Masanori Aihara

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.

High Frequency of Antibiotic-Associated Diarrhea due to Toxin A-Negative, Toxin B-Positive Clostridium difficile in a Hospital in Japan and Risk Factors for Infection

Patients hospitalized in a hospital with a high incidence of antibiotic-associated diarrhea due to toxin A-negative, toxin B-positive (A−/B+) Clostridium difficile were retrospectively investigated to determine the clinical manifestations and risk factors for infection. Of 77 Clostridium difficile isolates obtained from 77 patients during the 1-year investigation period, 30 were A−/B+ and 47 were toxin A-positive, toxin B-positive (A+/B+). By pulsed-field gel electrophoresis analysis, 23 of the 30 A−/B+ strains were outbreak-related, suggesting nosocomial spread of a single type of bacterium, which mainly affected patients in the wards of respiratory medicine, hematology and neurology. Using regression analysis, three factors were found to be associated with infection by A−/B+ isolates: (i) exposure to antineoplastic agents (P=0.01, odds ratio [OR]=5.1), (ii) the use of nasal feeding tubes (P=0.008, OR=5.2), and (iii) assignment to a certain internal medicine ward (P=0.05, OR=3.0). Between patients with Clostridium difficile-associated diarrhea caused by A−/B+ strains and those with A+/B+ strains, no statistically significant difference was found in body temperature, serum concentration of C-reactive protein, leukocyte count in whole blood, frequency of diarrhea, or type of underlying disease. These results indicate that A−/B+ strains of Clostridium difficile can cause intestinal infection in humans and they spread nosocomially in the same manner as A+/B+ strains.

Evaluation of MicroScan ESBL confirmation panel for Enterobacteriaceae-producing, extended-spectrum β-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.

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.

Polymerase chain reactionおよびアルカリホスファターゼ標識オリゴヌクレオチドプローブを用いた3菌種の抗酸菌及び属の検出同定法

Rapid detection and identification method of mycobacteria from specimen with infected mycobacteriosis was established by the combination of polymerase chain reaction and alkaline phosphatase labeled oligonucleotide probe (PCR-ALPDH). We prepaired four kinds of probes: Mycobacterium tuberculosis complex, M. avium, M. intracellulare, and mycobacterial-probe other than tuberculosis. PCR-ALPDH was compared with conventional methods using 234 specimens which suspected mycobacteriosis. In culture, 68 specimens (29.1%) were positive and 166 specimens (70.9%) were negative. In PCR-ALPDH, 88 specimens (37.6%) were positive and 146 specimens (62.4%) were negative. In 68 specimens which were positive in culture, the agreement of results of conventional identification and PCR-ALPDH for each probes were: 39/40 (PCR-ALPDH/Culture, 97.5%) in M. tuberculosis 5/9 (55.6%) in M. avium, 6/6 (100%) in M. intracellulare and 22/28 (88.6%) in MOTT isolation. Among 166 negative culture specimens, 27 specimens were positive in PCR-ALPDH. The results indicated that PCR-ALPDH method was applicable for the establishment of rapid and sensitive mycobacterial diagnostic system.