes (ARGs) in fish farm tanks that may provide selective pressure and co-selection for acquisition and horizontal transfer of ARGs. The appearance of multidrug resistance plasmid carrying resistance to heavy metals is alarming. The presence of plasmid-bearing MDR Vibrio isolates in fish farm effluent water may contribute to the dissemination of MDR genes to the environments, which ultimately poses threat to human health.