Original Article. Ratri Hortiwakul, M.Sc.*, Pantip Chayakul, M.D.*, Natnicha Ingviya, B.Sc.**

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1 Original Article In Vitro Activity of Cefminox and Other β-lactam Antibiotics Against Clinical Isolates of Extended- Spectrum-β-lactamase-Producing Klebsiella pneumoniae and Escherichia coli Ratri Hortiwakul, M.Sc.*, Pantip Chayakul, M.D.*, Natnicha Ingviya, B.Sc.** ABSTRACT This study aimed to determine in vitro activity of cefminox (CMN), cefepime (CFP), imipenem (IMP), meropenem (MER), piperacillin/tazobactam (PTZ), and cefoperazone/sulbactam (CPS) against clinical isolates of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella pneumoniae and Escherichia coli, obtained from hospitalized patients in Songklanagarind Hospital, Songkhla, Thailand, from The clinical isolates of K. pneumoniae and E. coli, 100 isolates of each, were processed according to the standard microbiological procedures, and the combination-disk method was applied to detect the production of ESBL. The minimal inhibitory concentrations (MIC) of CFP, IMP, MER, PTZ, and CPS were determined by the agars dilution technique, and CMN by the disk-diffusion method. The percentage of susceptibility of CMN against ESBL-producing K. pneumoniae and E. coli was 95 percent and 87 percent respectively. The percentage of susceptibility of other β-lactam antibiotics varied between 71 percent and 100 percent, with both IMP and MER showing 100 percent susceptibility. Cetepime had 71 percent and 72 percent susceptibility against K. pneumoniae and E. coli, respectively. In conclusion, IMP, and MER showed the most potent activity against both ESBL-producing K. pneumoniae and E. coli while cefepime was the least active antibiotic. CMN, PTZ, and CPS had a good activity against these bacterial isolates. (J Infect Dis Antimicrob Agents 2006;23:9-14.) INTRODUCTION An expression of plasmid-encoded extendedspectrum-β-lactamases (ESBLs) can be widespread in Gram-negative bacteria, especially in Klebsiella spp. and Escherichia coli. 1 These enzymes confer resistance to both monobactams and oxyiminocephalosporins such *Infectious Disease Unit, Department of Medicine, Faculty of Medicine, Prince of Songkla University, Songkhla 90110, Thailand. **Department of Pathology, Faculty of Medicine, Prince of Songkla University, Songkhla 90110, Thailand. Received for publication: September 5, Reprint request: Ratri Hortiwakul, M.Sc., Infectious Disease Unit, Department of Medicine, Faculty of Medicine, Prince of Songkla University, Songkhla 90110, Thailand. Keywords: Extended-spectrum β-lactamase, ESBL-producing Klebsiella pneumoniae, ESBL-producing Escherichia coli, cefminox, cefepime, imipenem, meropenem, piperacillin/tazobactam, cefoperazone/sulbactam 9

2 10 J INFECT DIS ANTIMICROB AGENTS Jan.-Apr as aztreonem, ceftazidime, ceftriaxone, and cefotaxime. The susceptibility pattern of clinically isolated Gramnegative bacteria in each hospital may be changing over time period, and a co-resistance to other antimicrobial agents often occurred in ceftazidime-resistant Klebsiella isolates. 2,3 Among the cephamycins, the cefoxitin has an in vitro activity against ESBL-producing K. pneumoniae and E. coli, with its MIC clustering around the minimal inhibitory concentration (MIC) breakpoint. However, it is not recommended for clinical use because of a lack of clinical trials. In addition, most ESBLproducing strains also have another mechanism of antibiotic resistance including a decrease of absence of expression of porins. 4 Porins are outer-membrane proteins (OMPs) that play role in the diffusion of a variety of molecules, including bacterial nutrients and antimicrobial agents. Cefminox is a β-lactamase-stable cephamycin which was reported to be active over other cephamycins against β-lactamase-producing bacteria. 5 Thus, cefminox may be useful for the treatment of infections caused by ESBL-producing K. pneumoniae and E. coli. This study aimed to determine the susceptibility of cefminox, cefepime, imipenem, meropenem, piperacillin/tazobactam, cefoperazone/sulbactam against ESBL-producing K. pneumoniae and E. coli isolates obtained from hospitalized patients in Songklanagarind Hospital, Songkhla, Thailand from 2003 to MATERIALS AND METHODS Bacteria All clinical isolates of K. pneumoniae and E. coli were obtained from 2003 to 2004, identified by the standard procedures 6, and kept frozen in glycerol-tryptic soy broth at -80 C until use. ESBL detection test K. pneumoniae and E. coli were tested for ESBL production by the combination-disk method. 7 Briefly, the bacterial inoculum were initially applied to the Mueller-Hinton agar as the standard disk-diffusion test. Six antimicrobial disks containing cefotaxime (30 µg), cefotaxime/clavulanic acid (30 µg/10 µg), ceftazidime (30 µg), ceftazidime/clavulanic acid (30 µg/ 10 µg), cefpodoxime (10 µg), and cefpodoxime/ clavulanic acid (10 µg/1 µg) were used in our study. The ESBL production was determined by a more-than-5 mm increase in an inhibition-zone diameter of each antimicrobial disk tested in combination with clavulanic acid, compared with the zone of that antimicrobial disk tested without clavulanic acid. K. pneumoniae ATCC and E. coli ATCC were used for a quality control. Antimicrobial agents Since the standard powder of cefminox was not available, the cefminox disk from Meiji Seika Pharma, Tokyo, Japan was used. The powder of other antimicrobial agents with known potencies were obtained from each pharmaceutical company in parenthesis as follows: cefepime (Bristol-Myers Squibb), imipenem (MSD), meropenem (AstraZeneca), piperacillin/tazobactam (Wyeth), and cefoperazone/ sulbactam (Pfizer). Susceptibility testing Cefminox susceptibility testing was performed by the disk-diffusion method. Cefepime, imipenem, meropenem, piperacillin/tazobactam, and cefoperazone/ sulbactam were tested for the MIC by the agar-dilution method as described by the National Committee for Clinical Laboratory Standards (NCCLS). 8 A quality control was performed by testing E. coli ATCC The MICs of each drug were reported as an MIC range, MIC 50, and MIC 90. The MIC 50 and MIC 90 were expressed as the nearest log 2 concentration of antibiotic

3 Vol. 23 No. 1 In vitro activity of β-lactam antibiotics against ESBL-producing K. pneumoniae and E. coli:- Hortiwakul R, et al. 11 that inhibits 50 percent and 90 percent of the strains. 9 RESULTS The clinical isolates of K. pneumoniae were obtained from both in sterile and non-sterile sites. The sterile site included 29 blood, 31 body fluid, 3 tissue, and 4 cerebrospiral fluid (CSF) specimens. The non-sterile sites included 10 pulurent discharge, 14 urine, and 9 sputum specimens, the clinical isolates of E. coli included 31 blood, 31 body fluid, 15 tissue, 1 CSF, 10 purulent discharge, 8 urine, and 4 sputum specimens. All tested isolates were ESBL positive. The susceptibility of cefminox and other β-lactam antibiotics are shown in Table 1 and 2, respectively. Cefminox Table 1. The results of cefminox disk susceptibility determination for ESBL-producing K. pneumoniae and E. coli. Species (No. of isolates) Inhibition zone (mm) (mean + S.D.) % susceptibility K. pneumoniae (100) Susceptible Intermediate Resistant E. coli (100) Susceptible Intermediate Resistant Zone size: susceptible + 20 mm, intermediate mm, resistant < 15 mm. Table 2. Activities of six antimicrobial agents clinical isolates of ESBL-producing K. pneumoniae and E. coli. Antimicrobial agents Range MIC 50 MIC 90 % susceptibility* ESBL-producing K. pneumoniae Cefminox Cefepime Imipenem Meropenem Cefoperazone/sulbactam Piperacillin/tazobactam ESBL-producing E. coli Cefminox Cefepime Imipenem Meropenem Cefoperazone/sulbactam Piperacillin/tazobactam *Susceptibility breakpoint defined by the NCCLS: cefepime MIC < 8 mg/l, imipenem and meropenem < 4 mg/l, and cefoperazone/ sulbactam and piperacillin/tazobactam MIC < 16 mg/l.

4 12 J INFECT DIS ANTIMICROB AGENTS Jan.-Apr was active against both ESBL-producing K. pneumoniae and E. coli, with 95 percent and 87 percent susceptibility, respectively. The cefepime MIC 50 for both K. pneumoniae and E. coli were 4 mg/l, while the MIC 90 were 16 and 32 mg/l, respectively. Both carbapenems, imipenem and meropenem, had 100 percent susceptibility, with the MIC 90 of 2 and 0.12 mg/ L against ESBL-producing K. pneumoniae, and the MIC90 of 0.5 and 0.12 mg/l against ESBL-producing E. coli, respectively. Among β-lactam/β-lactamas inhibitor antibiotics, the cefoperazone/sulbactam MIC 50 and MIC 90 were 8 and 32 mg/l, and the piperacillin/ tazobactam MIC 50 and MIC 90 were 6 and 32 mg/l, respectively against ESBL-producing K. pneumoniae. The cefoperazone/sulbactam MIC 50 and MIC were 2 90 and 16 mg/l, and the piperacillin/tazobactam MIC 50 and MIC 90 were 8 and 16 mg/l, respectively against ESBLproducing E. coli. Table 3. shows the percent susceptibility of all tested drugs against the bacteria obtained from sterile and non-sterile sites. The susceptibility of cefminox against ESBL-producing E. coli from the non-sterile sites was significantly better than that from the sterile sites, while both cefoperazone/sulbactam and piperacillin/ tazobactam had higher susceptibility in the sterile sites than those from the non-sterile sites. ESBL-producing K. pneumoniae showed no differences in the susceptibility patterns of all tested drugs. DISCUSSION Cefminox is a new bactericidal cephamycin with antibacterial activity against aerobic and anaerobic bacteria. It is as stable as most other cephamycins, regarding the action of the β-lactamases produced by K. pneumoniae, E. coli, and other bacteria. Watanabe and Omoto (1990) had reported the high-potent activity of cefminox against Gram-negative bacteria and anaerobes, but the weak activity against Staphylococcus aureus and Pseudomonas aeruginosa. 5 The results of our study also showed the potent in vitro activity of cefminox against ESBL- producing K. pneumoniae and E. coli, with 95 and 87 percent susceptibility, respectively. However, Anathan and colleagues reported the cephamycin (cefoxitin and Table 3 Percentage of susceptibility of ESBL-producing K. pneumoniae and E. coli from the sterile and non-sterile sites against various antimicrobial agents. Antimicrobial agents K. pneumoniae* (n=100) E. coli* (n=100) Sterile site (67) Non-sterile site (33) Sterile site (78) Non-sterile site (22) Cefminox Cefepime Imipenem Meropenem Cefoperazone/sulbactam Piperacillin/tazobactam * ESBL-producing K. pneumoniae Sterile sites: blood (29), body fluid (31), tissue (3), cerebrospinal fluid (4). Non-sterile sites: pus (10), urine (14), sputum (9). * * ESBL-producing E. coli Sterile sites: blood (31), body fluid (31), tissue (15), cerebrospinal fluid (1). Non-sterile sites: pus (10), urine (8), sputum (4).

5 Vol. 23 No. 1 In vitro activity of β-lactam antibiotics against ESBL-producing K. pneumoniae and E. coli:- Hortiwakul R, et al. 13 cefotetan) resistance in ESBL positive and negative isolates of K. pneumoniae and E. coli was due to a loss of an outer membrane protein (porin). 10 A recent study has shown that an inoculum size of bacteria does not effect on a bacteriolytic activity of cefminox and cefoxitin, in contrast to that of imipenem, cefotaxime, and ceftizoxime. 11 In our study, cefepime has a moderate in vitro activity against both K. pneumoniae and E. coli, with 71 and 72 percent susceptibility. Several previous in vitro studies reported a moderate activity of cefepime against ESBL-producing E. coli and K. pneumoniae, with a range of 61 and 100 percent. 12 In 1998, Biedenbach and the Thailand Antimicrobial Resistance Study Group reported 100 percent susceptibility of cefepime against E. coli and K. pneumoniae. 13 However, a debate continues regarding the clinical utility of cefepime for the treatment of infections caused by ESBL-producing strains. 14 Among the β-lactam/β-lactamase inhibitors antibiotics, our study showed that both cefoperazone/sulbactam and piperacillin/tazobactam were active against ESBLproducing K. pneumoniae and E. coli. An in vitro activity of a β-lactam/β-lactamase inhibitor antibiotics against ESBL-producing bacterial strains varied, depending on the subtypes of ESBL production. It revealed that the TEM and SHV subtypes of ESBLs were more susceptible to β-lactam/β-lactamase inhibitor antibiotics than the other subtypes. 15 In 1998, Pfaller and colleagues reported the susceptibility of piperacillin/ tazobactam against ESBL-producing E. coli and Klebsiella were 86 percent and 72 percent, respectively. 16 In our study, it had 92 percent and 86 percent susceptibility, respectively against ESBL-producing E. coli and K. pneumoniae. A multi-center study in Japan in 1999 reported the various susceptibility of cefoperazone/sulbactam against the clinical isolates of K. pneumoniae and E. coli, ranging from 70 to 100 percent, but only 3-9 percent of the isolates were ESBL positive. 17 In our study, cefoperazone/sulbactam had 90 percent and 95 percent susceptibility against ESBLproducing K. pneumoniae and E. coli. In conclusion, imipenem and meropenem were still the most active against ESBL- producing K. pneumoniae and E. coli, followed by cefminox and β-lactam-β-lactamase inhibitor, antibiotics. ACKNOWLEDGEMENT This project is supported by research grants from the Faculty of Medicine, Prince of Songkla University, Hat Yai,Songkhla, Thailand. References 1. Rice L. Evolution and clinical importance of extendedspectrum β-lactamases. Chest 2001;119 (2 Suppl): 391S-396S. 2. Jacoby GA. Genetics of extended-spectrum β-lactamases. Eur J Clin Microbiol Infect Dis 1994;13 Suppl 1:S Jacoby GA. Extended-spectrum β-lactamases and other enzymes providing resistance to oxyimino-β-lactams. Infect Dis Clin North Am 1997;11: Pangon B, Bizet C, Bure A, et al. In vivo selection of a cephamycin-resistant, porin-deficient mutant of Klebsiella pneumoniae producing a TEM-3 β-lactamase. J Infect Dis 1989;159: Watanabe S, Omoto S. Pharmacolgy of cefminox, a new bactericidal cephamycin. Drugs Exp Clin Res 1990;16: Farmer JJ. Enterobacteriaceae: introduction and identification. In: Murray PR, ed. Manual of clinical microbiology, 6 th ed. Washington DC: American Society for Microbiology, 1995: National Committee for Clinical Laboratory Standards (NCCLS). Performance standards for antimicrobial susceptibility testing. 12 th informational supplement (M-100-S12). Wayne, Pa: NCCLS, National Committee for Clinical Laboratory Standards

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