a Department of Pharmacy Practice and b Department of Internal Medicine, University of Nebraska Medical Center, 986045 Nebraska Medical Center, Omaha, NE 68198-6045, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Susceptibilities and timekill characteristics were evaluated for cefepime, imipenemcilastatin, meropenem and piperacillintazobactam. MICs were determined in cation-supplemented MuellerHinton broth (MHB) by broth microdilution,6 and verified in triplicate. Timekill studies were determined at 1x, 2x, 4x and 6x MIC in 10 mL MHB using bacteria in log-phase growth (6 x 105 cfu/mL). Bacterial density was assessed at specified time-points, samples were serially diluted and 100 µL aliquots were plated and incubated at 37°C for 24 h. Viable colonies were counted at a lower limit of detection of 102 cfu/mL.
Further MIC and timekill experiments were performed to evaluate regrowth observed in initial timekill experiments with cefepime and piperacillintazobactam. MIC values and bactericidal activity were assessed at time zero under the following conditions: (i) MHB containing antimicrobial (either piperacillintazobactam or cefepime at a known concentration); (ii) MHB containing antimicrobial and C600N; and (iii) MHB containing antimicrobial and C600N/SHV-5, C600N/TEM-10 or C600N/TEM-26. After 24 h incubation, bacteria were removed by 0.22 µm filtration, and C600N was added before 24 h MIC and bacterial density assessment. MIC values at baseline and 24 h were compared, to determine if antimicrobial concentration and activity were decreased in the presence of bacteria expressing an ESBL.
Statistical analysis
Graphical representation and statistical analysis were performed with GraphPad Prism (GraphPad Software, Inc, San Diego, CA, USA). A P value of <0.05 was considered significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bactericidal characteristics are shown at 4 x MIC (Figure 1). Mean time to
99.9% kill was 3.0, 1.5 and 4.5 h for antimicrobials tested against SHV-5, TEM-10 and TEM-26 transconjugants, respectively. Imipenemcilastatin and meropenem exhibited rapid and sustained bactericidal effect. Cefepime and piperacillintazobactam were initially bactericidal against C600N/TEM-10 and C600N/TEM-26. However, increasing bacterial counts after 6 h resulted in mean log10 cfu/mL increases of 2.75 and 1.72 for cefepime and piperacillintazobactam, respectively, at 24 h. Further study of bacterial regrowth demonstrated no increase in MIC under any experimental conditions comparing 0 h and 24 h incubations. The C600N/SHV-5 transconjugant did not survive at detectable concentrations at 24 h with any ß-lactam agent tested.
|
TEM-26 were recovered at 24 h in tests with imipenem cilastatin at the lower MICs studied. Meropenem was bactericidal for all transconjugants and growth was undetectable after 3 h at all concentrations.
Figure 2 represents the activity of cefepime and piperacillintazobactam at achievable serum concentrations against C600N/SHV-5, C600N/TEM-10 and C600N/ TEM-26. Cefepime and piperacillintazobactam exhibited differing bactericidal effects dependent upon the specific ESBL expressed. C600N/SHV-5 was completely killed by cefepime within 3 h, whereas piperacillintazobactam did not suppress C600N/SHV-5 bacterial growth at 24 h incubation. Bactericidal activity of imipenemcilastatin and meropenem was uninfluenced by the type of ESBL expressed (data not shown), as growth of all isolates was inhibited by 3 h and subsequently remained undetectable for 24 h.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous authors89 have reported that ß-lactam activity is related to the type and amount of specific ESBL enzyme produced. Our results support others suggesting that microbes expressing TEM-derived enzymes remain susceptible to piperacillintazobactam (MIC 16 mg/L) compared with organisms expressing SHV-5 (MIC 3264 mg/L).9 While others have reported cefepime MIC values at least two-fold lower for TEMthan for SHV-derived ESBLs,8 our investigation suggests the opposite (two-fold higher MICs for TEMcompared with SHV-derived ESBLs). Carbapenems are less influenced by type and amount of specific ESBL produced.8
We attempted to characterize better the regrowth phenomenon observed at 24 h when transconjugates were exposed to cefepime or piperacillintazobactam. Possible explanations included antimicrobial inactivation or resistant sub-population growth. Sub-populations of isolates that regrew at 24 h had MIC values identical to those of the original SHVor TEM-derived transconjugants. The clinical relevance of this regrowth is not known, as these drugs are administered at dosing intervals more frequent than every 24 h. Antimicrobial concentrations tested represent standard concentrations attained in humans during typical dosing regimens.
Limitations of in vitro susceptibility testing may influence the predictive value of clinical efficacy. Important considerations include high plasmid copy number,8 promoter efficiency,8 hyperproduction of ß-lactamases, decreased permeability and porin deficiencies.10 In creating an isogenic panel, we characterized the in vitro activity of a single enzyme (SHV-5, TEM-10 or TEM-26) against targeted antimicrobials so results would not be misrepresented by other possible resistance factors. Clinically, ESBLs are primarily expressed in Klebsiella pneumoniae which exhibit variable in vitro susceptibilities dependent upon the type of ESBL produced.
In conclusion, carbapenems retain full in vitro activity against ESBL-producing bacteria. Based on institution-specific susceptibilities, piperacillintazobactam may be a suitable alternative agent for TEM-derived enzymes, whereas cefepime represents a potential option for SHV-derived enzymes.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Fey, P. D., Moland, E. S., Iwen, P. C., Hinrichs, S. H. & Rupp, M. E. (1998). Outbreak of Klebsiella pneumoniae producing an extended-spectrum ß-lactamase in pediatric liver transplant patients. In Program and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 1998. Abstract K127, p. 538. American Society for Microbiology, Washington, DC.
3 . Quinn, J. P., Miyashiro, D., Sahm, D., Flamm, R. & Bush, K. (1989). Novel plasmid-mediated-ß-lactamase (TEM-10) conferring selective resistance to ceftazidime and aztreonam in clinical isolates of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 33, 14516.[ISI][Medline]
4 . Naumovski, L., Quinn, J. P., Miyashiro, D., Patel, M., Bush, K., Singer, S. B. et al. (1992). Outbreak of ceftazidime resistance due to a novel extended-spectrum-ß-lactamase in isolates from cancer patients. Antimicrobial Agents and Chemotherapy 36, 19916.[Abstract]
5 . Rice, L. B., Marshall, S. H. & Carias, L. L. (1992). Tn5381, a conjugative transposon identifiable as a circular form in Enterococcus faecalis. Journal of Bacteriology 174, 730815.[Abstract]
6 . National Committee for Clinical Laboratory Standards. (1997). Performance Standards for Antimicrobial Susceptibility TestingEighth Informational Supplement: Approved Standard M7-A4. NCCLS, Wayne, PA.
7 . Gautom, R. K. (1997). Rapid pulsed-field gel electrophoresis protocol for typing of E. coli 0157:H7 and other gram-negative organisms in 1 day. Journal of Clinical Microbiology 35, 297780.[Abstract]
8 . Jacoby, G. A. & Carreras, I. (1990). Activities of ß-lactam antibiotics against Escherichia coli strains producing extended-spectrum ß-lactamases. Antimicrobial Agents and Chemotherapy 34, 85862.[ISI][Medline]
9 . Pagani, L., Migliavacca, R., Luzzaro, F., Giacobone, E., Perilli, M., Micheletti, P. et al. (1998). Comparative activity of piperacillin/ tazobactam against clinical isolates of extended-spectrum ß-lactamase-producing Enterobacteriaceae. Chemotherapy 44, 37784.[ISI][Medline]
10
.
Ardanuy, C., Liñares, J., Domínguez, M. A., Hernández-Allés, S., Benedí, V. J. & Martínez-Martínez, L. (1998). Outer membrane profiles of clonally related Klebsiella pneumoniae isolates from clinical samples and activities of cephalosporins and carbapenems. Antimicrobial Agents and Chemotherapy 42, 163640.
Received 10 January 2000; returned 22 March 2000; revised 2 May 2000; accepted 8 May 2000