Antimicrobial susceptibility and plasmid profiles of Flavobacterium psychrophilum strains isolated in Chile

Abstract

Flavobacterium psychrophilum is one of the most threatening bacterial infections for freshwater fish species in the world and is of considerable economic significance to aquaculture producers. Up to now, antimicrobial therapies represent the only recourse to control the disease in farmed fish. In this study, a total of 40 F. psychrophilum isolates obtained from Chilean salmonid farms were tested to determine their antimicrobial susceptibility using minimum inhibitory concentration (MIC) determinations and disk diffusion tests, for oxytetracycline (OTC), florfenicol (FLO) and oxolinic acid (OXA) and also to examine the plasmid profile to assist in the characterization of the strains isolated from different salmonid species. In addition, the presence of mutations in the quinolone resistance-determining region (QRDR) was evaluated. Normalized resistance interpretation (NRI) of MIC data suggested cut-off values for non-susceptible at R>0.203, and 2.35μgmL -1 for OTC, and FLO, respectively. For OXA a slightly larger coefficient of variation in the calculations required a conservative estimate at R≥16μgmL -1. Of the 40 Chilean isolates studied, 90%, 92.5%, and 85% F. psychrophilum were resistant to the three antimicrobials. In the disk diffusion tests, all isolates produced zones of inhibition to OTC 30 and FLO 30. However, there was a low or no correlation between MIC results and inhibition zone diameter values and valid NRI calculations were not possible to perform. For OXA 2 disks, no zones were observed (<7mm). No correlation was found between quinolone resistance and PCR-RFLP analysis; whereas 5 isolates had mutations in the QRDR of gyrA. Thirty-nine of 40 isolates possessed a single plasmid (15 isolates with 3.5kb) or combinations of two plasmids (17 and 7 isolates of sizes 1.5 and 3.5kb and 2.7 and 3.5kb, respectively), but a relationship between plasmid and resistance could not be established. In summary, we demonstrate in this work by MIC assays that high levels of resistance to OTC, FLO and OXA exist among Chilean isolates, which could be associated with the high amounts of antimicrobials applied at the Chilean farms to control outbreaks caused by F. psychrophilum. Disk contents for disk diffusion tests were not suitable for this bacterium. Surveillance in the use of drugs is recommended in cultured freshwater fish to prevent resistance problem. © 2012 Elsevier B.V.