a Department of Biology, Area of Microbiology and IMEDEA-CSIC, University of the Balearic Islands, b Department of Microbiology, School of Medicine, University of Seville and c Department of Microbiology, School of Biology, University of Barcelona, Spain
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Abstract |
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Introduction |
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Outer membrane alterations have been related to resistance to antimicrobial agents in Escherichia coli and other Gram-negative bacteria,3 including K. pneumoniae.4 In most of those studies, however, the specific contribution of alterations in porin expression is difficult to determine, because other mechanisms of resistance are commonly present in porin-deficient strains (such as production of ß-lactamases or aminoglycoside-modifying enzymes, modified topoisomerases or energy-dependent efflux systems).4
In order to evaluate the relationship between alterations of the K. pneumoniae outer membrane and its resistance to antimicrobial agents, we determined the activity of several groups of antimicrobial agents against a K. pneumoniae strain and several mutants with defined outer membrane alterations derived from it. As described previously, K. pneumoniae C3 does not exhibit mutations in the gyrA or parC genes, and energy-dependent efflux of fluoroquinolones does not occur in this strain.5 In this study we have also observed that this strain expresses very low levels of ß-lactamase and is susceptible to aminoglycosides, chloramphenicol and tetracycline. All these factors make this strain ideal for evaluating the contribution made by various alterations in cell envelopes to the resistance to antimicrobial agents.
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Materials and methods |
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The parental strain K. pneumoniae C3 (an environmental isolate, serotype O1:K66) expresses three major outer membrane proteins, OmpA, OmpK35 porin (analogous to E. coli OmpF) and OmpK36 porin (analogous to E. coli OmpC), with apparent molecular weights of 34, 35 and 36 kDa,6 respectively. Isogenic mutants were derived from K. pneumoniae C3 (Table). Strain KT791 is a capsuledeficient mutant (O1:K) obtained after UV mutagenesis and selection with anticapsular serum.7 Mutant KT793 is a serum-sensitive double mutant (O:K) derived from KT791, obtained by selection with bacteriophage FC3-2, specific for lipopolysaccharide (LPS) serotype O1.7 Mutant KT707 (O:K66) is a rough LPS, serum-sensitive strain derived from the wild-type K. pneumoniae C3 deficient in porin OmpK35, but still expressing porin OmpK36.6 Mutant KT5001 (OmpK35+, OmpK36) was derived from KT793 as a mutant resistant to phage FC3-11, specific for porin OmpK36.8 Plasmid pSUV100 (ompK36::Tn5) was used to replace by double recombination the wild-type ompK36 gene of a spontaneous rifampin-resistant mutant from KT793, giving strain KT5002 (OmpK35+, OmpK36); plasmid pPH1JI (containing gentamicin and chloramphenicol resistance genes) was used to provoke gene recombination.8 Mutant KT5002-2 (OmpK35, OmpK36) was obtained from strain KT5002 by selection on Mueller Hinton agar containing cefoxitin 4 mg/L. When KT5002-2 was tested for susceptibility to cefoxitin using Etest strips (see below), one colony grew within the inhibition zone; it was cultured in antibiotic-free medium and named mutant KT5002-22. Mutant KT5003P (OmpK35, OmpK36) was obtained from mutant KT5001 by selection on Mueller Hinton agar containing cefoxitin 64 mg/L (Sigma, Madrid, Spain).
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Susceptibility testing
A microdilution assay according to NCCLS10 guidelines was used. In some cases, Etest strips (AB Biodisk, Solna, Sweden) were used, according to the manufacturer's instructions, to confirm microdilution results. The following antimicrobial agents were evaluated: amoxycillin (Sigma), amoxycillin plus clavulanic acid (SmithKline Beecham, Madrid, Spain) at a proportion of 2:1, cephalothin (Sigma), cefoxitin (Sigma), cefotaxime (Sigma), ceftazidime (Glaxo, Madrid, Spain), cefepime (Bristol MyersSquibb, Madrid, Spain), imipenem (Merck Sharp and Dohme, Madrid, Spain), meropenem (Zeneca, Madrid, Spain), chloramphenicol (Sigma), tetracycline (Sigma), ciprofloxacin (Bayer, Leverkusen, Germany), pefloxacin (RhônePoulenc, St Antoine, France), sparfloxacin (Rhône Poulenc), gentamicin (Sigma), tobramycin (Sigma) and amikacin (BristolMyersSquibb). The combinations cefotaxime plus clavulanic acid and ceftazidime plus clavulanic acid (clavulanic acid at a fixed concentration of 2 mg/L) were also tested against some strains.
Analysis of outer membrane proteins
Bacterial cells were grown in nutrient broth (beef extract (Difco, Detroit, MI, USA) 3 g and proteose peptone no. 3, 5 g (Difco) in 1 L of distilled water) to improve OmpK35 expression. Cells in logarithmic phase were lysed by sonication and cell membranes were recovered by ultracentrifugation. Outer membrane proteins (OMPs) were obtained after treatment of cell membranes with sodium lauryl sarcosinate (Sigma) and subsequent ultracentrifugation. OMP profiles were determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS PAGE), using 11% acrylamide, 0.5% bis-acrylamide and 0.1% SDS in the running gel.
Determination of ß-lactamase activity
ß-Lactamase activity was determined spectrophotometrically using crude supernatants of sonicated cells. Cephaloridine (Sigma, 0.1 mM) and ampicillin (Sigma, 0.5 mM) were used as substrates in independent assays. One unit of enzyme was defined as the amount of enzyme that hydrolysed 1 µmol of substrate per minute at 30°C at 295 nm (cephaloridine) or 232 nm (ampicillin). Enzyme activity was standardized against protein concentration in the supernatant, as determined by the Bradford assay.11
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Results and discussion |
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The activities of antimicrobial agents against the tested strains are presented in the Table. K. pneumoniae C3 was susceptible to amoxycillin, as expected from its low ß-lactamase activity. Cefotaxime was as active as cefepime, and four times more active than ceftazidime. Meropenem was eight times more active than imipenem. Alterations affecting capsule or antigen O expression did not result in relevant changes in susceptibility to the different groups of antimicrobial agents tested. Similarly, when the organisms lacked expression of either OmpK35 (KT707) or OmpK36 (KT5001, KT5002) no significant changes were observed.
Strains that lacked both OmpK35 and OmpK36 porins showed significant (four-fold or more) increases in MIC of all ß-lactams except imipenem and (in two out of three mutants) meropenem. This was the case for strains KT5002-2 and KT5002-22, which showed increased resistance to amoxycillin, cephalothin, cefoxitin, cefotaxime and ceftazidime. These results support previous studies indicating that ß-lactam resistance in K. pneumoniae increases significantly when its two major porins are lost simultaneously.4 The MICs of cephalothin and cefoxitin were higher in strains KT5002-22 and KT5003P than in strain KT5002-2, suggesting that other mechanisms besides porin loss and ß-lactamase production, may be involved in ß-lactam resistance in K. pneumoniae. This was most clearly observed in strain KT5003P, in which the MICs of all ß-lactams, except imipenem and, to a lesser extent, meropenem, were considerably increased. The MICs of cefotaxime and ceftazidime did not change (mutants KT5002 and KT5002-22) or decreased by only one dilution step (mutants KT5002-2 and KT5003P) in the presence of clavulanic acid. The role of the increased ß-lactamase production observed in strain KT5003P is not known. Further studies on the nature of the ß-lactamase produced by the parental strain C3 and the mutant KT5003P would be needed to clarify this point. It is possible that selection of mutants KT5002-2, KT5002-22 and KT5003P with different concentrations of cefoxitin could produce pleiotropic mutation(s) affecting not only the ompK35 gene, but also some other gene(s) coding for other mechanisms responsible for the decreased susceptibility to ß-lactams observed in these strains (such as pencillin-binding proteins or efflux pumps). New studies are in progress to evaluate this possibility. It is not known if these mechanisms will also turn out to be involved in basal resistance to ß-lactams of clinical isolates of K. pneumoniae.
Susceptibility to chloramphenicol, tetracycline and aminoglycosides did not change after capsule, antigen O or porin mutations, even when expression of both major porins was lost (Table). The decreased activity of gentamicin and chloramphenicol observed in strain KT5002 and mutants derived from it was caused by the presence of plasmid pPH1JI, used to integrate the ompK36::Tn5 gene from plasmid pSUV100 within the native ompK36 gene of strain KT793 (see Materials and methods).
K. pneumoniae C3 is highly susceptible to fluoroquinolones, and loss of capsule or antigen O does not alter the MICs of three fluoroquinolones with different hydrophobicity. Moreover, loss of porins did not result in increased MICs of any of the three agents evaluated (including the most hydrophilic compound, ciprofloxacin). Other studies have shown that, in the porin-deficient mutant KT5003P, there is a four-fold increase in the MIC of norfloxacin5 and that in some clinical porin-deficient isolates of K. pneumoniae, MICs of fluoroquinolones increase by a factor of two or four4,5,13 in comparison with clonally related strains expressing porin(s). Cloning and expression of OmpK36 in the latter strains restore fluoroquinolone activity.4 Fluoroquinolone-resistant clinical isolates also contain topoisomerase mutations or show decreased drug accumulation, both of which can cause fluoroquinolone resistance.5,13 It seems likely that, as previously discussed with regard to ß-lactams, porin deficiency is more relevant when the drug shows lower intrinsic activity or when other mechanisms of resistance are also present within the same cell.
In conclusion, loss of expression of one porin, either OmpK35 or OmpK36, did not alter the MICs for K. pneumoniae deficient in other mechanisms of resistance. Only when both porins were lost was a decrease in the activity of a few ß-lactams observed. This suggested that, in K. pneumoniae C3, several mutations are needed for the organism to become resistant to antimicrobial agents of clinical importance. Unfortunately this is not the case for most clinical isolates of K. pneumoniae, which normally produce a penicillinase or occasionally express extended-spectrum ß-lactamase or AmpC-type enzymes or harbour topoisomerase mutation(s); these may facilitate the appearance of clinically significant resistance to antimicrobial agents.
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Acknowledgments |
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Notes |
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References |
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2 . Bradford, P. A., Urban, C., Mariano, N., Projan, S. J., Rahal, J. J. & Bush, K. (1997). Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrobial Agents and Chemotherapy 41, 5639.[Abstract]
3 . Nikaido, H. (1989). Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrobial Agents and Chemotherapy 33, 18316.[ISI][Medline]
4 . Martínez-Martínez, L., Hernández-Allés, S., Albertí, S., Tomás, J. M., Benedí, V. J. & Jacoby, G. A. (1996). In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy 40, 3428.[Abstract]
5
.
Martínez-Martínez, L., García, I., Ballesta, S., Benedí, V. J., Hernández-Allés, S. & Pascual, A. (1998). Energy-dependent accumulation of fluoroquinolones in quinolone-resistant Klebsiella pneumoniae strains. Antimicrobial Agents and Chemotherapy 42, 18502.
6 . Tomás, J. M., Benedí, V. J., Ciurana, B. & Jofre, J. (1986). Role of capsule and O antigen in resistance of Klebsiella pneumoniae to serum bactericidal activity. Infection and Immunity 54, 859.[ISI][Medline]
7 . Benedí, V. J., Ciurana, B. & Tomás, J. M. (1989). Isolation and characterization of Klebsiella pneumoniae unencapsulated mutants. Journal of Clinical Microbiology 27, 827.[ISI][Medline]
8 . Hernández-Allés, S., Albertí, S., Rubires, X., Merino, S., Tomás, J. M. & Benedí, V. J. (1995). Isolation of FC3-11, a bacteriophage specific for the Klebsiella pneumoniae porin OmpK36, and its use for the isolation of porin-deficient mutants. Canadian Journal of Microbiology 41, 399406.[ISI]
9 . Albertí, S., Rodríguez-Quiñones, F., Schirmer, T., Rummel, G., Tomás, J. M., Rosenbusch, J. P. et al. (1995). A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding. Infection and Immunity 63, 90310.[Abstract]
10 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.
11 . Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analytical Biochemistry 72, 24854.[ISI][Medline]
12 . Hernández-Allés, S., Albertí, S., Alvarez, M. D., Domenech-Sanchez, A., Martínez-Martínez, L., Gil, J. et al. (1999). Porin expression in clinical isolates of Klebsiella pneumoniae. Microbiology 145, 6739.[Abstract]
13 . Deguchi, T., Kawamura, T., Yasuda, M., Nakano, M., Fukuda, H., Kato, H. et al. (1997). In vivo selection of Klebsiella pneumoniae strains with enhanced quinolone resistance during fluoroquinolone treatment of urinary tract infections. Antimicrobial Agents and Chemotherapy 41, 160911.[Abstract]
Received 26 May 1999; returned 12 October 1999; revised 22 December 1999; accepted 22 March 2000