Monte Carlo simulation for evaluation of the efficacy of carbapenems and new quinolones against ESBL-producing Escherichia coli.

Extended-spectrum beta-lactamase (ESBL)-producing bacteria are known to be resistant to penicillins, cephalosporins, and monobactams because of their substrate specificity, and these bacteria are sensitive only to a narrow range of antimicrobial agents. The present study was undertaken to evaluate the efficacy of carbapenems and the new quinolones against ESBL-producing Escherichia coli, using a Monte Carlo simulation based on the pharmacokinetic/pharmacodynamic (PK/PD) theory. The time above MIC (TAM, %) served as the PK/PD parameter for carbapenems, with the target level set at 40%. The AUC/MIC served as the PK/PD parameter for the new quinolones, with the target level set at more than 125. In the analysis of drug sensitivity, the MIC50 of all carbapenems other than imipenem was low (0.03 microg/ml), while the MIC50 of the new quinolones was higher (1-2 microg/ml). The probability of achieving the PK/PD target with carba penems after two doses at the usual dose level, as determined by the Monte Carlo simulation, was high for each of the carbapenems tested (99.0% for biapenem, 99.60% for meropenem, and 95.03% for doripenem), except for imipenem. Among the new quinolones, the highest probability of achieving the PK/PD target was obtained with pazufloxacin (42.90%). Thus, the results of the present study have revealed that carbapenems are effective at the regular dose and can be used as the first-choice antibiotics for ESBL-producing E. coli because the resistance ratios for carbapenems are low compared to those of the new quinolones.

[Extended-spectrum-beta-lactamase-producing Proteus mirabilis: laboratory-based surveillance in cooperation with 12 clinical laboratories in the Kinki Region of Japan].

We studied 247 strains of Proteus mirabilis collected during the 6 months from November 2003 to April 2004 from 12 clinical laboratories in the Kinki region of Japan for the production of extended-spectrum beta-lactamase (ESBL). Eighteen strains (7.3%) showed MICs for cefpodoxime of > or = 2 microg/mL and 13 strains (5.2%) were positive for the double-disk synergy test. Susceptibility depended on genotype. MICs for cefepime, cefozopran, and cefpirome were high (> or = 8 microg/mL), and that for ceftazidime was low (0.12-0.5 microg/mL). Meropenem showed the lowest MIC (< or = 0.03-0.25 microg/mL) of the calbapenems, while other calbapenems showed somewhat higher values (0.5-2 microg/mL). The MIC of tazobactam/piperacillin was also relatively low (< or = 0.25-1 microg/mL). Analysis of the ESBL genotype by the polymerase chain reaction showed that 12 of 13 strains were CTX-M2 types. CTX-M9 was detected in a single laboratory. The clinical background showed 5 strains in urine samples. Twelve of 13 strains were detected in patients with minimal devices use. No symptoms were found in most cases of established syndrome. Analysis of PCR fingerprint profiles of random amplified polymorphic DNA patterns showed that 6 of 7 strains from hospital 1 showed the same pattern, and 5 of 5 strains from hospital 13 showed the same pattern, suggesting the nosocomial spread of P. mirabilis in each hospital.

Metallo-beta-lactamase-producing gram-negative bacilli: laboratory-based surveillance in cooperation with 13 clinical laboratories in the Kinki region of Japan.

A total of 19,753 strains of gram-negative rods collected during two 6-month periods (October 2000 to March 2001 and November 2001 to April 2002) from 13 clinical laboratories in the Kinki region of Japan were investigated for the production of metallo-beta-lactamases (MBLs). MBLs were detected in 96 (0.5%) of the 19,753 isolates by the broth microdilution method, the 2-mercaptopropionic acid inhibition test, and PCR and DNA sequencing analyses. MBL-positive isolates were detected in 9 of 13 laboratories, with the rate of detection ranging between 0 and 2.6% for each laboratory. Forty-four of 1,429 (3.1%) Serratia marcescens, 22 of 6,198 (0.4%) Pseudomonas aeruginosa, 21 of 1,108 (1.9%) Acinetobacter spp., 4 of 544 (0.7%) Citrobacter freundii, 3 of 127 (2.4%) Providencia rettgeri, 1 of 434 (0.2%) Morganella morganii, and 1 of 1,483 (0.1%) Enterobacter cloacae isolates were positive for MBLs. Of these 96 MBL-positive strains, 87 (90.6%), 7 (7.3%), and 2 (2.1%) isolates carried the genes for IMP-1-group MBLs, IMP-2-group MBLs, and VIM-2-group MBLs, respectively. The class 1 integrase gene, intI1, was detected in all MBL-positive strains, and the aac (6')-Ib gene was detected in 37 (38.5%) isolates. Strains with identical PCR fingerprint profiles in a random amplified polymorphic DNA pattern analysis were isolated successively from five separate hospitals, suggesting the nosocomial spread of the organism in each hospital. In conclusion, many species of MBL-positive gram-negative rods are distributed widely in different hospitals in the Kinki region of Japan. The present findings should be considered during the development of policies and strategies to prevent the emergence and further spread of MBL-producing bacteria.

Evaluation of MicroScan ESBL confirmation panel for Enterobacteriaceae-producing, extended-spectrum beta-lactamases isolated in Japan.

We assessed use of the MicroScan ESBL confirmation panel (Dade Behring, Tokyo, Japan) for the detection of eight Enterobacteriaceae-producing extended-spectrum beta-lactamases (ESBL) species. Of 137 bacterial strains isolated from patients in 32 hospitals in the Kinki area of Japan, 91 produced ESBL and comprised 60 bacteria (of E. coli, K. oxytoca, and K. pneumoniae) targeted by the NCCLS ESBL test and 31 non-target bacteria such as chromosomal AmpC-producing bacteria (e.g., Serratia marcescens, Enterobacter spp.). Sensitivity and specificity of the MicroScan panel for the target bacteria were 92% and 93%, respectively; sensitivity and specificity for non-target bacteria were 52% and 100%, respectively. There were 20 ESBL-positive strains that were not inhibited by clavulanic acid in the MicroScan panel (3 of 32 ESBL-producing E. coli strains, 1 of 24 K. pneumoniae, 1 of 4 K. oxytoca, 8 of 13 E. cloacae, and 7 of 14 S. marcescens), and most of them were bacteria not targeted by the NCCLS test. In 19 of the 20 strains, the synergy effect of clavulanic acid was observed in the modified-double-disk synergy test using only the cefepime-disk. Because these strains had high MICs of > or = 16 microg/ml for cephamycins such as cefoxitin and cefmetazole, these strains might produce high levels of AmpC in addition to ESBL. The MicroScan ESBL confirmation panel showed excellent performance in detecting target, but not other bacteria. Addition of cefepime and clavulanic acid to the MicroScan panel may significantly improve detection of non-target bacteria.

[Application of the MIC breakpoints based on pharmacokinetics and pharmacodynamics parameter in the clinical laboratory].

The effectiveness of time-dependent antibiotics such as beta-lactams is related to the time above the MIC (TAM, %). We constructed a program to calculate the TAMs of beta-lactams using the pharmacokinetic parameters of the Japanese dosing regimen of a phase I study of the Japanese Society for Antimicrobial Chemotherapy (JSAC), and compared them with the MIC breakpoints published by the National Committee for Clinical Laboratory Standards (NCCLS) and JSAC. If the effective TAM was assumed to be more than 40% of the dosing interval, the pharmacokinetic/pharmacodynamic (PK/PD) breakpoints calculated by our program were in agreement with the JSAC breakpoints for pneumonia within 1 dilution MIC. When comparing with the NCCLS breakpoints for Enterobacteriaceae or Staphylococcus, the PK/PD breakpoints dosing three times per day of ampicillin (1 g, intravenous dose; i.v.), piperacillin (2 g, i.v.), cefotaxime (1 g, i.v.) and cefmetazole (1 g, i.v.) were calculated to be less than 2-fold dilution MIC, and those of amoxicillin (0.25 g, oral dose; p.o.) and cefaclor (0.5 g, p.o.) were calculated to be less than 3- to 4-fold dilution of MIC. Our program could calculate TAMs and PK/PD breakpoints by inputting the two factors of MIC and dosing interval. If this information is routinely reported to physicians from clinical laboratories, an appropriate dosing schedule could be proposed for various infectious cases.

[In vitro activity of a new semisynthetic echinocandin, micafungin, against clinical isolates of Candida species isolated in Tenri Hospital].

We have examined the antifungal activities of the available antifungal agents including micafungin (MCFG), one of the echinocandin antifungal group, against 92 yeast-like fungi isolated at our hospital during a 3-month period from November 2002 to February 2003. Determination of the antifungal susceptibility was conducted in conformity with the Standards of the Japanese Society for Medical Mycology. The MIC 80% of the antifungal agents against 4 fungi species including C. albicans (55 strains), C. tropicalis (20 strains), C. glabrata (8 strains), C. krusei (5 strains) were as follows; MCFG: 0.03-0.125 microgram/ml, amphotericin-B: 0.125-0.25 microgram/ml, 5-fluorocytosine: 0.125-16 micrograms/ml, itraconazole: 0.25-2 micrograms/ml, fluconazole: 0.5-32 micrograms/ml. The isolation rate of the drug-resistant fungi was 20% for the fluconazole (FLCZ)-resistant C. tropicalis and 33% when including the susceptible dose dependent (S-DD) class. The rate was 5% for FLCZ-resistant strains of C. albicans and 11% when including the S-DD class. However, MCFG was shown to have an excellent antifungal activity against those azole-resistant strains of Candida species. An analysis of the randomly amplified polymorphic DNA pattern (RAPD) was carried out to assess the fingerprinting of the azole-resistant strains. The results demonstrated a common pattern in 3 of the 6 strains of C. tropicalis that showed MIC of > or = 16 micrograms/ml for fluconazole, while all of the 6 strains of C. albicans demonstrated their respective patterns.

[Antimicrobial susceptibility and beta-lactamase types among clinical isolates during January and February 2000 in the Kinki area of Japan].

We studied antimicrobial susceptibility and beta-lactamase types among clinical isolates in the Kinki area of Japan. Eight hundreds isolates of eight organisms were collected by seven medical institutions during January and February 2000. The rates of beta-lactamase producing by using the chromogenic nitrocephin test were 68.0% against Staphylococcus aureus isolates, 6.0% against Haemophilus influenzae isolates, 98.0% against Moraxella catarrhalis isolates. The rate of beta-lactamase negative ampicillin-resistant H. influenzae was 4.0% (4 out of 100). The result of beta-lactamase producing by using the acid-metric method were as follows the penicillinase and cephalosporinase: 27.0% and 37.0% against Escherichia coli isolates, 37.0% and 1.0% against Klebsiella pneumoniae isolates, 21.8% and 100% against Enterobacter cloacae isolates, 24.2% and 96.0% against Serratia marcescens isolates, 7.0% and 22.0% against Pseudomonas aeruginosa isolates. We identified beta-lactamase type of each isolate detected by polymerase chain reaction: SHV-derived extended-spectrum beta-lactamase (ESBLs) (1 isolate of E. coli and 1 isolate of K. pneumoniae), CTX-M-1-derived ESBLs (1 isolate of K. pneumoniae, 1 of E. cloacae and 4 of S. marcescens), and IMP-1-derived metallo beta-lactamases (2 isolates of S. marcescens).