Evidence for Active Efﬂux as the Primary Mechanism of Resistance to Ciproﬂoxacin in Salmonella enterica Serovar Typhimurium

The occurrence of active efﬂux and cell wall modiﬁcations were studied in Salmonella enterica serovar Typhimurium mutants that were selected with enroﬂoxacin and whose phenotypes of resistance to ﬂuoro- quinolones could not be explained only by mutations in the genes coding for gyrase or topoisomerase IV. Mutant BN18/21 exhibited a decreased susceptibility to ciproﬂoxacin (MIC 5 0.125 m g/ml) but did not have a mutation in the gyrA gene. Mutants BN18/41 and BN18/71 had the same substitution, Gly81Cys in GyrA, but exhibited different levels of resistance to ciproﬂoxacin (MICs 5 2 and 8 m g/ml, respectively). None of the mutants had mutations in the parC gene. Evidence for active efﬂux was provided by a classical ﬂuorimetric method, which revealed a three- to fourfold decrease in ciproﬂoxacin accumulation in the three mutants compared to that in the parent strain, which was annuled by addition of the efﬂux pump inhibitor carbonyl cyanide m -chlorophenylhydrazone. In mutant BN18/71, a second ﬂuorimetric method also showed a 50% reduction in the level of accumulation of ethidium bromide, a known efﬂux pump substrate. Immunoblotting and enzyme-linked immunosorbent assay experiments with an anti-AcrA antibody revealed that the resistance phenotype was strongly correlated with the expression level of the AcrAB efﬂux pump and suggested that decreased susceptibility to ciproﬂoxacin due to active efﬂux probably related to overproduction of this pump could occur before that due to gyrA mutations. Alterations were also found in the outer membrane protein and lipopolysaccharide proﬁles of the mutants, and these alterations were possibly responsible for the decrease in the permeability of the outer membrane that was observed in the mutants and that could act synergistically with active efﬂux to decrease the level of ciproﬂoxacin accumulation. plates supplemented with increas- ing concentrations of enroﬂoxacin (9). Our previous study showed that the QRDR sequences of gyrA , gyrB , parC , and parE of mutant BN18/21 were iden- tical to those of parent strain BN18 (9). Mutants BN18/41 and BN18/71 were shown to have a single mutation in the QRDR of gyrA (Gly81Cys) but none in the QRDRs of gyrB , parC , and parE . samples were then centrifuged at 5,600 3 g for 10 min. The ﬂuorescence of the supernatant was measured with a Quanta Master C-60 spectroﬂuorimeter (Photon Technology International, Monmouth Junction, N.J.) at excitation and emission wavelengths of 279 and 447 nm, respectively (3). The concentration of ciproﬂoxacin in the supernatant was calculated by comparison with a standard curve for ciproﬂoxacin (0.02 to 2.5 m g/ml) in 0.1 M glycine hydrochloride (pH 3.0). The results were expressed as nanograms of ciproﬂoxacin incorporated per milligram (dry weight) of bacteria. The experiments were performed at least three times to make sure of the reproducibility. (ii) Accumulation of ethidium bromide. A previously described ﬂuorimetric method (1) was used, with slight modiﬁcations, to measure the accumulation of ethidium bromide. Brieﬂy, cells were grown overnight, pelleted, and resuspended to an A 600 of 0.2 in sodium phosphate buffer (pH 7.0). For demonstration of active efﬂux, ethidium bromide was added to the suspension at a ﬁnal concen- tration of 2 m g/ml. Fluorescence was used as a measure of the amount of ethidium bromide incorporated by the cells and was recorded with a spectroﬂuo- rimeter at excitation and emission wavelengths of 530 and 600 nm, respectively. CCCP was added to the suspension (ﬁnal concentration, 100 m M) after 400 s of incubation. The natural ﬂuorescence of the cells was subtracted, and the ﬂuorescence intensity was expressed in arbitrary units. For demonstration of differ- ences in outer membrane permeability, cells were incubated with CCCP for 3 min before addition of ethidium bromide. Results were expressed as the ﬂuo- rescence intensity of the cells incubated with ethidium bromide alone subtracted from that of the cells incubated with CCCP and ethidium bromide. Acr pump expression analysis by immunoblotting and ELISA. Bacteria were grown overnight at 37°C centrifugation, pended one-half Laemmli proteins separated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) transferred at 0.8 mA/cm nitrocellulose membrane. The three Tris-buffered 0.15% 30 with 1% skim incubated overnight at room temperature an anti-AcrA polyclonal diluted 1/2,000 containing 0.05% Tween 20 (TBS-T) and 0.33% skim milk. After three washes in TBS-T, the incubated for 1 h with peroxidase conjugated to protein A diluted 1/1,000 in TBS-T St. Louis, Mo. ). after three washes in TBS-T, blot developed by incubation at room temperature in a solution of TBS containing 0.06% 4-chloro-1-naphthol 5 mM H 2 O 2 The stopped by washes in distilled enzyme-linked immunosorbent assay (ELISA), resuspended an of 1.0 in phosphate-buffered saline (PBS; pH 7.2) A total of m of the used to each well of 18 h at room anti-AcrA serially diluted from 1/500 1/256,000 containing 0.05% Tween (PBS-T) incubated r 2 h Binding of the anti-AcrA detected by a further incubation with the peroxidase-protein A conjugate diluted 1/1,000