Expression of the adeB gene and responsiveness to 1-(1-naphthylmethyl)-piperazine and phenylalanyl-arginyl-β-naphthylamide in clinical isolates of Acinetobacter baumannii.

Sir, AdeABC, a resistance–nodulation–cell division (RND)-type efflux pump, is an attractive research target as a cause of multiple antibiotic resistance in Acinetobacter baumannii. The overexpression of the adeB gene encoding the AdeB efflux protein has previously been associated with multidrug resistance in various A. baumannii strains. – 3 These reports led us to perform a study in a group of clinical isolates of A. baumannii, to assess the relative expression of the adeB gene and responsiveness to the potential efflux pump inhibitors (EPIs), 1-(1-naphthylmethyl)-piperazine (NMP) and phenylalanyl-arginyl-b-naphthylamide (PAbN). – 4 Fourteen multidrug-resistant A. baumannii isolates obtained from different patients in an intensive care unit and grouped previously into three clones were included in the study (Table 1). A. baumannii ATCC 19606, A. baumannii SBMox2, A. baumannii U10247 and Escherichia coli ATCC 25922 were used as reference strains. The MICs of ciprofloxacin, gentamicin, erythromycin, chloramphenicol, trimethoprim and tetracycline were determined by the broth microdilution test in the absence and presence of NMP and PAbN, at final concentrations of 100 mg/L. For the detection of adeB transcripts, RT–PCR was performed (RNeasy Kit and Omniscript RT Kit from Qiagen, Hilden, Germany; and FastStart DNA Master SYBR Green I Kit from Roche, Mannheim, Germany) using LightCycler System 2 (Roche) with the primers previously described by Higgins et al. Eight isolates (MU-2, -5, -7, -9, -10, -12, -13 and -14) showed reductions in various antibiotic MICs after the addition of EPIs (Table 1). This demonstrates the ability of NMP and PAbN to partially reverse drug resistance in a significant portion of the isolates. We can conclude that the phenotypic changes in the drug susceptibilities of our EPI-responsive isolates might be associated with the inhibition of functional RND-type drug efflux. – 4 In general, NMP was more active than PAbN. Such differences in the activities of the two compounds have been previously reported and associated with the different mechanisms of action of NMP and PAbN. Significant effects of NMP were observed on ciprofloxacin, erythromycin and trimethoprim MICs (Table 1). Moreover, NMP switched the resistance phenotype to susceptible in two isolates (MU-7 and A. baumannii SBMox2) for trimethoprim and/or tetracycline. This inhibitor may have affinity sites similar to those for these antibiotics inside the efflux pump. The adeB expression of EPI-responsive isolates was as high as that found in A. baumannii SBMox2, which has been previously shown to be an adeB overexpresser (Table 1). This supports our conclusion that the responsiveness of the isolates to EPIs might be due to the inhibition of functional RND-type drug efflux, particularly the AdeB pump. Earlier experiments have also highlighted the causal connection between adeB overexpression and responsiveness to EPIs in various A. baumannii strains. Despite the fact that the aminoglycosides have been shown to be good substrates of the AdeB pump, the gentamicin MICs for most adeB overexpressers did not markedly change in the presence of EPIs. – 3,7,9,10 This could be due to the presence of other resistance mechanisms, such as aminoglycoside modification enzymes and/or some recently recognized efflux pumps, which may have low affinity for the EPIs tested here. Alternatively, the affinity constant for the efflux pump sites could be stronger for gentamicin in comparison with EPIs. In terms of changes in quinolone susceptibility, despite the fact that NMP decreased the ciprofloxacin MICs from 16–32 mg/L to 2–8 mg/L, the adeB overexpressers were still resistant to this antibiotic (Table 1). The persistence of ciprofloxacin resistance in these isolates might be associated with mutations in the gyrA gene. Interestingly, the adeB expression of A. baumannii ATCC 19606 was high and similar to that of A. baumannii SBMox2. Although this strain was initially found to be susceptible to ciprofloxacin and tetracycline, the addition of NMP caused reductions in their MICs (Table 1). This supports earlier findings suggesting the efficiency of the AdeB pump alone was not sufficient to increase various antibiotic MICs to