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

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.

Objective: Mycoplasma hominis usually colonizes the lower urogenital tract and has been occasionally associated with pelvic inflammatory disease, postpartum fever, preterm labor in pregnant females. The aim of this study was to investigate the incidence and antimicrobial susceptibilities of M. hominis isolated from the urogenital tracts of pregnant females.

Methods: Specimens were obtained from the urogenital tract of pregnant females at Department of Obstetrics and Gynecology, Ehime University Hospital, between November 2014 and December 2017. The identification of M. hominis was confirmed by the polymerase chain reaction (PCR) methods. The minimum inhibitory concentrations (MICs) of antibiotics were measured using a broth microdilution assay.

Results: Of the 1074 specimens tested, 63 (5.9%) were positive for M. hominis. The M. hominis-positive rate was highest at 21.3% between 18 and 24 years old. The 21 (25.6%) of 82 patients with bacterial vaginosis were positive for M. hominis. The 17 (40.5%) of 42 patients delivered by cesarean section that occurred infections including of intrauterine infection and pelvic abscess were positive for M. hominis. They were all administered β-lactam antibiotics before and after cesarean section. All patients recovered immediately following administration of clindamycin (CLDM). β-lactam antibiotics, macrolides and fosfomycin (FOM) were all resistant against M. hominis strains. In contrast, M. hominis strains were susceptible to CLDM, minocycline (MINO) and quinolones.

Conclusions: Our data suggests that the prevalence of genital M. hominis in pregnant females is high at younger age, bacterial vaginosis and infections after cesarean section with β-lactam antibiotics administration. CLDM, MINO and quinolones may be recommended against M. hominis infection. Especially, CLDM can be used as the adequate agent for pregnant females because tetracycline and quinolones are undesirable during pregnancy and lactation.

There is a report that an infection by medicine resistant bacteria will be the number one cause of death in 2050 according to the recommendation of WHO, and the CPE (carbapenem-producing Enterobacteriaceae) infection is regarded as a problem in particular. When detecting CPE, it is important how to detect stealth type CPE sensitive to carbapenem series medicines. So we used the 2 types of screening culture medium, "KBM" CRE-JU culture medium ‹KOJINBAIO› (CRE-JU culture medium) and the FRPM culture medium, and tried to detect drug-resistant gram-negative bacilli such as CPE, stealth type CPE, ESBL-producing bacteria, and excess AmpC-producing bacteria (AmpC-producing bacteria), etc. in combination of this culture mediums. As a result, CRE-JU culture medium showed a difference in the growth of CPE depending on the amount of inoculated bacteria while β-lactamase non-producing strain and other strains except for high concentration ESBL-producing bacteria and AmpC-producing bacteria were un-growing. Most of the CRE, stealth type CPE, ESBL-producing bacteria and AmpC-producing bacteria grew in the FRPM culture medium while most of the β-lactamase non-producing strains with a MIC value of meropenem (MEPM) of 2 µg/mL or less were un-growing. From these results, it was suggested that when a strain grown on CRE-JU and FRPM culture mediums, it could be distinguished as CPE, and when strains grown on FRPM culture medium which were un-grown on CRE-JU culture medium, it could be distinguished as drug-resistant bacteria such as stealth type CPE, ESBL-producing bacteria, and AmpC-producing bacteria. When strains not grown on CRE-JU and FRPM culture mediums, it could be distinguished as sensitive.

The PCR-based open reading frame typing (POT) is an important method for analyzing outbreak information. Many institutions use POT as a molecular epidemiological method for analyzing horizontal transmission in methicillin-resistant Staphylococcus aureus (MRSA). However, typing and analyzing MRSA only based on POT, with high detection frequency, has some limitations. In this study, we analyzed 62 strains of MRSA, isolated at Ehime University Hospital between January 2018 to December 2018 based on six POT types, toxin type, and antimicrobial susceptibility. Types of POT and strains used were as follows: 106-183-37 (28 strains), 106-137-80 (7 strains), 106-77-113 (7 strains), 106-9-80 (7 strains), 70-18-81 (7 strains), 106-247-33 (6 strains). Based on antimicrobial susceptibility patterns, 5 types of MRSA were detected, including types susceptible to gentamicin (GM), clarithromycin (CAM), and levofloxacin (LVFX). Strains belonging to the same POT type, showed differential antimicrobial susceptibility patterns and had different toxin productivity. These findings suggest that the combination of POT method with antimicrobial susceptibility patterns and toxin type may be a useful technique for MRSA typing.

We carried out performance evaluation tests using clinical isolates in a modified yeast fungal drug susceptibility test kit ASTY plus caspofungin (CPFG) (Kyokuto Pharmaceutical Industry Co., Ltd., Tokyo, Japan). We used 51 strains of clinical isolates (Candida albicans, 30 strains; C. dubliniensis, 2 strains; C. glabrata, 4 strains; C. tropicalis, 4 strains; C. guilliermondii, 4 strains; C. parapsilosis, 4 strains; C. krusei, 3 strains). In this evaluation, minimum inhibitory concentration values of clinical isolates using the control CLSI method (Yeast-like fungi FP; Eiken Chemical, Tokyo, Japan), ASTY method, and another method (Yeast-like fungus DP; Eiken Chemical) were compared. The drugs with a concordance rate of 90% or more within ±1 tube were CPFG, amphotericin-B, 5-flucytosine, voriconazole, and those with a concordance rate of 80% or more were micafungin, fluconazole, and itraconazole. The above results clarified that the ASTY method correlated highly with the CLSI method.

Genetic testing is widely used as a rapid diagnostic method to identify microorganisms and detect antibiotic resistance genes. The nucleic acid to be analyzed is located inside the cell wall, the cell membrane or nuclear envelope. Therefore, it is essential to disassemble them in nucleic acid extraction operation. It is also necessary to remove or inactivate interfering substances by exposing cytoplasmic components accompanying cell disruption. Nucleic acid extraction is an indispensable task, but depending on the selected method, it may have a significant effect on the genetic test results. However, the DNA extraction method that is actually selected tends to emphasize work efficiency, and the appropriate evaluation of the extraction operation is neglected. In this study, we focused on the purity of the extracted DNA, and examined six existing extraction methods and original extraction methods using Gram-negative bacilli as a simple model. As a result, there was a large difference in DNA purity depending on the extraction method. When used in a qualitative gene amplification test, there was a difference in the shading of the bands. However, the detection of resistance genes all gave similar results. Furthermore, as a result of using the original extraction method, the extraction method using sodium decylbenzenesulfonate (SDBS) was the most excellent extraction method from the viewpoint of recovered DNA and operability.

Identification of bacteria by using MALDI Biotyper is relevant at the species category if Score Value (SV) is not less than 2.000. However, in practical examination, the analysis by MALDI Biotyper frequently produces the multiple candidate bacterial species with SV ≥2.000. In this study, we analyzed the ratio of multiple results among 10,081 specimens and identified the species of bacteria with high frequency of multiple results. Our analysis indicated that 8,129 strains out of 10,081 strains examined from July 2015 to July 2017, showed multiple identification results with MALDI Biotyper, and that multiple result was obtained in 4.9% of gram positive cocci analysis, 5.8% of gram positive rods, 25.4% of gram negative cocci, 16% of gram negative rod, none of fungus. In particular, MALDI Biotyper analysis of Enterobacter spp. (E. cloacae, E. asburiae, E. kobei, etc.), Acinetobacter spp. (A. baumannii, A. nosocomialis, A. pittii etc.), Neisseria spp. (N. flavescens, N. perflava etc.) had high ratios of multiple results. Our data suggests that genetic homology among bacteria results in multiple results of bacteria identification. The mass spectrometer method is the rapid test for bacteria identification. However, for obtaining higher specificity, it is required to combine with other methods. Furthermore, systematic annotation of bacteria is highly recommended.

Streptococcus pneumoniae is one of the major bacterial pathogens of community-acquired pneumonia. Immunochromatographic assay tests are used to detect pneumococcal capsular antigen. In many cases, They can be read visually. The Alere™ reader (Reader), which was developed in October 2018 by Alere Medical Co., Ltd. (currently, Abbott Diagnostics Medical Co., Ltd.) for interpreting BinaxNOW™ Streptococcus pneumoniae test (BinaxNOW™), quickly displays the results of the immunochromatographic tests, objectively and accurately, as it was launched for the purpose of streamlining laboratory workflow. The performance of the reader was evaluated by using urine samples from 100 patients, who were ordered pneumococcal urine antigen test from September 2018 to February 2019 at our hospital. Of the 100 samples, 14 were visually positive and 19 were reader positive. All visually positive samples generated reader positive result. Because 1 of the 5 cases which indicated a negative visual determination and positive reader determination was a sample with strong viscosity and turbidity, it was retested after centrifugation at 3,000×g for 10 min, resulting in negative reader determination. In 2 cases, S. pneumoniae were detected in sputum gram stains and culture tests. 5 discrepant samples were all visually and reader positive after concentration by centrifugal ultrafiltration. A questionnaire about visual interpretation was conducted among 31 individuals, by using urine from day 0 to day 4 collected from the patients whose test result was visually negative, reader positive and sputum culture positive at day 0. As a result, the number of operators who determined visually positive was 0 on day 0 (0%), 16 on day 1 (51.6%), 13 on day 2 (41.9%), 2 on day 3 (6.5%), and 0 on day 4 (0%). There were individual differences in ability to interpret low level positive result visually. On the other hand, reader can remove individual differences among operators from the interpretation of BinaxNOW™ and interpret positive result earlier than visual interpretation. Therefore reader was considered to be useful tool in clinical settings.