Genetic Characterization of the Tetracycline-Resistance Gene tet(X) Carried by Two Epilithonimonas Strains Isolated from Farmed Diseased Rainbow Trout, Oncorhynchus mykiss in Chile

Abstract

The main objective of this study was to characterize the tet(X) genes, which encode a monooxygenase that catalyzes the degradation of tetracycline antibiotics, carried by the resistant strains FP105 and FP233-J200, using whole-genome sequencing analysis. The isolates were recovered from fin lesion and kidney samples of diseased rainbow trout Oncorhynchus mykiss, during two Flavobacteriosis outbreaks occurring in freshwater farms located in Southern Chile. The strains were identified as Epilithonimonas spp. by using biochemical tests and by genome comparison analysis using the PATRIC bioinformatics platform and exhibited a minimum inhibitory concentration (MIC) of oxytetracycline of 128 µg/mL. The tet(X) genes were located on small contigs of the FP105 and FP233-J200 genomes. The sequences obtained for the tet(X) genes and their genetic environment were compared with the genomes available in the GenBank database of strains of the Chryseobacterium clade belonging to the Flavobacterium family, isolated from fish and carrying the tet(X) gene. The Tet(X) proteins synthesized by the Chilean Epilithonimonas strains showed a high amino acid similarity (range from 84% to 100%), with the available sequences found in strains belonging to the genus Chryseobacterium and Flavobacterium isolated from fish. An identical neighborhood of tet(X) genes from both Chilean strains was observed. The genetic environment of tet(X) observed in the two strains of Epilithonimonas studied was characterized by the upstream location of a sequence encoding a hypothetical protein and a downstream located alpha/beta hydrolase-encoding gene, similar to the observed in some of the tet(X) genes carried by Chryseobacterium and Flavobacterium strains isolated from fish, but the produced proteins exhibited a low amino acid identity (25–27%) when compared to these synthesized by the Chilean strains. This study reports for the first time the carriage of the tet(X) gene by the Epilithonimonas genus and their detection in fish pathogenic bacteria isolated from farmed salmonids in Chile, thus limiting the use of therapies based on oxytetracycline, the antimicrobial most widely used in Chilean freshwater salmonid farming. This results suggest that pathogenic strains of the Chryseobacterium clade occurring in Chilean salmonid farms may serve as important reservoirs of tet(X) genes.

Dissemination and prevalence of plasmid-mediated high-level tigecycline resistance gene tet (X4)

“…In addition, five tet (X4)-positive Acinetobacter were also isolated from the bar-headed goose samples in Qinghai ( Chen et al, 2020 ). In the latest report, researchers also detected the tet (X) variant genes in wild fish and shrimp ( Li et al, 2019 ; Concha et al, 2021 ). Wild animals were not directly exposed to clinical antibiotics, but more and more ARGs were detected in them, indicating wild animals including migratory birds, were likely to be involved in the large-scale exchange of ARGs, especially long-distance transmission of cross species ( Allen et al, 2010 ; Wang et al, 2017 ; Zeballos-Gross et al, 2021 ; Luo et al, 2022 ).…”

Phenotypic and genomic characterization of a non‐pathogenic Epilithonimonas ginsengisoli isolated from diseased farmed rainbow trout (Oncorhynchus mykiss) in Chile

“…Considering that oxytetracycline is the most widely used antimicrobial in the freshwater phase of salmonid farming in Chile, the increasing detection of tet(X)-carrying flavobacterial representatives suggests a growing risk of therapeutic failure and prompts the need for continuous monitoring of this gene, which could spread easily in the freshwater fish-farming environment (Shin et al, 2023). In a previous study, the tet(X) gene was detected in the bacterial isolates FP105 and FP233-J200 isolated from a fin lesion (FP105) and the kidney (FP223-J200) of diseased rainbow trout positively diagnosed with flavobacteriosis and sampled from two freshwater Chilean farms located in the south of Chile (Llanquihue Lake and Cude River, respectively) (Ayala et al, 2017;Concha et al, 2021).…”

“…The genus type strain Epilithonimonas tenax was recovered from freshwater epilithon (O'Sullivan et al., 2006). To date, three isolates collected from flavobacterial outbreaks occurring in freshwater Chilean rainbow trout ( Oncorhynchus mykiss ) farms have been described as belonging to this genus, but the exact taxonomic position, prior to this study, was not identified (Ayala et al., 2017; Concha et al., 2021). Interestingly, apart from identification at the genus level and antibiotic resistance, the pathogenicity of Epilithonimonas spp.…”

Antibiotic Resistance in the Finfish Aquaculture Industry: A Review

“…There are multiple examples: tet B, tet M, tet W were firstly isolated in the intestine and rearing water of red seabream ( Pagrus major ) [ 105 ]; tet A, tet B, tet E, tet H, tet l, tet 34, tet 35 and 10 others had unknown tet genes isolated from Chilean salmon ( Salmo salar ) farms [ 106 ]; furthermore, Higuera-Llanten and coworkers [ 107 ] also detected the presence of tet 34, tet 35, tet A, tet B, tet E, tet H, tet L, and tet M genes in the same matrixes. Among seafoods (including finfish and crustaceans), Concha et al [ 108 ] discovered tet X gene in Epilithonimonas strains from rainbow trout ( Oncorhynchus mykiss ) and Han et al [ 109 ] amplified, in shrimp samples, that the tet B gene was carried in a single copy plasmid, named pTetB-VA1, comprising 5162-bp. The whole genome analysis revealed that this plasmid consists of 9 ORFs (overlapping open reading frames) encoding tetracycline-resistant repressor proteins, transcriptional regulatory proteins, and transposases and showed a 99% sequence identity to other tet gene plasmids ( pIS04-68 and pAQU2 ).…”

“…ARGs for sulfonamides resistance were also discovered in many commensal bacterial strains in Japanese mariculture areas [ 122 ], in Vietnamese freshwater farms [ 108 ], China Hainan, Guangdong, Tianjin, Hangzhou, Yantai, and Taihu Lake [ 113 , 123 , 124 , 125 ].…”