The efflux pump inhibitor Phe-Arg-ß-naphthylamide does not abolish the activity of the Stenotrophomonas maltophilia SmeDEF multidrug efflux pump

Patricia Sánchez, Uyen Le and José L. Martínez*

Centro Nacional de Biotecnología (CSIC), Biotecnología Microbiana, 28049 Madrid, Spain

Keywords: MDR inhibitors, antibiotic efflux pumps, Stenotrophomonas maltophilia, antibiotic resistance

Sir,

Multidrug (MDR) efflux pumps are important elements for intrinsic and acquired antibiotic bacterial resistance. Although MDR pumps are usually down-regulated, at least under laboratory growing conditions, mutants overexpressing such pumps are easily selected. Inhibition of MDR pumps might have several benefits, including a reduction in the level of resistance to several drugs, a reduction in the number of antibiotic-resistant mutants to be selected in the presence of antibiotics and a reduction in the level of intrinsic resistance displayed by bacterial pathogens. The first potentially useful MDR inhibitor so far described is MC-207,110, which is active against the Pseudomonas aeruginosa efflux pumps MexAB-OprM, MexCD-OprJ and MexEF-OprN, as well as against the Escherichia coli efflux pump AcrAB.1 In a letter recently published in the present journal,2 the effect of this inhibitor on the MIC of quinolones for a collection of Acinetobacter baumannii and Stenotrophomonas maltophilia strains, in which the activity of efflux pumps was not previously determined, was also analysed.2

In order to gain some more insight into the effect of this efflux pump inhibitor over S. maltophilia MDR determinants, we have tested the influence of MC-207,110 (purchased from Sigma as Phe-Arg-ß-naphthylamide) on the susceptibility of a collection of S. maltophilia strains previously characterized with respect to the level of production of the MDR pump SmeDEF.3 SmeDEF, the first efflux pump described in S. maltophilia, is capable of extruding tetracycline, chloramphenicol, erythromycin and quinolones.4 SmeDEF contributes to intrinsic5 and acquired antibiotic resistance in this bacterial species. In fact 33% clinical isolates of S. maltophilia have been demonstrated to overexpress SmeDEF.3 The collection includes the strain D457R, a spontaneous SmeDEF-overproducing mutant obtained from the wild-type strain D457, and two E. coli strains, one with a deletion of the acrAB MDR pump, and the other a derivative of the former harbouring a plasmid (pAS2), which contains the smeDEF genes and expresses SmeDEF in an active form. Antibiotic susceptibilities were tested by the broth microdilution method in Mueller–Hinton broth at 37°C with a standard inoculum of 104 cells/mL, in either the presence or absence of Phe-Arg-ß-naphthylamide at a concentration of 20 mg/L. The results of the analysis are shown in Table 1.


View this table:
[in this window]
[in a new window]
 
Table 1.  Effect of Phe-Arg-ß-naphthylamide on the MICs of antibiotics for S. maltophilia isolates producing different amounts of SmeDEF
 
The MDR inhibitor Phe-Arg-ß-naphthylamide did not reduce the MIC of ciprofloxacin or tetracycline. However, a slight decrease in MICs was observed for chloramphenicol and erythromycin. This effect was higher on the MICs of nalidixic acid. In a recent letter, it was stated that Phe-Arg-ß-naphthylamide reduced the MIC of nalidixic acid but not of ciprofloxacin for A. baumannii and S. maltophilia.2 On the other hand, it has been reported that this inhibitor could reduce the MIC of ciprofloxacin for quinolone-resistant E. coli.6 Careful analysis of published data indicates, however, that this reduction is small, and the effect is only detected in four of 10 analysed strains. Our data then confirm that Phe-Arg-ß-naphthylamide cannot inhibit ciprofloxacin efflux. In the previous report, there was no independent evidence of the activity of efflux pumps in the S. maltophilia strains tested. Herein we have analysed the effect of Phe-Arg-ß-naphthylamide on bacterial strains overproducing SmeDEF. Since we did not detect any difference in the MICs, it is clear that Phe-Arg-ß-naphthylamide cannot inhibit the efflux of ciprofloxacin and tetracycline by SmeDEF, at least under the conditions tested.

For some of the strains, the MIC of tetracycline was slightly higher in the presence of Phe-Arg-ß-naphthylamide. Although the differences are very small, this reduction in tetracycline susceptibility has been confirmed by analysing the effect of tetracycline (4 mg/L), in either the presence or absence of Phe-Arg-ß-naphthylamide, on the growth kinetics of strain E999. As expected, tetracycline produced a strong reduction of bacterial growth rate. Nevertheless, the antibiotic did not affect bacterial growth when Phe-Arg-ß-naphthylamide was included in the culture medium. This indicates that Phe-Arg-ß-naphthylamide is antagonistic to tetracycline. A possible explanation for this paradoxical effect could be that Phe-Arg-ß-naphthylamide might inhibit the active entrance of the antibiotic inside the cells.

As can be seen in Table 1, there is no correlation between SmeDEF overproduction and a reduction in MICs by Phe-Arg-ß-naphthylamide. This suggests that Phe-Arg-ß-naphthylamide might inhibit some other MDR systems expressed by S. maltophilia independently of SmeDEF. It was previously observed that the magnitude of the effect of Phe-Arg-ß-naphthylamide was dependent on the structure of each particular antibiotic. The authors discussed that this could indicate that different antibiotics may have different binding sites, and that the effect of Phe-Arg-ß-naphthylamide is binding site specific.1 Herein, we found that the same occurred for S. maltophilia. The effect was dependent on the structure of the antibiotic and was also strain dependent, which probably reflects the variability in the level of expression of the different MDR pumps that clinical isolates of S. maltophilia might have.

Acknowledgements

This research was aided in part by grant CAM 08.2/0020.1/2001.

Footnotes

* Corresponding author. Tel: +34-91-5854571; Fax: +34-91-5854506; E-mail: jlmtnez{at}cnb.uam.es Back

References

1 . Lomovskaya, O., Warren, M. S., Lee, A., Galazzo, J., Fronko, R., Lee, M. et al. (2001). Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: Novel agents for combination therapy. Antimicrobial Agents and Chemotherapy 45, 105–16.[Abstract/Free Full Text]

2 . Ribera, A., Ruiz, J., de Anta, M. T. J. & Vila, J. (2002). Effect of an efflux pump inhibitor on the MIC of nalidixic acid for Acinetobacter baumannii and Stenotrophomonas maltophilia clinical isolates. Journal of Antimicrobial Chemotherapy 49, 697–8.[Free Full Text]

3 . Alonso, A. & Martinez, J. L. (2001). Expression of multidrug efflux pump SmeDEF by clinical isolates of Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 45, 1879–81.[Abstract/Free Full Text]

4 . Alonso, A. & Martinez, J. L. (2000). Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 44, 3079–86.[Abstract/Free Full Text]

5 . Zhang, L., Li, X. Z. & Poole, K. (2001). SmeDEF multidrug efflux pump contributes to intrinsic multidrug resistance in Stenotrophomonas maltophilia. Antimicrobial Agents and Chemotherapy 45, 3497–503.[Abstract/Free Full Text]

6 . Mazzariol, A., Tokue, Y., Kanegawa, T. M., Cornaglia, G. & Nikaido, H. (2000). High-level fluoroquinolone-resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA. Antimicrobial Agents and Chemotherapy 44, 3441–3.[Abstract/Free Full Text]