Rinsho Biseibutsu Jinsoku Shindan Kenkyukai shi = JARMAM : Journal of the Association for Rapid Method and Automation in Microbiology最新文献

Extended-Spectrum beta-Lactamase (ESBL)-producers in the family Enterobacteriaceae are recognized worldwide as nosocomial pathogens, however it is difficult to screen them in the routine laboratory processing. ChromID ESBL agar newly developed for screening ESBL-producing Enterobacteriaceae was released in Japan in April, 2007. We evaluated the clinical assessment of ChromID ESBL agar in routine microbiology laboratory. The 47 strains investigated were clinical isolates belonging to the family Enterobacteriaceae with the MICs of cefpodoxime greater than 2 mug/ml. The 27 ESBL-producers examined were comprising of 19 Escherichia coli, 3 Klebsiella oxytoca, 1 Citrobacter freundii, 3 Enterobacter cloacae, and 1 S. marcescens (ESBL group) and 20 ESBL non-producers consiating of 5 K. oxytoca, 1 Proteus mirabilis, 1 P. vlugaris, 2 Serratia marcescens, 8 C. freundii, 2 Enterobacter cloacae, and 1 E. aerogenes (non-ESBL group). Characterization of beta-lactamase genes was carried out by use of polymerase chain reaction. As the results, the sensitivity and the specificity of ChromID ESBL agar plates after incubation for 18 hours was 100% and 20%, respectively. It should be noted that the values of specificity was extremely low compared with those of the sensitivity. These findings clearly suggested that in cases of utilizing ChromID ESBL agar plates, it should be important to consider its characteristic properties, as even the ESBL-non-producers could grow on these media only when they were resistant to CPDX.

In recent years, the detection of specific pathogens has been remarkably speedy with the development of new nucleic acid amplification methods such as real-time PCR, which is expected to be a great contribution to clinical diagnosis. Real-time PCR was introduced in our hospital, 2004 with the aim of detecting various pathogens. The system has been established and used for rapid bacteriological diagnosis for outpatient services in our hospital. This system can contribute greatly to achieving reliable and quick diagnosis, particularly for atypical pneumonia and hemorrhagic colitis caused by Escherichia coli. This system requires only 45 minutes on average for atypical pneumonia diagnosis, from receiving a specimen to the reporting of its results, which shortens the diagnosis time to about one-tenth of conventional methods. Survey on the type of initial dose in cases of Mycoplasma pneumoniae PCR positives shows that any administration of beta-lactam antibiotics, not effective to M. pneumoniae, has not been reported. Concerning the diagnosis of hemorrhagic colitis caused by Escherichia coli, it has been possible for us to report the results within one and a half hours, or within two hours including the legal notification of a third class infectious disease to a public health center. The SYBR Green Idetection system used in our hospital is superior to other detection systems comparing with versatility and cost-effectiveness. This report advocates that real-time PCR can contribute greatly to a rapid and cost-effective diagnostic system making full use of the characteristics of conventional bacteriological rapid-diagnostic methods, such as Gram staining and immuno-chromatography.

Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha-toxin using ESI/MS has not been reported. In this study, we report the successful cloning, expression, secretion, identification and sequence determination of the C. perfringens alpha-toxin.

The serotyping of staphylocoagulase is widely used in Japan. However, the conventional immunoassay based on neutralization of the antisera is so laborious and time-consuming that it is not widely used in the other countries. In order to overcome these drawbacks we developed a novel staphylocoagulase serotyping method based on a microplate format using polystyrene latex particles. Addition of latex particles promotes the formation of fibrin complexes, which represents a more rapidly and easily detected endpoint. For 83 strains, 90% were classified into serotypes within 3 h, and there was no discrepancy in the results between our method and the conventional method. These results indicate that the present microplate method is rapid, simple, and interpretable.

Because the detection of bacteremia is an important indication for subsequent treatment and prognosis, blood culture-positive results should be reported as soon as possible. Here, we present our attempts to promptly report the results of blood culture-positive samples and successive support systems for medical examinations. The BacT/ALERT system is used for culturing blood in our hospital. If a positive blood culture is obtained, medical technologists at the microbiological laboratory of our university hospital immediately contact the corresponding ward regarding the results of Gram staining and simultaneously inform the infectious disease physicians at the Department of Infection Control and Prevention. By using the positive blood cultures obtained from January 2003 to December 2004 (the period during which only day shifts on weekdays was supported), we determined the time required from blood specimen collection to obtaining positive samples from the culture system. When the samples were inserted into the system on the day following blood collection or later, the average time required was 48 hours. On the other hand, when the samples were inserted into the system on the day of blood collection, the average time required was 33 hours. After consideration of the importance of blood cultures, the examination system at the microbiological laboratory was changed to support day shifts every day in May 2008. The time taken from blood specimen collection for blood cultures to obtaining positive samples from the system was 40 hours on an average when day shifts were supported only on weekdays (during the above mentioned period), whereas the average time required was 31 hours when day shifts were supported every day (from May to June 2008). Since January 2002, the Department of Infection Control and Prevention have supported medical examinations for infectious diseases, including blood culture-positive cases. Compared between before and after the establishment of the support system (in 2001 and 2007), the 30-day mortality rates from bloodstream infections with Staphylococcus aureus and Candida spp. have improved from 30% to 17% and 56% to 23%, respectively. As indicated in our results, it is important to immediately deliver blood culture samples to the laboratory, start blood cultures (insert into the system), promptly report the results of Gram staining after the positive samples are obtained, and administer suitable antibiotic treatment.

Diarrhea caused by the Escherichia coli with held adhesion came to attention. We performed an adhesion-related gene and relation of diarrhea. Subjects were 77 outpatients with diarrhea from June 2003 to December 2005. A total of 102 E. coli strains randomly isolated from stool specimens. All the toxigenic examinations were negative, and there were not the relations. Adhesion-related gene were 10 strains found. As for the contents, astA was 5 strains, 2 strains of aggR, 2 strains of eaeA, 1 strain of eaeA plus astA. Of these, we were able to classify 5 strains in serological typing, but remain 5 strains did not typing. Only one strain of O157 was VT positive. There is not it with causative E. coli of diarrhea even if serological typing is negative. Therefore it was thought that an adhesion-related gene test was important.

Minimum inhibitory concentrations (MICs) of vancomycin (VCM) and teicoplanin (TEIC) were measured using a novel susceptibility test based on the chemiluminescence assay method (CA) (Rapid-Lumi Eiken; Eiken Chemicals, Tokyo, Japan) against 84 strains of Staphylococcus aureus, consisting of 82 strains of methicillin-resistant S. aureus (MRSA) from clinical isolated, S. aureus Mu3 involving beta-lactam antibiotic induced vancomycin (VCM) resistant MRSA (BIVR) and methicillin-susceptible S. aureus ATCC 29213. The results were in good accordance with the values determined by Clinical and Laboratory Standards Institute (CLSI): i.e., 100% (84/84) of consistency for VCM and 95% (80/84) for TEIC, respectively. In addition, BIVR strains were properly estimated from the results of the CA method and using the BIVR detection method with Mu3 agar (Mu3 Agar method), even though the incubation times was very short (2-4 h). In conclusion, it was found that the new method is reliable and rapid to detect BIVR strains in clinical laboratories.

During the period from January through December 2007, a total of 1,814 stool specimens from the inpatients of nine regional hospitals in mainland of the Ryukyus, were tested to identify vancomycin-resistant enterococci (VRE). All the stool specimens were primarily cultured onto the VRE selective agar plates, and a total of 195 specimens yielded positive enterococcal growth. Of 195 isolates, VRE screening agar tests identified 106 phenotypic VRE isolates, consisting 24 isolates of Enterococcus casseliflavus, 12 of E. faecalis, 4 of E. faecium, and 66 of E. gallinarum. Then, the 106 VRE isolates were tested for vanA and vanB genes by polymerase chain reaction, the results indicating none of the isolates were positive for vanA or vanB genes. With these results, it can be concluded that, at present, mainland of the Ryukyus is VRE-free area, and it is necessary to continue careful search for incoming and spreading of VRE positive for vanA and vanB genes in Okinawa.