Introduction

Up to now, usage of antibiotics in aquaculture played a pivotal role in achieving major advances against bacterial infections. Still, Bacteria may gain antibiotic resistance and spread as a consequence of inappropriate and extensive use of antibiotics. The rapid emergence of antibiotic resistant bacteria endangers the efficacy of antibiotic and leads to the spread of resistance genes. In the present study, phenotypic antibiotic resistance against 12 antibiotics and the presence of 14 antibiotic resistance genes were evaluated in archival Lactococcus garvieae strains isolated from small-scale rainbow trout farms in Turkey, Italy, Greece, and Spain.

Material and methods

Twenty archival L. garvieae strains of each country were previously isolated from diseased rainbow trout (Oncorhynchus mykiss). Antibiotic susceptibility tests were performed by the disk diffusion method using kanamycin (K; 30 μg), ampicillin (AMP; 10 μg or 25 μg), gentamycin (CN; 10 μg or 30 μg), florphenicol (FFC; 30 μg), streptomycin (S; 10 μg), erythromycin (E; 15 μg), oxolinic acid (OA; 2 μg or 10 μg), flumequine (FLM; 30 μg), penicillin (P; 10 μg), amoxicillin (AMX; 20 μg or 25 μg), neomycin (N;30 μg), and oxytetracycline (OT; 30 μg) discs. Presence of florfenicol (floR), tetracycline (tetA, tetB, tetC, tetG), erythromycin (ermA, ermB, ermC), streptomycin (strB), β-lactamase ampicillin (blaSHV, blaOXA, blaTEM), quinolones oxalinic acid (gnrA), and quinolones flumequine (gyrA) resistance genes was tested by PCR.

Results

Overall, antimicrobial susceptibility tests indicated that 93.8% of the isolates were resistant to oxolinic acid, followed by flumequine (91.3%), streptomycin (88.8%), kanamycin (72.5%), and neomycin (66.3%). Whereas, the highest susceptibilities were seen toward gentamycin (100%), oxytetracycline (97.5%), florfenicol (75%), ampicillin (73.8%), and amoxicillin (72.5%). Among all strains (n=80), 96.3% (n=77) had at least one resistance gene and 57.5% (n=46) had 2 or more antibiotic resistance genes. Among the screened tetracycline genes, tetC (57.5%) was the most prevalent gene followed by tetG (33.8%), ermC (23.8%) and tetB (21.3%), whereas floR (2.5%), ermA (2.5%), tetA (3.8%), and blaSHV (6.3%) were among the least detected genes. Antibiotic susceptibility differences and variation in antibiotic resistance genes were recorded among Turkish, Italian, Greek, and Spanish archival strains which indicates possible genetic and phenotypic differences among strains. Prevalence of resistance to commonly used antibiotics in aquaculture and detection of resistance genes in strains may lead to prolonged treatments and to the increase L. garvieae related mortalities. This study provides a starting point for comprehensive monitoring of antimicrobial resistance of L. garvieae strains in rainbow trout aquaculture in the Mediterranean Region.