1 Prince of Songkla University, Department of Clinical Pharmacy, Songkhla, Thailand; 2 University of Houston, Department of Clinical Sciences and Administration, Houston, TX; 3 Rush University, College of Medicine, Chicago, IL; 4 University of Illinois at Chicago, Microbiology Research Laboratory, Department of Pharmacy Practice, 833 South Wood Street, Chicago, IL, USA
Keywords: ESBL, Klebsiella pneumoniae, in vitro, combination activity
Sir,
The carbapenems are generally regarded as the drugs of choice for serious infections caused by extended-spectrum ß-lactamase (ESBL)-producing organisms. These agents have demonstrated excellent in vitro activity as well as favourable clinical outcomes. In clinical practice, combination therapy is often utilized despite minimal in vitro and in vivo data. The purpose of this study was to examine the activity of imipenem alone and in combination with amikacin or ciprofloxacin against ESBL-producing strains of K. pneumoniae.
Four clinical isolates of ESBL-producing K. pneumoniae were obtained from the University of Illinois at Chicago Medical Center (Chicago, IL, USA). To determine bacterial relatedness, chromosomal DNA for pulsed-field gel electrophoresis (PFGE) was prepared as described by Murray et al.1 Restriction endonuclease digestion by XbaI was performed at 37°C for 18 h. PFGE was performed using a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA, USA) as described previously.2 Ethidium bromide-stained agar gels were examined visually, with strains defined as different if PFGE patterns differed in more than six chromosomal restriction fragment positions. PFGE typing revealed that the strains were unrelated.
Confirmation of ESBL production was determined by a 5 mm increase in zone diameter with discs containing ceftazidime/clavulanate and ceftriaxone/clavulanate versus ceftazidime and ceftriaxone, respectively (Becton Dickinson, Cockeysville, MD, USA). Isoelectric focusing (IEF) was performed by the method of Matthew et al.3 using an Isobox (Hoefer Scientific Instruments, San Francisco, CA, USA) with pre-made (pH range 3.59.5) Ampholine PAGplates (Amersham Pharmacia Biotech USA, Piscataway, NJ, USA). Gels were stained for ß-lactamase activity with nitrocefin (Becton Dickinson). IEF revealed a band in all isolates that co-migrated with a known TEM-10 control. Three isolates (strains 3, 4 and 9) also contained an SHV ß-lactamase.
For MIC and timekill studies, the organisms were incubated in broth overnight at 35°C to obtain log-phase growth. The organisms were diluted with saline until the turbidity matched a 0.5 McFarland standard using a spectrophotometer at 625 nm. Each suspension was further diluted in MuellerHinton broth (Difco, Detroit, MI, USA) supplemented with 22.5 mg/L calcium and 11.25 mg/L magnesium to obtain a final inoculum of 5 x 105 cfu/mL.
Imipenem, amikacin and ciprofloxacin (United States Pharmacopeia, Rockville, MD, USA) were prepared according to the NCCLS guidelines.4 MICs were determined in duplicate using the microdilution broth method.4 Microtitre plates were incubated at 35°C and read at 1620 h. All isolates were susceptible to imipenem and amikacin. One isolate (strain 4) was susceptible to ciprofloxacin, whereas three isolates were only intermediately susceptible.
The bactericidal activity of the agents was determined in duplicate using the timekill method.5 The concentration of each antibiotic was 2 x MIC. The final inoculum was confirmed at time 0; subsequent viable counts were performed at 1, 1.5, 2, 6, 12 and 24 h. Dilutions were utilized to minimize antibiotic carryover. Samples were plated on blood agar plates and colonies were counted after 24 h incubation at 35°C. The lower limit of detection was 1.3 log cfu/mL. Bactericidal activity was defined as a 3 log decrease in cfu/mL. Synergy was defined as a
2 log decrease in cfu/mL demonstrated with the combination of agents versus the single most active agent. Additivity was defined as a
1 log decrease with the combination compared with the single most active agent. Antagonism was defined as
2 log increase when in combination as opposed to the single most active agent.
The bactericidal activities of the agents are shown in Figure 1. Imipenem demonstrated bactericidal activity against all isolates at 6 h. However, re-growth was observed at 12 and 24 h in two isolates and one isolate, respectively. MICs for colonies that re-grew at 24 h did not differ significantly (defined as a >2-fold increase in MIC) from initial MICs. Therefore, re-growth was likely to be due to inactivation of the antibiotic and not to the development of resistance. The clinical relevance of re-growth at 1224 h is unknown, since the dosing interval of imipenem is every 68 h. Re-growth did not occur with the other agents.
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Limited data are available on combination activity of imipenem and aminoglycosides or fluoroquinolones against ESBL-producing organisms. Paterson et al.6 performed an Etest synergy study with 16 blood isolates of ESBL-producing K. pneumoniae. Similar to our results, synergy and antagonism were not demonstrated when imipenem was combined with either amikacin or ciprofloxacin. Additive effects were reported with two and three isolates when imipenem was combined with amikacin and ciprofloxacin, respectively. The use of Etest strips for synergy testing has not been validated, making it difficult to draw firm conclusions from their results.
Our in vitro results are in agreement with clinical data that have demonstrated imipenem to be effective as single treatment in most infections. A broader selection of ESBL-producing organisms should be studied to determine whether combination therapy may present theoretical advantages in the treatment of serious infections in critically ill patients.
Acknowledgements
The authors acknowledge the technical assistance of Jennifer L. Prause and Swathi Kelkar. This work was presented in part as a poster at the Annual Meeting of the American College of Clinical Pharmacy, Los Angeles, CA, USA, 2000.
Footnotes
* Corresponding author. Tel: +1-312-996-8639; Fax: +1-312-413-1797; E-mail: pendland{at}uic.edu
References
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3 . Matthew, M., Harris, A. M., Marshall, M. J. & Ross, G. W. (1975). The use of isoelectric focusing for detection and identification of beta-lactamases. Journal of General Microbiology 88, 16978.[ISI][Medline]
4 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
5 . National Committee for Clinical Laboratory Standards. (1999). Methods for Determining Bactericidal Activity of Antimicrobial AgentsApproved Guideline M26-A. NCCLS, Wayne, PA, USA.
6 . Paterson, D. L., Bolmstrom, A., Karlsson, A. & Goransson, E. (1999). Activity of antibiotic combinations against extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, 1999. Abstract 2338, p. 291. American Society for Microbiology, Washington, DC, USA.