Abstract
Our previous studies showed that particular antibiotic resistance genes (ARGs) were enriched locally in sediments below fish farms in the Northern Baltic Sea, Finland, even when the selection pressure from antibiotics was negligible. We assumed that a constant influx of farmed fish feces could be the plausible source of the ARGs enriched in the farm sediments. In the present study, we analyzed the composition of the antibiotic resistome from the intestinal contents of 20 fish from the Baltic Sea farms. We used a high-throughput method, WaferGen qPCR array with 364 primer sets to detect and quantify ARGs, mobile genetic elements (MGE), and the 16S rRNA gene. Despite a considerably wide selection of qPCR primer sets, only 28 genes were detected in the intestinal contents. The detected genes were ARGs encoding resistance to sulfonamide (sul1), trimethoprim (dfrA1), tetracycline [tet(32), tetM, tetO, tetW], aminoglycoside (aadA1, aadA2), chloramphenicol (catA1), and efflux-pumps resistance genes (emrB, matA, mefA, msrA). The detected genes also included class 1 integron-associated genes (intI1, qacEΔ1) and transposases (tnpA). Importantly, most of the detected genes were the same genes enriched in the farm sediments. This preliminary study suggests that feces from farmed fish contribute to the ARG enrichment in farm sediments despite the lack of contemporaneous antibiotic treatments at the farms. We observed that the intestinal contents of individual farmed fish had their own resistome compositions. Our result also showed that the total relative abundances of transposases and tet genes were significantly correlated (p = 0.001, R2 = 0.71). In addition, we analyzed the mucosal skin and gill filament resistomes of the farmed fish but only one multidrug-efflux resistance gene (emrB) was detected. To our knowledge, this is the first study reporting the resistome of farmed fish using a culture-independent method. Determining the possible sources of ARGs, especially mobilized ARGs, is essential for controlling the occurrence and spread of ARGs at fish farming facilities and for lowering the risk of ARG spread from the farms to surrounding environments.
Highlights
Fish farms have been suggested as one reservoir of antibiotic resistance genes (ARGs) in the environment due to the prophylactic and therapeutic use of antibiotics (Cabello et al, 2013; Miranda et al, 2013)
Of the 364 primer sets; 307 primer sets were used for ARGs encoding resistance to the nine main classes of antibiotics (aminoglycoside, beta-lactam,/(chlor)/(am)phenicol, macrolide (MSLB), multidrug-efflux, sulfonamide, tetracycline, trimethoprim, and vancomycin) and covering the three major mechanisms of antibiotic resistance; 21 primer sets for other genes encoding resistance to antibacterial compounds, such as antiseptic and antibacterial peptides; 34 primer sets for genes associated with mobile genetic elements (MGE), such as plasmids, transposons, and insertion sequences (IS) as well as integrons; one primer set for a mercury resistance gene to study a possible co-selection of ARGs with mercury resistance gene; and one primer set for the 16S rRNA genes
The WaferGen qPCR array used 307 primer sets to detect and quantify ARGs, 21 primer sets for antibacterial or antiseptic resistance genes, 34 primer sets for MGE-associated genes, and one primer set for a mercury resistance gene
Summary
Fish farms have been suggested as one reservoir of antibiotic resistance genes (ARGs) in the environment due to the prophylactic and therapeutic use of antibiotics (Cabello et al, 2013; Miranda et al, 2013). The use of antibiotics in fish farming in Finland is controlled by the Finnish Food Safety Authority (EVIRA) and requires a guideline from veterinary professionals. Oxytetracycline, a combination of sulfonamide-trimethoprim, and florfenicol are the antibiotics authorized for use in Finnish fish farms (EVIRA, 2015a,b). From 2001 to 2014, ∼2.3 metric tons of sulfonamide, 0.6 metric ton of trimethoprim, 1.2 metric tons of oxytetracycline, and 0.04 metric ton of florfenicol were used in fish farming in Finland (EVIRA, 2015a,b). The antibiotics are used mainly against fish pathogens, such as Aeromonas salmonicida, Flavobacterium psychrophilum, and Flavobacterium columnare, and pathogens which occur only in sea farms, such as Vibrio anguillarum, Yersinia ruckeri, Pseudomonas anguilliseptica, and Pseudomonas edwardsielloosi (ViljamaaDirks, 2016)
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