AAC
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Padilla, E.
Right arrow Articles by Borrell, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Padilla, E.
Right arrow Articles by Borrell, N.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, June 2006, p. 2258-2260, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01513-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Effect of Porins and Plasmid-Mediated AmpC ß-Lactamases on the Efficacy of ß-Lactams in Rat Pneumonia Caused by Klebsiella pneumoniae

Emma Padilla,1,3 Diana Alonso,3 Antonio Doménech-Sánchez,3 Cristina Gomez,2 José Luis Pérez,1,3 Sebastián Albertí,3 and Nuria Borrell1,3*

Servicio de Microbiología,1 Anatomía Patológica, Hospital Son Dureta, IUNICS,2 Departmento de Biología, Universitat de les Illes Balears, Palma de Mallorca, Spain3

Received 25 November 2005/ Returned for modification 12 January 2006/ Accepted 21 March 2006


   ABSTRACT
Top
 Abstract
Text
 References
 
The in vivo activities of imipenem, meropenem, and cefepime were studied in a model of rat pneumonia caused by a plasmid-mediated AmpC ß-lactamase ACT-1-producing Klebsiella pneumoniae strain (K. pneumoniae strain 12) and a derivative porin-deficient mutant (K. pneumoniae strain 12dp). No differences between these activities were seen with K. pneumoniae 12. Only meropenem showed an activity slightly better than that of imipenem with K. pneumoniae 12dp.


   TEXT
Top
 Abstract
 Text
References
 
The number of infections caused by gram-negative bacteria producing plasmid-mediated AmpC-type ß-lactamases (PACBL) has increased in the last 10 years (3, 7, 15, 18), including three reports of outbreaks (9, 15, 20). The only active ß-lactams in those cases are carbapenems (imipenem, meropenem, and ertapenem) and zwitterionic cephalosporins (cefepime and cefpirome). However, cooperation with other mechanisms of resistance, such as decreased permeability, results in the subsequent elevation of the MICs of antibiotics with good activity, in some cases up to resistance level (2, 3, 10).

The aim of this study was to evaluate the in vivo efficacies of human regimens of imipenem, meropenem, and cefepime against a PACBL ACT-1-producing Klebsiella pneumoniae strain (K. pneumoniae 12) and its derived in vitro-obtained mutant deficient in porins (K. pneumoniae 12dp) by using a model of experimental pneumonia in immunocompetent rats.

K. pneumoniae strain 52145 is a clinical isolate that has been described previously (14). K. pneumoniae strain 12 is a derivative of K. pneumoniae 52145 containing the PACBL ACT-1 by conjugation with K. pneumoniae C2 (pMG246) (1). K. pneumoniae strain dp is a porin-deficient mutant derived from K. pneumoniae strain 52145, and it was obtained by overnight culture containing cefoxitin at 64 mg/ml and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis as previously described (8). K. pneumoniae strain 12dp is a porin-deficient derivative of K. pneumoniae 52145 expressing ACT-1. Expression of the AmpC ß-lactamase in both K. pneumoniae 12 and K. pneumoniae 12dp was verified by nitrocefin hydrolysis.

Imipenem-cilastatin and cilastatin (Merck Sharp & Dohme-Chibret, Madrid, Spain), meropenem (Astra Zeneca Pharma, Madrid, Spain), and cefepime (Bristol-Myers Squibb, Madrid, Spain) were used according to the manufacturer's instructions. MICs of imipenem, meropenem, cefepime, and ertapenem were determined by the Etest method (AB Biodisk, Solna, Sweden) and are summarized in Table 1.


View this table:
[in this window]
[in a new window]
  		TABLE 1. In vitro susceptibilities of K. pneumoniae 52145 and its derivative strains

 
Pharmacokinetic studies were done by a single intraperitoneal dose in male Wistar rats (Harlan Ibérica S. L., Barcelona, Spain) of 40 mg/kg of body weight for imipenem, 40 mg/kg for meropenem, or 60 mg/kg for cefepime. Antibiotic concentrations in blood were determined by the disk plate bioassay method (5). Since rats produce dehydropeptidase, which is able to hydrolyze meropenem (6), cilastatin (1:1) was given together with meropenem. Estimations of the maximum antibiotic plasma concentration (in mg/liter), half-life (in h), and time during which the plasma concentration remained above the MIC (in h) were calculated by a linear regression analysis in an open one-compartment model (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Pharmacokinetic parameters of the antimicrobials used in animal model and t>MIC of the antibiotics for K. pneumoniae strains 12 and 12dpa

 
The pneumonia model used was a modification of a model previously developed by Mimoz et al. (12, 13). Rats were intraperitoneally anesthetized with ketamine (Pfizer, Madrid, Spain) (100 mg/kg of body weight) plus xylazine (Sigma-Aldrich, Madrid, Spain) (10 mg/kg of body weight) and transtracheally inoculated with 106 CFU of K. pneumoniae 12 or 107 CFU of K. pneumoniae 12dp plus 0.5% agar (Oxoid SA, Madrid, Spain). Bilateral pneumonia was observed by standard techniques (19).

Treatment groups, according to pharmacokinetic parameters, received intraperitoneal injections of 40 mg/kg/2 h for imipenem or meropenem-cilastatin or 60 mg/kg/4 h for cefepime. Therapy was started 4 h after bacterial inoculation and continued for 3 days. Animal sacrifice was performed 8 h after the last antibiotic dose. The lungs were quantitatively cultured, and viable bacteria were expressed as log10 CFU/g of lung. Survival at day 3 was also recorded. A Kruskal-Wallis test and a Mann-Whitney U test were used for statistical analysis by the SPSS 8.0 statistical package (SPSS Inc., Chicago, IL). Emergence of resistance during ß-lactam therapy was examined at the end of each experiment by plating 400 µl of the lung homogenates onto MacConkey agar with 4x MIC (mg/liter) of imipenem, cefepime, or meropenem.

In our study, the introduction of the PACBL ACT-1 in the K. pneumoniae 52145 strain (K. pneumoniae strain 12) did not change the MICs of carbapenems but slightly increased the cefepime MIC from 0.03 to 0.25 µg/ml. The same strain with the loss of OmpK35 and OmpK36 porins (K. pneumoniae strain 12dp), however, had an increase in MICs of imipenem from 0.25 to 16 mg/liter, of meropenem from 0.03 to 8 mg/liter, and of cefepime from 0.25 to 2 mg/liter (Table 1). Bradford et al. (4) demonstrated for the first time in vitro imipenem resistance in K. pneumoniae strains with the combination of ACT-1 and the loss of an outer membrane protein. Similar results were later reported with other PACBL (11).

Mortality and bacterial counts in the lungs of the animals in the different groups and with the different strains are summarized in Table 3. The efficacy results in rats with pneumonia infected with K. pneumoniae 12 and K. pneumoniae 12dp strains show that antimicrobial activities of imipenem, meropenem, and cefepime seem to be adequate for the treatment of pneumonia caused by AmpC ß-lactamase ACT-1-producing strains with or without porin deficiency, with a reduction of 5 log10 CFU/g and 0% mortality, with no differences between them (P > 0.05). However, for treated animals infected with K. pneumoniae plasmid AmpC without OmpK35 and OmpK36 porins, meropenem seems to be more active than imipenem (P ≤ 0.05). Recently, Pichardo et al. (16, 17) have also demonstrated that carbapenems and cefepime produce similar reductions of the bacterial counts, with imipenem producing the best clearance in an animal model of pulmonary infection with a porin (OmpK35 and OmpK36)-deficient K. pneumoniae strain or with the same strain with a plasmid-mediated AmpC ß-lactamase (FOX-5 or CMY-2). In those studies, only true stable mutants were selected by cefepime treatment. We did not observe this phenomenon, even though it may be also possible that the bacterial density of the lungs in treated animals was too low to detect such subpopulations, as these mutants are reported to occur in 1 of 105 to 108 wild-type strains possessing AmpC ß-lactamases (11).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Bacterial counts in lungs and mortality for bacterial strains studied with different therapeutic groups and placeboa

 
We conclude that imipenem, meropenem, and cefepime decrease bacterial lung counts in K. pneumoniae strains producing ACT-1 with or without the presence of porins, probably due to good antibiotic tissue concentrations (over MIC) by the compounds. However, little difference could be seen regardless of the AmpC-type ß-lactamase strain used for infection, since some antibiotics could be slightly more effective in decreasing lung bacterial burden.


   ACKNOWLEDGMENTS
 
We thank L. Martínez-Martínez (Hospital Marqués de Valdecillas, Spain) for his critical review of the manuscript.

This work was supported by Instituto Carlos III of Spain through grants from Fondo de Investigacion Sanitaria (01/0034-02), Red Española de Investigación en Patología Infecciosa (REIPI, C03/14), and Red Respira (RTIC C03/11).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Hospital Son Dureta, Andrea Doria no. 55, 07014 Palma de Mallorca, Illes Balears, Spain. Phone: 34-71-175238. Fax: 34-71-175185. E-mail: nborrell{at}hsd.es. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Alvarez, M., J. H. Tran, N. Chow, and G. A. Jacoby. 2004. Epidemiology of conjugative plasmid-mediated AmpC ß-lactamases in the United States. Antimicrob. Agents Chemother. 48:533-537.[Abstract/Free Full Text]
  2. Ardanuy, C., J. Liñares, M. A. Domínguez, S. Hernández-Allés, V. J. Benedí, and L. Martínez-Martínez. 1998. Outer membrane profiles of clonally related Klebsiella pneumoniae isolates from clinical samples and activities of cephalosporins and carbapenems. Antimicrob. Agents Chemother. 42:1636-1640.[Abstract/Free Full Text]
  3. Babini, G. S., and D. M. Livermore. 2000. Antimicrobial resistance amongst Klebsiella spp. collected from intensive care units in Southern and Western Europe in 1997-1998. J. Antimicrob. Chemother. 45:183-189.[Abstract/Free Full Text]
  4. Bradford, P. A., C. Urban, N. Mariano, S. J. Projan, J. J. Rahal, and K. Bush. 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. Antimicrob. Agents Chemother. 41:563-569.[Abstract]
  5. Chapin-Robertson, K., and S. Edberg. 1991. Measurement of antibiotics in human body fluids: techniques and significance, p. 295-366. In V. Lorian (ed.), Antibiotics in laboratory medicine. Williams & Wilkins, Baltimore, Md.
  6. Fukasawa, M., Y. Sumita, E. T. Harabe, T. Tanio, H. Nouda, T. Kohzuki, T. Okuda, H. Matsumura, and M. Sunagawa. 1992. Stability of meropenem and effect of 1ß-methyl substitution on its stability in the presence of renal dehydropeptidase I. Antimicrob. Agents Chemother. 36:1577-1579.[Abstract/Free Full Text]
  7. Hanson, N. D. 2003. AmpC beta-lactamases: what do we need to know for the future? J. Antimicrob. Chemother. 52:2-4.[Free Full Text]
  8. Hernández-Allés, S., S. Albertí, D. Álvarez, A. Doménech-Sánchez, L. Martínez-Martínez, J. Gil, J. M. Tomás, and V. J. Benedí. 1999. Porin expression in clinical isolates of Klebsiella pneumoniae. Microbiology 145:673-679.[Abstract]
  9. Lee, J. H., H. I. Jung, J. H. Jung, J. S. Park, J. B. Ahn, S. H. Jeong, B. C. Jeong, J. H. Lee, and S. H. Lee. 2004. Dissemination of transferable AmpC-type beta-lactamase (CMY-10) in Korean hospital. Microb. Drug Resist. 10:224-230.[Medline]
  10. Martínez-Martínez, L., S. Hernández-Allés, S. Albertí, J. M. Tomás, V. J. Benedí, and G. A. Jacoby. 1996. In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum cephalosporins. Antimicrob. Agents Chemother. 40:342-348.[Abstract]
  11. Martínez-Martínez, L., A. Pascual, S. Hernández-Allés, D. Álvarez-Díaz, A. I. Suárez, J. Tran, V. J. Benedí, and G. A. Jacoby. 1999. Roles of ß-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrob. Agents Chemother. 43:1669-1673.[Abstract/Free Full Text]
  12. Mimoz, O., A. Jacolot, S. Leotard, N. Hidri, K. Samii, P. Nordmann, and O. Petitjean. 1998. Efficacies of cefepime, ceftazidime, and imipenem alone or in combination with amikacin in rats with experimental pneumonia due to ceftazidime-susceptible or -resistant Enterobacter cloacae strains. Antimicrob. Agents Chemother. 42:3304-3308.[Abstract/Free Full Text]
  13. Mimoz, O., S. Leotard, A. Jacolot, C. Padoin, K. Louchahi, O. Petitjean, and P. Nordmann. 2000. Efficacies of imipenem, meropenem, cefepime, and ceftazidime in rats with experimental pneumonia due to a carbapenem-hydrolyzing ß-lactamase-producing strain of Enterobacter cloacae. Antimicrob. Agents Chemother. 44:885-890.[Abstract/Free Full Text]
  14. Nassif, X., J.-M. Fournier, J. Arondel, and P. J. Sansonetti. 1989. Mucoid phenotype of Klebsiella pneumoniae is a plasmid-encoded virulence factor. Infect. Immun. 57:546-552.[Abstract/Free Full Text]
  15. Papanicolaou, G. A., A. A. Medeiros, and G. A. Jacoby. 1990. Novel plasmid-mediated ß-lactamase (MIR-1) conferring resistance to oxyimino- and {alpha}-methoxy ß-lactams in clinical isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 34:2200-2209.[Abstract/Free Full Text]
  16. Pichardo, C., M. C. Conejo, M. Bernabéu-Wittel, A. Pascual, M. E. Jiménez-Mejías, M. de Cueto, M. E. Pachón-Ibáñez, I. García, J. Oachón, and L. Martínez-Martínez. 2005. Activity of cefepime and carbapenems in experimental pneumonia caused by porin-deficient Klebsiella pneumoniae producing FOX-5 ß-lactamase. Clin. Microbiol. Infect. 11:31-38.[Medline]
  17. Pichardo, C., J. M. Rodríguez-Martínez, M. E. Pachón-Ibañez, C. Conejo, J. Ibáñez-Martínez, L. Martínez-Martínez, J. Pachón, and A. Pascual. 2005. Efficacy of cefepime and imipenem in experimental murine pneumonia caused by porin-deficient Klebsiella pneumoniae producing CMY-2 ß-lactamase. Antimicrob. Agents Chemother. 49:3311-3316.[Abstract/Free Full Text]
  18. Podschun, R., and U. Ullmann. 1998. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microbiol. Rev. 11:589-603.[Abstract/Free Full Text]
  19. Prophet, E. B., B. Mills, J. B. Arringtonand, and L. H. Sobin (ed.). 1992. Laboratory methods in histotechnology. American Registry of Pathology, Washington, D.C.
  20. Yan, J.-J., W.-C. Ko, Y.-C. Jung, C.-L. Chuang, and J.-J. Wu. 2002. Emergence of Klebsiella pneumoniae isolates producing inducible DHA-1 ß-lactamase in a university hospital in Taiwan. J. Clin. Microbiol. 40:3121-3126.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, June 2006, p. 2258-2260, Vol. 50, No. 6
0066-4804/06/$08.00+0     doi:10.1128/AAC.01513-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Padilla, E.
Right arrow Articles by Borrell, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Padilla, E.
Right arrow Articles by Borrell, N.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Clin. Vaccine Immunol. Clin. Microbiol. Rev.
J. Clin. Microbiol. ALL ASM JOURNALS