Effect of the efflux pump inhibitor Phe-Arg-ß-naphthylamide on the MIC values of the quinolones, tetracycline and chloramphenicol, in Escherichia coli isolates of different origin

Yolanda Sáenz1, Joaquim Ruiz2, Myriam Zarazaga1, Meritxell Teixidó3, Carmen Torres1 and Jordi Vila2,*

1 Área Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño; 2 Servei de Microbiología, Institut Clínic d’Infeccions i Immunología, IDIBAPS, Hospital Clínic, Facultat de Medicina, Universitat de Barcelona, Barcelona; 3 Institut de Recerca Biomédica de Barcelona, Parc Científic de Barcelona, Universitat de Barcelona, Spain

Keywords: quinolone resistance, E. coli, GyrA, ParC

Sir,

The main mechanisms of quinolone resistance in Escherichia coli include mutations in the gyrA and parC target genes and decreased intracellular accumulation.1 Active efflux pumps are important for intrinsic and acquired antibiotic resistance, and overexpression of efflux pumps affecting quinolones is becoming increasingly common in E. coli. Recently, efflux pump inhibitors have been investigated with a view to improving and potentiating the activity of exported antibiotics.2 Phe-Arg-ß-naphthylamide has been described as a broad-spectrum efflux pump inhibitor in Pseudomonas aeruginosa.2 The effect of this inhibitor on fluoroquinolone susceptibilities in Enterobacter aerogenes,3 E. coli,4 P. aeruginosa,2 Acinetobacter baumannii5 and Stenotrophomonas maltophilia5 has also been studied, but not for nalidixic acid and other antimicrobial agents in E. coli. Therefore in this study, we analysed the influence of the Phe-Arg-ß-naphthylamide (Sigma) inhibitor on the activity of quinolones, chloramphenicol and tetracycline against a collection of 60 E. coli isolates previously characterized with respect to amino acid changes in both GyrA and ParC proteins.6,7 The collection includes the strain Nor5, an in vitro selected quinolone-resistant mutant, in which the overexpression of MarA, SoxS and AcrA has been shown (J. Vila, B. Peter, J. Sánchez-Cespedes & M. Tario, unpublished data). The remaining 59 isolates were recovered from foods (n = 11), humans (n = 25) and animals (n = 23).

Susceptibilities to nalidixic acid, norfloxacin, chloramphenicol, tetracycline and ciprofloxacin were studied by agar dilution in the presence or absence of 20 mg/L of Phe-Arg-ß-naphthylamide, in accordance with NCCLS guidelines. All of the 60 E. coli isolates showed an MIC value of >256 mg/L to Phe-Arg-ß-naphthylamide. Table 1 shows the effect of 20 mg/L of the inhibitor Phe-Arg-ß-naphthylamide on the MIC values of the five tested antibiotics in 60 E. coli isolates with different numbers of amino acid changes in GyrA and ParC proteins. The results show that the use of 20 mg/L of Phe-Arg-ß-naphthylamide decreased the MIC of nalidixic acid (from two to five serial two-fold dilution steps) in all isolates tested, even in those with the nalidixic acid-susceptible phenotype. This high reduction in the MIC values was observed independent of the number of amino acid changes in GyrA and ParC. This effect was especially noticeable among isolates carrying one single GyrA substitution, in which the MIC50 of nalidixic acid decreased from resistant to susceptible levels (from 128 to 8 mg/L). This result indicates that one amino acid change in GyrA by itself (without an additional mechanism of resistance) is unable to confer a resistant phenotype to nalidixic acid in E. coli. On the other hand, almost no decreases in the MIC50 were detected in the fluoroquinolones tested. Nevertheless, Phe-Arg-ß-naphthylamide affected the MIC of ciprofloxacin in 10 of the 60 E. coli isolates whose MIC values were lowered by at least two serial dilution steps. Eight of these 10 isolates also decreased the MIC of norfloxacin (in both cases including mutant Nor5). Interestingly, fluoroquinolone MIC values for these 10 isolates were higher than the MIC50 that corresponds with the number of substitutions present in the targets. The addition of the inhibitor decreased these high MIC values to expected MIC50 values (for example, the MIC of ciprofloxacin was decreased from 1 to 0.25 mg/L in one isolate with only a mutation in GyrA). The presence of 20 mg/L of Phe-Arg-ß-naphthylamide also affected the MIC of chloramphenicol in 51 isolates whose MICs decreased by two or three serial dilution steps. Finally, no effect was observed when the MIC of tetracycline was analysed.


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Table 1. MIC values (in mg/L) of the five tested antibiotics in the presence or absence of 20 mg/L of Phe-Arg-ß-naphthylamide in a series of 60 E. coli isolates organized by number of amino acid changes in GyrA and ParC proteins
 
The effect of different concentrations of Phe-Arg-ß-naphthylamide (40, 80 or 160 mg/L) on nalidixic acid, ciprofloxacin and norfloxacin MICs was also studied in 25 of the E. coli isolates. The influence of the Phe-Arg-ß-naphthylamide was homogeneous in the range 20–160 mg/L of the inhibitor in all the isolates tested.

These results might be explained by the activity of the inhibitor against two or more efflux systems. The effect on the MIC of nalidixic acid in all the strains (both susceptible and resistant) suggests the presence of a constitutive efflux pump able to discharge the antibacterial agent and confer a basal level of resistance. Moreover, chloramphenicol might be another substrate of this hypothetical efflux pump. The Phe-Arg-ß-naphthylamide only affects the MIC of fluoroquinolones in isolates with levels of resistance higher than those expected according to the presence of alterations in their targets. These high MIC values might be explained by the overexpression of the AcrAB efflux pump, as previously described,2,3 although the overexpression of other efflux pumps cannot be ruled out. A proposal to be considered is the possible inhibitor effect over the outer membrane protein of the efflux system.

In summary, the inhibitor Phe-Arg-ß-naphthylamide produces a decrease in the MIC of nalidixic acid in E. coli isolates with different susceptibilities to this quinolone. This result suggests the inability of a single amino acid substitution in the GyrA protein to confer resistance phenotypes to this antimicrobial agent. Moreover, the presence of a constitutive efflux pump able to discharge nalidixic acid easily is strongly suggested. These facts may pave the way to the design of novel combination therapies that could be used against strains with amino acid substitutions in GyrA.

Acknowledgements

This work was supported in part by grants from the Fondo de Investigaciones Sanitarias of Spain (FIS 01/973 and FIS 02/0535). Yolanda Sáenz has an FPI fellowship from the University of La Rioja (FPI-UR-00/72785864).

Footnotes

* Correspondence address. Servei de Microbiología, Hospital Clínic i Provincial de Barcelona,Villarroel 170, 08036, Barcelona, Spain. Tel: +34-932-275522; Fax: +34-932-275454; E-mail: vila{at}medicina.ub.es Back

References

1 . Everett, M. J., Fang Jin, Y., Ricci, V. et al. (1996). Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrobial Agents and Chemotherapy 40, 2380–6.[Abstract]

2 . Lomovskaya, O., Warren, M. S., Lee, A. 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]

3 . Malléa, M., Mahamoud, A., Chevalier, J. et al. (2003). Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates. Biochemical Journal 376, 801–5.[CrossRef][ISI][Medline]

4 . Mazzariol, A., Tokue, Y., Kanegawa, T. M. et al. (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]

5 . Ribera, A., Ruiz, J., Jiménez de Anta, M. T. et al. (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]

6 . Sáenz, Y., Zarazaga, M., Briñas, L. et al. (2003). Mutations in gyrA and parC genes in nalidixic acid-resistant Escherichia coli strains obtained from food products, humans and animals. Journal of Antimicrobial Chemotherapy 51, 1001–5.[Abstract/Free Full Text]

7 . Vila, J., Ruiz, J., Goñi, P. et al. (1996). Detection of mutations in parC in quinolone-resistant clinical isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 40, 491–3.[Abstract]