Energy-dependent accumulation of fluoroquinolones in quinolone- resistant Klebsiella pneumoniae strains

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 0066-4804/98/$04.0010July 1998, p. 1850–1852 Vol. 42, No. 7 Copyright © 1998, American Society for Microbiology. All Rights Reserved. Energy-Dependent Accumulation of Fluoroquinolones in Quinolone-Resistant Klebsiella pneumoniae Strains LUIS MARTI ´NEZ-MARTI ´NEZ, 1 * ISABEL GARCI ´A, 1 SOFI ´A BALLESTA, 1 VICENTE JAVIER BENEDI ´, 2 SANTIAGO HERNA ´NDEZ-ALLE ´S, 2 AND ALVARO PASCUAL 1 Department of Microbiology, School of Medicine, University of Seville, Seville, 1 and Area of Microbiology, Department of Environmental Biology and IMEDEA (CSIC-UIB), University of Balearic Island, Palma de Mallorca, 2 Spain Received 29 October 1997/Returned for modification 8 January 1998/Accepted 9 March 1998 The intracellular accumulation of norfloxacin and pefloxacin in Klebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents. Several mechanisms are involved in the resistance to fluoroquinolones (FQ) by gram-negative bacteria. In Escherichia coli, point mutations in the quinolone resistance-determining region (QRDR) of gyrA (21) are the most important cause of resistance. Mutations in gyrB are uncommon in clinical isolates (22), while mutations in parC (19) or (much less frequently) in parE (4) contribute to increased levels of resistance. The gyrA gene of Klebsiella pneumoniae has been cloned and sequenced and is similar (about 85% at the nucleotide level and about 90% at the amino acid level) to its E. coli homolog (5). The role of gyrA mutations in FQ-resistant K. pneumoniae has been demonstrated, these mutations being analogous to those previously observed in E. coli (6, 7). Decreased activity of FQ against E. coli has also been related to a reduced intracellular drug accumulation due to lipopolysaccharide or porin alterations impairing uptake or because of enhanced efflux (10, 11). Cross-resistance between FQ and beta-lactams in K. pneumoniae mutants has been related to outer membrane protein alterations (12, 17). Expression of the ramA locus from K. pneumoniae in E. coli confers resistance to norfloxacin, determines the active efflux of tetracycline and chloramphenicol, and causes almost complete loss of OmpF (8). It has been reported that the high hydrophobicity of FQ is generally associated with decreased accumulation in both E. coli and Pseudomonas aeruginosa (14). The lipopolysaccharide of the outer leaflet of the outer membrane in some bacteria blocks the access of the drug to the microorganism. Other reports indicate that hydrophylic compounds may cross the outer membrane through porins (11). The objectives of this study were to evaluate the accumulation of two FQ with different hydrophobicities (norfloxacin [NFX] and pefloxacin [PFX]) in K. pneumoniae by studying FQ-susceptible isogenic strains with known outer membrane alterations and FQ-resistant clinical isolates. (This work was presented in part at the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., 15 to 18 September 1996 [13].) An environmental isolate, K. pneumoniae C3 (18), and the derived mutants KT717, KT793, and KT5003 (1–3, 9) were used. K. pneumoniae LB2 and LB4 (12) and K. pneumoniae CSUB10S and CSUB10R (kindly provided by J. Lin˜ares [Barcelona, Spain]) are clinical isolates; LB and CSUB isolates are epidemiologically unrelated, as determined by pulse-field gel electrophoresis (data not shown). The outer membrane characteristics of all strains are shown in Table 1. MICs of NFX (Sigma, Madrid, Spain) and PFX (Rhoˆne-Poulenc, Antony, France) were determined by microdilution, according to National Committee for Clinical Laboratory Standards NCCLS guidelines (16). To assess FQ accumulation in K. pneumoniae C3, bacterial cells were incubated with 2, 5, 10, or 50 mg of each FQ per ml at 37°C for different periods of time. For the other strains a concentration of 10 mg/ml was used. The cells were separated from the extracellular solution by centrifugation through a silicon oil barrier (1.029 g/cm 3 ). The entire cell pellet was placed in 2 ml of 0.1 M glycine-HCl buffer (pH 3.0), vortexed, and centrifuged for 5 min at 12,800 3g. The amount of FQ in the supernatant was determined by spectrofluorometry. Bacterially associated FQ was expressed as nanograms of FQ per milligram [dry weight] of cells. The effects of the energy inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) (0.1 mM; Sigma) and 2,4-dinitrophenol (Sigma) were also evaluated. Data are expressed as means 6standard deviations. Differences among groups were compared by analysis of variance, which was used to assess statistical significance at Pvalues of #0.05. To analyze alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV, the QRDR of gyrA and the analogous region of parC were sequenced with previously described primers (6). MICs of PFX were identical for K. pneumoniae C3 and the derived mutants. A two-dilution step increase in the MIC of NFX for mutant KT5003 was observed in comparison with the parental strain K. pneumoniae C3. MICs of both quinolones for K. pneumoniae LB4 and CSUB10R were higher than for K. pneumoniae LB2 and CSUB10S, respectively (Table 1). The intracellular accumulation of NFX and PFX in K. pneumoniae C3 was rapid (Fig. 1) and not saturable at extracellular concentrations ranging from 2to50mg/ml. Nonsignificant differences in the accumulation of FQ were observed in isogenic strains KT717, KT793, and KT5003. These results indicate that the roles of cell wall components in the intrabacterial accumulation of FQ with different grades of hydrophobicity are small or nonexistent. The high susceptibilities of the nonclinical strains, K. pneumoniae C3 and the mutants, to FQ are probably related to the absence of specific mechanisms of resistance to FQ. The accumulations of * Corresponding author. Mailing address: Department of Microbiology, School of Medicine, Apdo 914, 41080 Seville, Spain. Phone: 34-5-4557448. Fax: 34-5-4377413. E-mail: [email protected]. 1850 on August 1, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://aac.asm.org/Downloaded from on August 1, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://aac.asm.org/Downloaded from on August 1, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://aac.asm.org/Downloaded from both FQ were significantly lower in the clinical K. pneumoniae isolates LB4 and CSUB10R. Preincubation of strains in the presence of CCCP did not modify the accumulation of NFX in K. pneumoniae C3, but it significantly increased the accumulation of NFX in both K. pneumoniae LB4 and CSUB10R (Table 1). Similar results were obtained with 2,4-dinitrophenol. This suggests that, at least in part, the reduced accumulation observed in both clinical isolates could be caused by an energydependent mechanism that pumps FQ out of the bacterial cell. Deguchi et al. have also described an active efflux system for NFX in FQ-resistant K. pneumoniae (7). Different authors have found discrepant amino acids at position 83 of the GyrA protein in FQ-susceptible K. pneumoniae: Thr (accession no. X16817 [5] and strain C3 [this work]) and Ser (ATCC 13833 [6]). In either case, a change in this position was observed in FQ-resistant strains LB2-LB4 (Tyr) and CSUB10S-CSUB10R (Phe). Both pairs of strains also have changes at position 112 (Val to Ile). These changes should contribute to the observed resistance to FQ, as similar changes have been observed in other gram-negative organisms (6, 20, 21). No changes in the QRDRs of the parC genes of the four clinical isolates studied were observed, in comparison with the sequence for K. pneumoniae C3. New studies are in progress to elucidate the roles of porin deficiency, efflux, and topoisomerase modification in the resistance of K. pneumoniae to FQ. Deguchi et al. have suggested that the system responsible for active efflux of NFX may contribute to increased resistance to beta-lactams, chloramphenicol, and tetracycline. The relative importance of the efflux systems of K. pneumoniae LB4 and CSUB10R to antimicrobial agents other than FQ is not known. Both strains produce broad- and extended-spectrum beta-lac- tamases. Loss of porins may explain the increased resistance to beta-lactams in comparison with their respective parental strains LB2 and CSUB10S (12), but this does not discount the importance of the efflux mechanism as a contributor to betalactam resistance in K. pneumoniae, as has previously been shown for P. aeruginosa (15). As soon as the genetics of the efflux mechanisms in these two strains are determined, their importance as factors contributing to resistance to antimicrobial agents other than FQ can be defined. This work was supported by grants from Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT) of the Spanish government (PB96- 0197). S.H.-A. was supported by a predoctoral fellowship from CICYT (FP94-41497233). REFERENCES 1. Albertı´, S., G. Marque´s, S. Camprubı´, S. Merino, J. M. Toma´s, F. Vivanco, and V. J. Benedı´. 1993. C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins. Infect. Immun. 61:852–860. 2. Albertı´, S., F. Rodrı´guez-Quin˜ones, T. Schirmer, G. Rummel, J. M. Toma´s, J. P. Rosenbusch, and V. J. Benedı´. 1995. A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding. Infect. Immun. 63:903–910. 3. Benedı´, V. J., B. Ciurana, and J. M. Toma´s. 1989. Isolation and characterization of Klebsiella pneumoniae unencapsulated mutants. J. Clin. Microbiol. 27:82–87. 4. Breines, D. M., S. Ouabdesselam, E. Y. Ng, J. Tankovic, S. Shah, C. J. Soussy, and D. Hooper. 1997. 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Agents Chemother. 35:1647–1650. 1852 NOTES ANTIMICROB.AGENTS CHEMOTHER. on August 1, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://aac.asm.org/Downloaded from ERRATUM Energy-Dependent Accumulation of Fluoroquinolones in Quinolone-Resistant Klebsiella pneumoniae Strains LUIS MARTI ´NEZ-MARTI ´NEZ, ISABEL GARCI ´A, SOFI ´A BALLESTA, VICENTE JAVIER BENEDI ´, SANTIAGO HERNA ´NDEZ-ALLE ´S, AND ALVARO PASCUAL Department of Microbiology, School of Medicine, University of Seville, Seville, and Area of Microbiology, Department of Environmental Biology and IMEDEA (CSIC-UIB), University of Balearic Island, Palma de Mallorca, Spain Volume 42, no. 7, p. 1850–1852, 1998. Page 1851, Table 1: “76 69” for accumulation of NFX after CCCP treatment for strain CSUB10R should read “161 618.” 2772