Polymorphism in gyrA is associated to quinolones resistance in Chilean Piscirickettsia salmonis field isolates.

Abstract

Keywords: antibiotic resistance, gyrA, Piscirickettsiasalmonis, quinolones.Piscirickettsia salmonis is a gram-negative, pleomor-phic coccoid-shaped and facultative intracellularfish pathogenic bacterium, that causes piscirickettsi-osis, a disease also known as salmonid rickettsialsepticaemia (SRS) (Fryer et al. 1990). In Chile,P. salmonis was isolated for the first time in 1989,but probably has been present since 1983 (Bravo C Rozas &Enriquez 2013). SRS has also been reported inimportant salmon producing countries such asCanada, Ireland, Scotland and Norway, but with alower economic impact (Rozas & Enriquez 2013).P. salmonis cells are slow growing and hard to culti-vate in free-cell media, which has hindered its isola-tion and further characterization. However, recentlydescribed supplemented media (Mikalsen et al.2008) facilitate their growth and the antibiotictesting assays to evaluate effective antimicrobial ther-apy. In this regard, quinolones were the former mostwidely used antimicrobial compounds in Chileanaquaculture, especially as a first line drug againstSRS.In Chile, a large proportion of antibiotics is usedfor prophylaxis rather than for chemotherapy. Forinstance, in comparison with Norway and Canada,1500 and eight times more antimicrobials wereneeded to obtain similar amounts of salmon inChile in 2007 (Cabello et al. 2013). The same year,930 metric tons of antibiotics were mostly used insalmon production. Of those antibiotics, 24% werequinolones (Cabello et al. 2013). This practice hasalready altered the microbiota of sediments in closeproximity to aquaculture sites, where a significantfraction of isolated bacteria is antibiotic resistant(Buschmann et al. 2012). Therefore, the emer-gence and potential spread of antibiotic-resistantP. salmonis isolates cannot be ruled out.Twenty P. salmonis isolates cultured from kid-ney samples of diseased fish collected from diversegeographic origin (Fig. 1) between 2010 and2012 were phenotypically characterized for quinol-ones susceptibility. The isolates were grown onagar (Mikalsen et al. 2008), at 18 °C for 10 daysand kept frozen in liquid nitrogen. For antibioticsensitivity testing, the minimal inhibitory concen-tration (MIC) for enrofloxacin, flumequine andoxolinic acid were assessed according to theinstructions given by the Clinical and LaboratoryStandards Institute (CLSI), guide M49-A. 96-wellmicroplates containing twofold serial dilutions(0.03–64 lgmL