Masaru Komatsu

Metallo-β-Lactamase-Producing Gram-Negative Bacilli: Laboratory-Based Surveillance in Cooperation with 13 Clinical Laboratories in the Kinki Region of Japan

ABSTRACT 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-β-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.

Successful Treatment of Breakthrough Trichosporon asahii Fungemia with Voriconazole in a Patient with Acute Myeloid Leukemia

We describe a 55-year-old man with acute myelogenous leukemia who developed breakthrough Trichosporon asahii fungemia during 5 days of micafungin treatment. Although the patients clinical condition improved considerably after the start of voriconazole treatment, blood culture results remained positive for T. asahii for 3 days, and fever persisted for 7 days thereafter. The patient achieved complete hematological remission, and he received successful consolidation chemotherapy without developing Trichosporon infection with the prophylactic use of voriconazole therapy. This case report illustrates that voriconazole may be useful in the treatment of disseminated T. asahii infection in neutropenic patients.

Epidemiology of Escherichia coli, Klebsiella species, and Proteus mirabilis strains producing extended-spectrum β-lactamases from clinical samples in the Kinki Region of Japan.

In the present study, nonduplicate, clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella spp, and Proteus mirabilis were collected during a 10-year period from 2000 to 2009 at several hospitals in the Kinki region, Japan. The detection rate of E coli markedly increased from 0.24% to 7.25%. The detection rate of Klebsiella pneumoniae increased from 0% to 2.44% and that of P mirabilis from 6.97% to 12.85%. The most frequently detected genotypes were the CTX-M9 group for E coli, the CTX-M2 group for K pneumoniae, and the CTX-M2 group for P mirabilis. E coli clone O25:H4-ST131 producing CTX-M-15, which is spreading worldwide, was first detected in 2007. The most common replicon type of E coli was the IncF type, particularly FIB, detected in 466 strains (69.7%). Of the K pneumoniae strains, 47 (55.3%) were of the IncN type; 77 P mirabilis strains (96.3%) were of the IncT type. In the future, the surveillance of various resistant bacteria, mainly ESBL-producing Enterobacteriaceae, should be expanded to prevent their spread.

Laboratory Surveillance for Prospective Plasmid-Mediated AmpC β-Lactamases in the Kinki Region of Japan

ABSTRACT Extended-spectrum β-lactamases, plasmid-mediated AmpC β-lactamases (PABLs), and plasmid-mediated metallo-β-lactamases confer resistance to many β-lactams. In Japan, although several reports exist on the prevalence of extended-spectrum β-lactamases and metallo-β-lactamases, the prevalence and characteristics of PABLs remain unknown. To investigate the production of PABLs, a total of 22,869 strains of 4 enterobacterial species, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis, were collected during six 6-month periods from 17 clinical laboratories in the Kinki region of Japan. PABLs were detected in 29 (0.13%) of 22,869 isolates by the 3-dimensional test, PCR analysis, and DNA sequencing analysis. PABL-positive isolates were detected among isolates from 13 laboratories. Seventeen of 13,995 (0.12%) E. coli isolates, 8 of 5,970 (0.13%) K. pneumoniae isolates, 3 of 1,722 (0.17%) K. oxytoca isolates, and 1 of 1,182 (0.08%) P. mirabilis isolates were positive for PABLs. Of these 29 PABL-positive strains, 20 (69.0%), 6 (20.7%), 2 (6.9%), and 1 (3.4%) carried the genes for CMY-2, DHA-1, CMY-8, and MOX-1 PABLs, respectively. Pattern analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoretic analysis revealed that the prevalence of CMY-2-producing E. coli strains was not due to epidemic strains and that 3 DHA-1-producing K. pneumoniae strains were identical, suggesting their clonal relatedness. In conclusion, the DHA-1 PABLs were predominantly present in K. pneumoniae strains, but CMY-2 PABLs were predominantly present in E. coli strains. The present findings will provide significant information to assist in preventing the emergence and further spread of PABL-producing bacteria.

Analysis of molecular epidemiologic characteristics of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli colonizing feces in hospital patients and community dwellers in a Japanese city.

Infectious diseases caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are prevalent because of nosocomial infection. In addition, colonization of ESBL-producing E. coli in the intestinal tract of community dwellers due to the contamination of meat or environmental water is assumed to be one of the sources, but the causes have not been clarified. To analyze these factors, we investigated the difference in clonal groups using a combination of phylogenetic groups and multilocus sequence typing of ESBL-producing E. coli, which were obtained from the feces of an inpatient group in our hospital and a community-dwelling group living in a Japanese city. The carriage rate of ESBL-producing E. coli in the inpatient group was 12.5% (32/257), similar to that of 8.5% (42/496) in the community dwellers (P = 0.082). Of the ESBL clonal groups detected from the community dwellers, 52% (22/42) were clonal groups, including D-ST1485, D-ST70, D-ST2847, B2-ST550, B2-ST3510, A-ST93, A-ST580, A-ST716 and B1-ST2787, that have not been detected from human pathogens, meat, companion animals and environmental water, whereas all clonal groups detected from the inpatients were those that had already been reported. The rate of fluoroquinolone-resistant ESBL clonal groups colonizing the intestinal tract of the inpatient group rose as the number of hospital days increased. These results indicated that different factors were related to colonization of ESBL-producing E. coli in the feces of the inpatient group and the community-dwelling group.

Rapid detection of B2-ST131 clonal group of extended-spectrum β-lactamase–producing Escherichia coli by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry: discovery of a peculiar amino acid substitution in B2-ST131 clonal group

One reason for the spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli worldwide is the global pandemic of the B2-ST131 clonal group. We searched for the specific biomarker peaks to distinguish between the B2-ST131 clonal group and other sequence type (ST) clonal groups isolated from clinical specimens obtained in our hospital. Biomarker peaks at m/z 7650 in the B2-ST131 group (sensitivity of 100% and specificity of 89.7%) and m/z 7707 in the other ST clonal groups showed the highest discrimination abilities. We further verified reproducibility against other Japanese clinical isolates obtained in another area of Japan. Differences between the molecular mass at the 7650m/z and 7707m/z peaks indicated an E34A amino acid substitution by proteomic and genomic analysis. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry rapidly and simply identified the B2-ST131 clonal group in routine examinations and will allow for adequate empirical therapy and the possibility to control both hospital infections and the global pandemic.

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

Extended-spectrum β-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 μg/ml), while the MIC50 of the new quinolones was higher (1–2 μg/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.

Spread of OXA-23-producing Acinetobacter baumannii ST2 and ST246 in a hospital in Japan

A total of 1085 strains of Acinetobacter sp. obtained from 280 medical institutions in the Chugoku and Shikoku areas of Japan were investigated between 2011 and 2013. Among these strains, 20 (1.84 %) showing meropenem or imipenem resistance with a MIC of >4 μg ml- 1 were detected. Of these 20 strains, the bla(OXA-23) gene was detected in 17 strains isolated from the same institution. The PFGE patterns of the 17 strains were separated into two clusters, and multi-locus sequence typing showed the sequence types (STs) to be ST2 and ST246. This investigation demonstrated that A. baumannii ST2 of international clone II, which has rarely been isolated in Japan, has not yet spread nationwide.

ラテックス凝集法およびイムノクロマトグラフィーを用いた糞便中Escherichia coli O157: H7の迅速検出法について

The rapid detection method of Escherichia coli O157 in feces by using the latex agglutination test kit (Prolex; Pro Lab Diagnostics) and the immunochromatic assay kit (NOW EH. E. coli; Binax) was studied. 176 fecal samples obtained from 154 healthy men and 22 patients who had diarrhea by E. coli O157 were examined. Tellurite cefixim sorbitol MacConkey (TC-SMAC), Prolex and NOW were used for studies and also these were done after the enriched culture by tellurite cefixime vancomycin toriptic soy broth (TCV-TSB). In the direct inoculation, 5 (23%) of 22 samples, 7 (32%) and 9 (41%), and after the enriched culture, 7 (32%), 10 (46%) and 11 (50%) were positive by culture, Prolex and NOW, respectively. On the other hand, 154 samples from healthy men were all negative in the direct inoculation, but in the enriched culture, 7 of 40 (18%) were positive by NOW. These samples were negative by boiled (100 degrees C, 15 minutes). Our results indicated that Prolex and NOW were useful for accurate and rapid diagnosis of E. coli O157 enteritis, and more sensitivity results were obtained with the enrichment broth.

Evaluation of the modified carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide. Rapid and accurate detection of CPE is necessary for appropriate antimicrobial treatment and hospital infection control. However, CPE contains some strains that are difficult to detect depending on genotype and MIC value of carbapenem, and a detection method has not been established. The recently reported modified carbapenem inactivation method (mCIM) has been developed in CLSI M100-S27 as a phenotypic technique for detecting carbapenemase activity. In the present study, we examined mCIM as a new CPE detection method using 207 Enterobacteriaceae isolates in comparison with the three existing screening methods of modified Hodge test, Carba NP test and carbapenem inactivation method and evaluated its performance. Consequently, both the sensitivity and specificity of mCIM were 100%, indicating better results than the conventional screening methods. The mCIM is a useful tool for microbiology laboratories due to its simplicity, clear criteria, cost-effectiveness and availability at any laboratory.