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Antimicrobial Agents and Chemotherapy, July 1998, p. 1850-1852, Vol. 42, No. 7
Department of Microbiology,
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. Liñares [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 (Rhône-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 µg of each FQ per ml at 37°C for different periods of
time. For the other strains a concentration of 10 µg/ml was used. The
cells were separated from the extracellular solution by centrifugation through a silicon oil barrier (1.029 g/cm3). 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 × g. 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 ± standard deviations. Differences among
groups were compared by analysis of variance, which was used to assess
statistical significance at P values of 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 2 to 50 µg/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 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 energy-dependent 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).
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Energy-Dependent Accumulation of Fluoroquinolones
in Quinolone-Resistant Klebsiella pneumoniae
Strains
ABSTRACT
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0.05.
TABLE 1.
Susceptibilities to and accumulation of NFX in four
isogenic and four clinical K. pneumoniae strains
View larger version (13K):
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FIG. 1.
Time-dependent accumulation of NFX (*) and PFX ( 
)
in K. pneumoniae C3.
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-lactamases. 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 beta-lactam 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.
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ACKNOWLEDGMENTS |
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This work was supported by grants from Comisión Interministerial de Ciencia y Tecnología (CICYT) of the Spanish government (PB96-0197). S.H.-A. was supported by a predoctoral fellowship from CICYT (FP94-41497233).
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FOOTNOTES |
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* 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: lmartin{at}cica.es.
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Antimicrobial Agents and Chemotherapy, July 1998, p. 1850-1852, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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