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

Recently, the global spread of carbapenemase-producing Enterobacterales has become a concern, and rapid detection methods are required. We have developed a rapid and inexpensive multiplex real-time PCR with melting curve analysis using the BD MAX™ system and evaluated it. We used 31 carbapenemase-producing Gram-negative bacteria (bla_IMP group, 12; bla_GES group, 6; bla_NDM group, 5; bla_VIM group, 3; bla_KPC group, 3; bla_OXA-48-like group, 1 strain; and bla_IMP group + bla_GES, 1 strain), 10 AmpC-producing Gram-negative bacteria, and 10 ESBLproducing Gram-negative bacteria. A BD MAX™ open platform system was used. Carbapenemase-positive and carbapenemase-negative strains were correctly identified 30 of 31 (excluding a bla_IMP group and bla_GES group co-coding strain) and all 20 of 20 isolates, respectively. Melting temperature (Tm) values of the various genes were as follows: bla_IMP group, 81.2±0.5°C; bla_VIM group, 91.8±0.4°C; bla_NDM group, 85.4±0.3°C; bla_GES group, 90.5±0.3°C; bla_KPC group, 94.1±0.5°C; and bla_OXA-48-like group, 82.1°C. Identification of the various genotypes was possible from the Tm values. However, only a peak derived from the bla_GES group could be detected in the strains producing both bla_IMP group and bla_GES group simultaneously, suggesting that only the genotype with the highest expression level could be captured in cases of simultaneous production. In the carbapenemase-negative strains, no obvious peaks were observed in the 20 AmpC and 20 ESBL-producing Gram-negative bacteria, and even when Tm values were detected, the dF/dT values were low and easily differentiated. This method appears to be very useful as a rapid and inexpensive test that can provide detection in about 2 hours.

Introduction: From 2018, IR Biotyper (IRBT; Bruker Daltonik GmbH, Germany) based on the Fourier transform infrared spectrophotometer has begun to be introduced as a new strain classification method in the field of clinical microbiological examination. We compared it with molecular epidemiology method to evaluate the usefulness of strain classification by IRBT.

Method: Homology of strain classification with molecular epidemiology method (Multilocus Sequencing Typing; MLST and PCR-based ORF Typing; POT) for 48 strains of Pseudomonas aeruginosa with different detection times from multiple institutions to evaluate the accuracy of IRBT was compared.

Results: IRBT used "KBM" SCD agar medium for preculture and was classified into 12 types when classified by Cut-off value 0.181, 8 types by MLST, and 13 types by POT. In the Adjusted Wallace between IRBT and molecular epidemiology method, MLST was 0.458 (95% CI; 0.295 to 0.620) and POT was 0.330 (95% CI; 0.135 to 0.525), indicating a discrepancy in strain classification.

Conclusion: No correlation was found between IRBT and the classification results by the molecular epidemiology method. In the molecular epidemiology method, strains are classified by matching only specific gene regions, but IRBT irradiates a sample with an infrared laser and classifies the strains according to the difference in absorption spectrum according to the molecular structure, so the measurement principle is different. When classifying strains by IRBT, it is desirable to grasp the clinical information of the detected strains and to target multiple strains isolated at the same facility at the same time.

Rapid detection of carbapenemase-producing Enterobacterales (CPE) is important in infection control, since it transmits plasmids carrying resistance genes. Here, we evaluated the rapid detection of CPE using the fully automated antimicrobial susceptability testing system "RAISUS S4". Sixty-two CPE strains including carbapenem-resistant Enterobacterales and 100 carbapenemase-non-producing Enterobacterales strains were used. RAISUS S4 was performed using both 18 hr and rapid methods. The sensitivity of CPE detection and decision time were evaluated using Meropenem results. The results showed that the sensitivity for CPE detection was 100% for both methods, with specificity of 97% for the 18 hr method and 95% for the rapid method. The mean CPE detection time for the 18 hr method was 7.2 hrs and 8.8 hrs for the rapid method. The mean MIC determination time of the 18 hr method for all 162 strains was 17.2 hrs and 8.8 hrs for the rapid method. In addition, we analyzed the absorbance values of the 18 hr method. Checking the growth curve at a drug concentration of 0.125 µg/ml and determining it to be positive when its absorbance reached 0.8 Abs, CPE could be detected in an average of 5.8 hrs with in sensitivity of 100% and specificity of 92%. The 18 hr method of RAISUS S4 allowed rapid detection of CPE, and the rapid method allowed earlier MIC determination, including sensitive isolates. These results suggest that RAISUS S4 can detect CPE rapidly without missing CPE by routine test even in Japan where the frequency of CPE isolation is low.

Campylobacter spp. has been the leading cause of bacterial food poisoning in Japan since 2000. The predominant Campylobacter spp. testing method employs selective medium to isolate Campylobacter spp. In the present study, we evaluated the Campylobacter-isolating capacity and clinical utility of BD^TM mCCDA Clear-HT, an agar medium containing a chromogenic substrate, on 230 diarrhea stool samples. After 48 hours incubation, 50 samples (21.7%) were positive with BD^TM mCCDA Clear-HT, while 61 samples (26.5%) were positive using modified Skirrow agar medium. In this study, BD^TM mCCDA Clear-HT had a lower detection rate of Campylobacter jejuni more than Vitalmedia modified Skirrow agar medium.

Man in his 80s. In March 20XX, the level of consciousness decreased at the admission facility, and he was transported as an emergency case. He was diagnosed as aspiration pneumonia, septic shock due to cholecystitis, and DIC, and was hospitalized for medical treatment. During the course of hospitalization, aspiration pneumonia continued to improve and worsen, but in January 20XX＋3, a fever of 38.7°C occurred, and Mucor circinelloides was detected in the blood culture collected at this time. In sputum 7 days before the blood culture was submitted, an image of suspicious zygomycosis was confirmed by Gram stain, so the patient was diagnosed with Mucor disease and started administration of amphotericin B. After that, the condition was temporarily stable, but due to recurrence of aspiration pneumonia and renal damage, he died 19 days after the start of amphotericin B administration. It is difficult to detect Mucor spp. in blood culture, however in this case, it was detected by the blood culture device; Versa TREK (Thermo Fisher Scientific K.K. Tokyo, Japan).

The emergence and dissemination of drug-resistant Gram-negative bacilli have been recognized as a serious health concern in worldwide. The isolation rates of Extended-Spectrum β-lactamases (ESBL) and AmpC β-lactamases (AmpC) producing gram negative rods are increasing in our hospital. In the present study, we evaluate the availability of the antimicrobial resistance testing by the direct disc methods using AmpC/ESBL differential discs. One hundred and ten strains of Enterobacterales were isolated during the observation period, of which 19 strains (17%) were ESBL-positive and 6 strains (5%) were AmpC-positive. The positive and negative coincidence rate between direct disc methods and standard disc methods were 100%. We conclude that the direct disc method is a useful and rapid detection method for ESBL and AmpC from blood culture samples.

Group B Streptococcus (hereinafter GBS) is the main pathogen in neonatal sepsis and meningitis, accounting for approximately one quarter of the cases. 1) Prevention of infection is therefore crucial. The GBS carriage testing of pregnant women is necessary to prevent infections. In this study, we examined the clinical utility of Pourmedia ViGBS agar medium (Eiken Chemical Co., Ltd.) in GBS screening, using a total of 197 vaginal and urine samples. Of these samples, 32 (16.2%) tested GBS positive with Pourmedia ViGBS agar medium, and 29 (14.7%) tested GBS positive with Nissui separated plate sheep blood agar/Drigalski agar medium (Nissui Pharmaceutical Co., Ltd.). These results indicate the usefulness of Pourmedia ViGBS agar medium as a GBS screening selective agar medium.

Making the antibiogram necessary for infectious disease treatment is an important operation of the microbiology laboratory. Antibiogram is required to be up-to-date and to keep up with the annual updates of the Clinical & Laboratory Standards Institute (CLSI). However, these operation and managements require a lot of effort. In addition, even in the surveillance and analysis comparison of multiple facilities, the difference in CLSI base year becomes a barrier, making unified analysis difficult. On the other hand the antibiogram by Japan Nosocomial Infections Surveillance (JANIS) has restrictions on the bacterial species, antibacterial agents, and date range. Accordingly we focused on the fact that the data transmitted to JANIS is in a common format, and attempt construction a system to making the antibiogram based on this. This system uses data transmitted to JANIS, is convenient, can use not only the latest but also past base year CLSI category, has no restrictions on bacterial species, antibacterial agents, date range, works on Microsoft Windows environment, pursuit of compliance with the guidelines, and automatically making the antibiogram.

Due to the increase in the number of companion animal breeders in Japan, there are more opportunities for companion animals to come into contact with humans than before. Therefore, we investigated the bacterial flora adhering to the skin of dogs and the bacterial flora was analyzed for the presence of zoonotic bacteria that infect humans from companion animals. With the cooperation of students enrolled in the Department of Medical Technology and Science, Faculty of Fukuoka Health Care, International University of Health and Welfare. 39 samples were collected from the abdomen, back and paws of 13 healthy dogs using sterile swabs by the scraping method. The isolation culture was carried out only for facultative anaerobic bacteria to obligate aerobic bacteria and Bacterial identification was determined by MALDI-TOF MS and 16S rRNA gene analysis. Among the identified strains were Pasteurella canis, Staphylococcus pseudintermedius, Staphylococcus intermedius, which were difficult to detect in humans. The overall ratio of detected bacteria was 35% for coagulasenegative staphylococci, 14% for coagulase-positive staphylococci, 5% for Enterobacteriaceae, and 45% for natural environment. In the future, it is expected that extended-spectrum β-lactamase producing bacteria and drug-resistant bacteria such as Carbapenem-resistant enterobacterales will also be transmitted to humans through contact with companion animals.

There are several problems associated with antimicrobial susceptibility testing (AST) of Haemophilus influenzae. β-Lactamase-negative ampicillin-resistant H. influenzae (BLNAR) isolates with minimum inhibitory concentration (MIC) of ampicillin (ABPC) <4 mg/l will be classified as susceptible according to the MIC breakpoint of the CLSI M100 criteria, in spite of harboring penicillin-binding protein (PBP) mutations that cause ABPC resistance. A total of 103 isolates were collected from clinical materials for analysis. The genotypes of the PBP mutations were analyzed by polymerase chain reaction. The WalkAway 96 Plus (WALKAWAY), dry plate Eiken (DP-EIKEN), and RAISUS S4 systems (RAISUS) were used for AST. HTM broth was used as the culture medium for WALKAWAY, Mueller‒Hinton broth with 5% lysed horse blood for DP-EIKEN, and HTM with 5% horse serum for RAISUS. The MIC concordance rates of ABPC for g-BLNAR, for RAISUS vs. DP-EIKEN, RAISUS vs. WALKAWAY, and DP-EIKEN vs. WALKAWAY were 96.1, 86.4, and 85.4%, respectively. WALKAWAY had a low correlation with the other two systems. Moreover, concordance rates of ABPC MIC ≥4 mg/l, which is considered as resistant, of 69 g-BLNAR isolates for the RAISUS, DP-EIKEN, and WALKAWAY systems were 68.1, 58.0, and 37.7%, respectively. Therefore, in Japan, where the BLNAR strain is isolated at a high frequency, it is necessary to understand the characteristics of the measuring systems to appropriately interpret the test results.