The intracellular accumulation of norfloxacin and perloxacin 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.Comisión Interministerial de Ciencia y Tecnología PB96-019

Ballesta Mudarra, Sofía

Benedí, Vicente J.

García Luque, Isabel

Hernández Allés, Santiago

Martínez Martínez, Luis

Pascual Hernández, Álvaro

English

idUS. Depósito de Investigación Universidad de Sevilla

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,0066-4804/98/$04.0010July 1998, p. 1850–1852 Vol. 42, No. 7Copyright © 1998, American Society for Microbiology. All Rights Reserved.Energy-Dependent Accumulation of Fluoroquinolones inQuinolone-Resistant Klebsiella pneumoniae StrainsLUIS MARTÍNEZ-MARTÍNEZ,1* ISABEL GARCÍA,1 SOFÍA BALLESTA,1 VICENTE JAVIER BENEDÍ,2SANTIAGO HERNÁNDEZ-ALLÉS,2 AND ALVARO PASCUAL1Department of Microbiology, School of Medicine, University of Seville, Seville,1 and Area of Microbiology, Departmentof Environmental Biology and IMEDEA (CSIC-UIB), University of Balearic Island, Palma de Mallorca,2 SpainReceived 29 October 1997/Returned for modification 8 January 1998/Accepted 9 March 1998The intracellular accumulation of norfloxacin and pefloxacin in Klebsiella pneumoniae was evaluated. Theroles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterialaccumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquin-olone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accu-mulation of the drugs enhanced their resistance to these agents.Several mechanisms are involved in the resistance to fluo-roquinolones (FQ) by gram-negative bacteria. In Escherichiacoli, point mutations in the quinolone resistance-determiningregion (QRDR) of gyrA (21) are the most important cause ofresistance. Mutations in gyrB are uncommon in clinical isolates(22), while mutations in parC (19) or (much less frequently) inparE (4) contribute to increased levels of resistance. The gyrAgene of Klebsiella pneumoniae has been cloned and sequencedand is similar (about 85% at the nucleotide level and about90% at the amino acid level) to its E. coli homolog (5). Therole of gyrA mutations in FQ-resistant K. pneumoniae has beendemonstrated, these mutations being analogous to those pre-viously observed in E. coli (6, 7). Decreased activity of FQagainst E. coli has also been related to a reduced intracellulardrug accumulation due to lipopolysaccharide or porin alter-ations impairing uptake or because of enhanced efflux (10, 11).Cross-resistance between FQ and beta-lactams in K. pneu-moniae mutants has been related to outer membrane proteinalterations (12, 17). Expression of the ramA locus from K.pneumoniae in E. coli confers resistance to norfloxacin, deter-mines the active efflux of tetracycline and chloramphenicol,and causes almost complete loss of OmpF (8). It has beenreported that the high hydrophobicity of FQ is generally asso-ciated with decreased accumulation in both E. coli and Pseudo-monas aeruginosa (14). The lipopolysaccharide of the outerleaflet of the outer membrane in some bacteria blocks theaccess of the drug to the microorganism. Other reports indi-cate that hydrophylic compounds may cross the outer mem-brane through porins (11).The objectives of this study were to evaluate the accumula-tion of two FQ with different hydrophobicities (norfloxacin[NFX] and pefloxacin [PFX]) in K. pneumoniae by studyingFQ-susceptible isogenic strains with known outer membranealterations and FQ-resistant clinical isolates.(This work was presented in part at the 36th InterscienceConference on Antimicrobial Agents and Chemotherapy, NewOrleans, La., 15 to 18 September 1996 [13].)An environmental isolate, K. pneumoniae C3 (18), and thederived mutants KT717, KT793, and KT5003 (1–3, 9) wereused. K. pneumoniae LB2 and LB4 (12) and K. pneumoniaeCSUB10S and CSUB10R (kindly provided by J. Liñares [Bar-celona, Spain]) are clinical isolates; LB and CSUB isolates areepidemiologically unrelated, as determined by pulse-field gelelectrophoresis (data not shown). The outer membrane char-acteristics of all strains are shown in Table 1. MICs of NFX(Sigma, Madrid, Spain) and PFX (Rhône-Poulenc, Antony,France) were determined by microdilution, according to Na-tional Committee for Clinical Laboratory Standards NCCLSguidelines (16). To assess FQ accumulation in K. pneumoniaeC3, bacterial cells were incubated with 2, 5, 10, or 50 mg of eachFQ per ml at 37°C for different periods of time. For the otherstrains a concentration of 10 mg/ml was used. The cells wereseparated from the extracellular solution by centrifugationthrough a silicon oil barrier (1.029 g/cm3). The entire cell pelletwas placed in 2 ml of 0.1 M glycine-HCl buffer (pH 3.0),vortexed, and centrifuged for 5 min at 12,800 3 g. The amountof FQ in the supernatant was determined by spectrofluorom-etry. Bacterially associated FQ was expressed as nanograms ofFQ per milligram [dry weight] of cells. The effects of theenergy inhibitors carbonyl cyanide m-chlorophenylhydrazone(CCCP) (0.1 mM; Sigma) and 2,4-dinitrophenol (Sigma) werealso evaluated. Data are expressed as means 6 standard devi-ations. Differences among groups were compared by analysisof variance, which was used to assess statistical significance atP values of #0.05.To analyze alterations in the GyrA subunit of DNA gyraseand the ParC subunit of topoisomerase IV, the QRDR of gyrAand the analogous region of parC were sequenced with previ-ously described primers (6). MICs of PFX were identical for K.pneumoniae C3 and the derived mutants. A two-dilution stepincrease in the MIC of NFX for mutant KT5003 was observedin comparison with the parental strain K. pneumoniae C3.MICs of both quinolones for K. pneumoniae LB4 andCSUB10R were higher than for K. pneumoniae LB2 andCSUB10S, respectively (Table 1). The intracellular accumula-tion of NFX and PFX in K. pneumoniae C3 was rapid (Fig. 1)and not saturable at extracellular concentrations ranging from2 to 50 mg/ml. Nonsignificant differences in the accumulationof FQ were observed in isogenic strains KT717, KT793, andKT5003. These results indicate that the roles of cell wall com-ponents in the intrabacterial accumulation of FQ with differentgrades of hydrophobicity are small or nonexistent. The highsusceptibilities of the nonclinical strains, K. pneumoniae C3and the mutants, to FQ are probably related to the absence ofspecific mechanisms of resistance to FQ. The accumulations of* Corresponding author. Mailing address: Department of Microbi-ology, School of Medicine, Apdo 914, 41080 Seville, Spain. Phone:34-5-4557448. Fax: 34-5-4377413. E-mail: lmartin@cica.es.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. pneumoniaeisolates LB4 and CSUB10R. Preincubation of strains in thepresence of CCCP did not modify the accumulation of NFX inK. pneumoniae C3, but it significantly increased the accumula-tion of NFX in both K. pneumoniae LB4 and CSUB10R (Table1). Similar results were obtained with 2,4-dinitrophenol. Thissuggests that, at least in part, the reduced accumulation ob-served in both clinical isolates could be caused by an energy-dependent mechanism that pumps FQ out of the bacterial cell.Deguchi et al. have also described an active efflux system forNFX in FQ-resistant K. pneumoniae (7).Different authors have found discrepant amino acids at po-sition 83 of the GyrA protein in FQ-susceptible K. pneumoniae:Thr (accession no. X16817 [5] and strain C3 [this work]) andSer (ATCC 13833 [6]). In either case, a change in this positionwas observed in FQ-resistant strains LB2-LB4 (Tyr) andCSUB10S-CSUB10R (Phe). Both pairs of strains also havechanges at position 112 (Val to Ile). These changes shouldcontribute to the observed resistance to FQ, as similar changeshave been observed in other gram-negative organisms (6, 20,21). No changes in the QRDRs of the parC genes of the fourclinical isolates studied were observed, in comparison with thesequence for K. pneumoniae C3. New studies are in progress toelucidate the roles of porin deficiency, efflux, and topoisomer-ase modification in the resistance of K. pneumoniae to FQ.Deguchi et al. have suggested that the system responsible foractive efflux of NFX may contribute to increased resistance tobeta-lactams, chloramphenicol, and tetracycline. The relativeimportance of the efflux systems of K. pneumoniae LB4 andCSUB10R 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 tobeta-lactams in comparison with their respective parentalstrains LB2 and CSUB10S (12), but this does not discount theimportance of the efflux mechanism as a contributor to beta-lactam resistance in K. pneumoniae, as has previously beenshown for P. aeruginosa (15). As soon as the genetics of theefflux mechanisms in these two strains are determined, theirimportance as factors contributing to resistance to antimicro-bial agents other than FQ can be defined.This work was supported by grants from Comisión Interministerialde Ciencia y Tecnologı́a (CICYT) of the Spanish government (PB96-0197). S.H.-A. was supported by a predoctoral fellowship from CICYT(FP94-41497233).REFERENCES1. Albertı́, S., G. Marqués, S. Camprubı́, S. Merino, J. M. Tomás, F. Vivanco,and V. J. Benedı́. 1993. C1q binding and activation of the complementclassical pathway by Klebsiella pneumoniae outer membrane proteins. Infect.Immun. 61:852–860.2. Albertı́, S., F. Rodrı́guez-Quiñones, T. Schirmer, G. Rummel, J. M. Tomás,J. P. Rosenbusch, and V. J. Benedı́. 1995. A porin from Klebsiella pneu-moniae: sequence homology, three-dimensional model, and complementbinding. Infect. Immun. 63:903–910.3. Benedı́, V. J., B. Ciurana, and J. M. Tomás. 1989. Isolation and character-ization 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. Quinolone resistance locus nfxD of Escherichiacoli is a mutant allele of the parE gene encoding a subunit of topoisomeraseIV. Antimicrob. Agents Chemother. 41:175–179.5. Dimri, G. P., and H. K. Das. 1990. Cloning and sequence analysis of gyrAgene of Klebsiella pneumoniae. Nucleic Acids Res. 18:151–156.6. Deguchi, T., A. Fukuoka, M. Yasuda, M. Nakano, S. Ozeki, E. Kanematsu,Y. Nishino, S. Ishihara, Y. Ban, and Y. Kawada. 1997. Alterations in theGyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV inquinolone-resistant clinical isolates of Klebsiella pneumoniae. Antimicrob.Agents Chemother. 41:699–701.7. Deguchi, T., T. Kawamura, M. Yasuda, M. Nakano, H. Fukuda, H. Kato, N.Kato, Y. Okano, and Y. Kawada. 1997. In vitro selection of Klebsiella pneu-moniae strains with enhanced quinolone resistance during fluoroquinolonetreatment of urinary tract infection. Antimicrob. Agents Chemother. 41:1609–1611.FIG. 1. Time-dependent accumulation of NFX (p) and PFX (Œ) in K. pneu-moniae C3.TABLE 1. Susceptibilities to and accumulation of NFX in four isogenic and four clinical K. pneumoniae strainsStrainPresence in strain of a: NFXMIC(mg/ml)Accumulation of NFX(ng/mg [dry weight])bO-Ag K-Ag OmpK34 OmpK35 OmpK36 Control CCCPIsogenic strainsC3 O1 K66 1 1 1 0.015 366 6 65 346 6 46KT717 O1 2 1 1 1 0.03 330 6 67 NDKT793 2 2 1 1 1 0.03 299 6 68 NDKT5003 2 2 1 2 2 0.06 314 6 54 NDClinical strainsLB2 ND ND 1 1 2 2 286 6 24 385 6 65LB4 ND ND 1 2 2 4 116 6 21 263 6 38CSUB10S ND ND 1 1 2 4 313 6 6 NDCSUB10R ND ND 1 2 2 16 66 6 20 76 6 9a OmpK34, OmpK35, and OmpK36 are the K. pneumoniae homologs of E. coli OmpA and porins OmpF and OmpC, respectively. O-Ag, O antigen; K-Ag, K antigen;ND, not determined.b Data are means 6 standard deviations of five experiments.VOL. 42, 1998 NOTES 1851 on August 1, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://aac.asm.org/Downloaded from 8. George, A. M., R. M. Hall, and H. W. Stokes. 1995. 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Methods fordilution susceptibility tests for bacteria that grow aerobically, 4th ed. Ap-proved standard M7-A4. National Committee for Clinical Laboratory Stan-dards, Wayne, Pa.17. Sanders, C. C., W. E. Sanders, R. V. Goering, and V. Vermer. 1984. Selectionof multiple antibiotic resistance by quinolones, b-lactams, and aminoglyco-sides with special reference to cross-resistance between unrelated drugclasses. Antimicrob. Agents Chemother. 26:787–801.18. Tomás, J. M., V. J. Benedı́, B. Ciurana, and J. Jofré. 1986. Role of capsuleand O antigen in resistance of Klebsiella pneumoniae to serum bactericidalactivity. Infect. Immun. 54:85–89.19. Vila, J., J. Ruiz, P. Goñi, and M. T. Jiménez De Anta. 1996. Detection ofmutations in parC in quinolone-resistant clinical isolates of Escherichia coli.Antimicrob. Agents Chemother. 40:491–493.20. Vila, J., J. Ruiz, F. Marco, A. Barcelo, P. Goñi, E. Giralt, and M. T. JiménezDe Anta. 1994. Association between double mutation in gyrA gene of cipro-floxacin-resistant clinical isolates of Escherichia coli and MICs. Antimicrob.Agents Chemother. 38:2477–2479.21. Yoshida, H., M. Bogaki, M. Nakamura, and S. Nakamura. 1990. Quinoloneresistance-determining region in the DNA gyrase gyrA gene of Escherichiacoli. Antimicrob. Agents Chemother. 34:1271–1272.22. Yoshida, H., M. Bogaki, M. Nakamura, L. Yamanaka, and S. Nakamura.1991. Quinolone resistance-determining region in the DNA gyrase gyrB geneof Escherichia coli. Antimicrob. Agents Chemother. 35:1647–1650.1852 NOTES ANTIMICROB. AGENTS CHEMOTHER. on August 1, 2017 by USE/BCTA.GEN UNIVERSITARIAhttp://aac.asm.org/Downloaded from ERRATUMEnergy-Dependent Accumulation of Fluoroquinolones inQuinolone-Resistant Klebsiella pneumoniae StrainsLUIS MARTÍNEZ-MARTÍNEZ, ISABEL GARCÍA, SOFÍA BALLESTA, VICENTE JAVIER BENEDÍ,SANTIAGO HERNÁNDEZ-ALLÉS, AND ALVARO PASCUALDepartment of Microbiology, School of Medicine, University of Seville, Seville, and Area of Microbiology, Department ofEnvironmental Biology and IMEDEA (CSIC-UIB), University of Balearic Island, Palma de Mallorca, SpainVolume 42, no. 7, p. 1850–1852, 1998. Page 1851, Table 1: “76 6 9” for accumulation of NFX after CCCP treatment for strainCSUB10R should read “161 6 18.”2772

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

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

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