Activity of nine antimicrobial agents against clinical isolates of Klebsiella pneumoniae producing extended-spectrum ß-lactamases and deficient or not in porins

A. Doménech-Sáncheza, A. Pascualb,c,*, A. I. Suárezc, D. Alvareza, V. J. Benedía and L. Martínez-Martínezb,c

a Area de Microbiología, Departamento de Biología, Universitat de les Illes Balears and IMEDEA (CSIC-UIB), Palma de Mallorca; b Department of Microbiology, School of Medicine, Seville; c University Hospital V. Macarena, Sevilla, Spain

Sir,

The most relevant single mechanism of resistance of Enterobacteriaceae to cephalosporins is the production of ß-lactamases.1 For Klebsiella pneumoniae, these enzymes are usually plasmid-mediated extended-spectrum ß-lactamases (ESBLs) or, less commonly, plasmid-mediated AmpC-type enzymes. K. pneumoniae produces two major porins, outer membrane proteins (Omps) K35 and K36, which are homologous to Escherichia coli porins OmpF and OmpC, respectively.2 In klebsiellae, simultaneous loss of both OmpK35 and OmpK36 causes increased resistance to cephalosporins and other ß-lactams when ESBL or AmpC-type ß-lactamase is also produced.3 ESBLs do not confer resistance to cephamycins and carbapenems.1 Cephamycins, however, are able to select for mutants that are deficient in porins, resulting in subsequent resistance to these agents.4 Cefepime and cefpirome are poorly hydrolysed by AmpC-type ß-lactamases but have less activity against ESBL- producing strains, particularly in porin-deficient organisms.3 ESBL-producing K. pneumoniae are more frequently resistant to aminoglycosides and fluoroquinolones than K. pneumoniae strains not producing these ß-lactamases. The purpose of this study was to evaluate the activity of cefepime, cefpirome, third-generation cephalosporins, carbapenems, amikacin and ciprofloxacin against clinical isolates of ESBL-producing K. pneumoniae with different porin profiles. Sixty-five non-consecutive clinical isolates of ceftazidime-resistant K. pneumoniae isolated at the Microbiology Laboratory of the University Hospital V. Macarena (Seville, Spain) in the period 1994–1997 were studied. Strains were selected to include 15 isolates deficient in porins (see below). E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and K. pneumoniae LB4 (producing SHV-5)4 were also included as controls for antimicrobial susceptibility testing. K. pneumoniae C3 (producing OmpK35 and OmpK36)5 and E. coli K-12 mutants deficient in OmpA, OmpF or OmpC were used as reference strains in the determination of porin profiles.

The activities of cefepime, cefpirome, cefotaxime, ceftazidime, cefoxitin, imipenem, meropenem, ciprofloxacin and amikacin were evaluated by a microdilution assay according to NCCLS guidelines.6 A >=8-fold decrease in the activities of either ceftazidime or cefotaxime with clavulanic acid (2 mg/L) compared with ceftazidime or cefotaxime alone was taken as an indication of ESBL production. Omps were obtained after sonication of bacteria and sarkosyl treatment of cell membranes. Omps were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) using 11% polyacrylamide in the running gel. Bacteria were categorized as porin producers (P+) if at least one protein band corresponding to either OmpK35 or OmpK36 was detected, and as porin-deficient (P-) if no major bands in the region of porins were observed.

Fifty of the 65 clinical isolates of ESBL-producing K. pneumoniae produced at least one porin (mostly OmpK36). The remaining 15 strains were deficient in both OmpK35 and OmpK36. The proportion of isolates expressing or not expressing porins does not reflect their actual frequency of isolation among clinical isolates, as K. pneumoniae strains deficient in both porins are much less common than would be predicted from these figures.

The activities of nine antimicrobial agents against P+ and P- strains are presented in the Table. The activity of cefepime and cefpirome against both P+ and P- strains is higher than that of cefotaxime and ceftazidime. Nevertheless, and according to the NCCLS recommendations, ESBL-producing K. pneumoniae are considered resistant to all cephalosporins, independently of the in vitro activity of the different compounds within this group. The MIC50s and MIC90s of cefoxitin against P+ and P- strains indicate that loss of porins is critical for resistance to this cephamycin in ESBL-producing K. pneumoniae. Porin loss contributes to increasing the level of resistance to the other cephalosporins in ESBL-producing strains.


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Table. MICs (mg/L) of nine antimicrobial agents against ESBL-producing clinical strains of K. pneumoniae expressing porins (P+) and deficient in porins (P-)
 
Both imipenem and meropenem were the most active agents against both bacterial groups. Meropenem was more active than imipenem against all P+ strains and most P- strains, but MIC ranges of meropenem against P- strains were broader than those of imipenem. These results agree with previous reports on laboratory7 and clinical4 strains of K. pneumoniae deficient in both OmpK35 and OmpK36, and indicate that a subset of ESBL-producing K. pneumoniae strains have mechanisms of resistance that affect meropenem more than imipenem. The results of this study indicate that in a subset of strains deficient in porins, the activity of meropenem is affected more than that of imipenem. Loss of porins seems not to affect the in vitro activity of amikacin in ESBL-producing strains. This should be expected from the ability of amikacin to use the porin-independent self-promoted pathway of penetration into the bacterial cell.

It has been observed that resistance to fluoroquinolones is higher in ESBL producers than in non-producers.8 The exact reasons for this relationship are not known. Previous reports considering clinical isolates and laboratory strains indicate that loss of porins causes a moderate increase in fluoroquinolone resistance.5 Surprisingly, MIC50s and MIC90s of ciprofloxacin in the strains herein evaluated were higher for P+ strains than for P- strains. This apparent paradox could be explained considering that target mutations contribute more significantly to fluoroquinolone resistance than loss of porins, and that the more numerous group of P+ strains can be expected to contain more organisms with multiple mutations in gyrA/parC than the group of P- strains. New studies are currently in progress to evaluate this phenomenon.

Notes

J Antimicrob Chemother 2000; 46: 858–860

* Corresponding author. Departamento de Microbiología, Facultad de Medicina, Apartado 914, 41080 Sevilla, Spain. Tel: +34-95-4557448; Fax: +34-95-4377413; E-mail: atomas{at}cica.es Back

References

1 . Livermore, D. M. (1995). ß-lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 557–84.[Abstract]

2 . Hernández-Allés, S., Alvarez, D., Domenech-Sánchez, A., Martínez-Martínez, L., Gil, J. et al. (1999). Porin expression in clinical isolates of Klebsiella pneumoniae. Microbiology 145, 673–9.[Abstract]

3 . Martínez-Martínez, L., Pascual, A., Hernández-Allés, S., Alvarez- Díaz, D., Suárez, A. I., Tran, J. et al. (1999). Roles of ß-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 43, 1669–73.[Abstract/Free Full Text]

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, 342–8.[Abstract]

5 . Tomás, J. M., Benedí, V. J., Ciurana, B. & Jofre, J. (1986). Role of capsule and O antigen in resistance of Klebsiella pneumoniae to serum activity. Infection and Immunity 54, 85–9.[ISI][Medline]

6 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Susceptibility Tests for Bacteria that Grow Aerobically; Fourth Edition—Approved Standard M7-A4. NCCLS, Wayne, PA.

7 . Pragai, Z. & Nagy, E. (1998). In-vitro selection of porin-deficient mutants of two strains of Klebsiella pneumoniae with reduced susceptibilities to meropenem but not to imipenem. Journal of Antimicrobial Chemotherapy 42, 821–4.[Abstract]

8 . Babini, G. S. & Livermore, D. M. (2000). Antimicrobial resistance amongst Klebsiella spp. collected from intensive care units in Southern and Western Europe in 1997–1998. Journal of Antimicrobial Chemotherapy 45, 183–9.[Abstract/Free Full Text]





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