Characterization of Mechanisms Lowering Susceptibility to Flumequine among Bacteria Isolated from Chilean Salmonid Farms

Abstract

Despite their great importance for human therapy, quinolones are still used in Chilean salmon farming, with flumequine and oxolinic acid currently approved for use in this industry. The aim of this study was to improve our knowledge of the mechanisms conferring low susceptibility or resistance to quinolones among bacteria recovered from Chilean salmon farms. Sixty-five isolates exhibiting resistance, reduced susceptibility, or susceptibility to flumequine recovered from salmon farms were identified by their 16S rRNA genes, detecting a high predominance of species belonging to the Pseudomonas genus (52%). The minimum inhibitory concentrations (MIC) of flumequine in the absence and presence of the efflux pump inhibitor (EPI) Phe-Arg-β-naphthylamide and resistance patterns of isolates were determined by a microdilution broth and disk diffusion assays, respectively, observing MIC values ranging from 0.25 to >64 µg/mL and a high level of multi-resistance (96%), mostly showing resistance to florfenicol and oxytetracycline. Furthermore, mechanisms conferring low susceptibility to quinolones mediated by efflux pump activity, quinolone target mutations, or horizontally acquired resistance genes (qepA, oqxA, aac(6′)-lb-cr, qnr) were investigated. Among isolates exhibiting resistance to flumequine (≥16 µg/mL), the occurrence of chromosomal mutations in target protein GyrA appears to be unusual (three out of 15), contrasting with the high incidence of mutations in GyrB (14 out of 17). Bacterial isolates showing resistance or reduced susceptibility to quinolones mediated by efflux pumps appear to be highly prevalent (49 isolates, 75%), thus suggesting a major role of intrinsic resistance mediated by active efflux.

Aquatic Environments as Hotspots of Transferable Low-Level Quinolone Resistance and Their Potential Contribution to High-Level Quinolone Resistance

“…It has been observed that bacteria carrying mutations in the quinolone action target enzymes can exhibit higher MIC values when efflux systems are expressed [ 18 , 282 , 290 ]. The same result was observed in Pseudomonas isolated from freshwater environments associated with fish farming [ 283 ]. Furthermore, it has been reported that enteric bacteria with high and intermediate resistance to quinolones exhibited the co-occurrence of qnr genes and mutations in the GyrA and ParC subunits [ 122 ].…”

“…The overexpression of various efflux pumps can lead to low-level resistance [ 277 , 278 , 279 ], lowering the cytoplasmic concentration of antimicrobials inside the cell and generating an advantage for the evolutionary selection of high-resistance strains [ 279 , 280 , 281 ]. In support of this trend, the combined activity of qnr genes with multidrug efflux systems drastically reducing the susceptibility to quinolones and providing a high-level of quinolone resistance has been reported [ 122 , 282 , 283 ], confirming the additive inhibitory activity of both antimicrobial mechanisms [ 18 ], as postulated in Figure 2 .…”

Flumequine-loaded titanate nanotubes as antibacterial agents for aquaculture farms

“…The hypothesis of bacterial resistance is ruled out because the development of FLUM resistance usually needs much longer time of exposure (more than 150 days). 59,60 Thus, we suggest that the degradation of FLUM in the broth and/or its metabolization within bacterial cell are the principle causes of its low antibacterial activity as reported. 60,61 The treatment with FLUM@TiNTs induced a progressive damage and cells death indicating that TiNTs enhances the drug efficacy thanks to their protection mechanism from degradation and progressive release during the experiment period.…”

“… 59,60 Thus, we suggest that the degradation of FLUM in the broth and/or its metabolization within bacterial cell are the principle causes of its low antibacterial activity as reported. 60,61 The treatment with FLUM@TiNTs induced a progressive damage and cells death indicating that TiNTs enhances the drug efficacy thanks to their protection mechanism from degradation and progressive release during the experiment period. Given the advantages of drug encapsulation which include (i) a sustained release that can reduce frequent doses administration and side effects, (ii) an enhanced absorption, 62 the use of TiNTs as FLUM nanocarrier appears as an attractive alternative for better FLUM release and antibacterial efficiency.…”

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

“…The MICs of oxytetracycline of FP105 and FP211-J200 isolates were determined by a microdilution procedure, as recommended by the CLSI guideline M07-A10 [ 60 ] and previously described [ 61 ]. Conical bottom microplates added with a cation-adjusted Mueller–Hinton broth (Difco Labs, NJ, USA) were inoculated with the antibiotic to obtain final series of two-fold concentrations in the range of 0.0625–512 µg/mL, and bacterial suspensions were inoculated in triplicate microplates, delivering approximately 10 4 colony-forming units per well and incubated at 28 °C for 24 h. The reference strains E. coli ATCC 25,922 and Aeromonas salmonicida ATCC 33,658 were included as quality controls, as was recommended [ 21 ].…”