[Evaluation of Screening Performance and Detection Time of Carbapenemase-Producing Enterobacterales Using RAISUS S4 in a Antimicrobial Sensitivity Test].

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.

[Comparative Verification of DNA Extraction Methods for Bacterial Nucleic Acid Amplification Test].

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.

Correlations between Resistance Classifications Based on Penicillin-Binding Protein Genotypes and Antimicrobial Susceptibility Test Results of Haemophilus influenzae.

There are several problems associated with antimicrobial susceptibility testing (AST) of Haemophilus influenzae. ß-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.

[Investigation of Skin Adherent Bacterial Flora in Dogs by MALDI-TOF MS and 16S rRNA Gene Analysis].

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 ß-lactamase producing bacteria and drug-resistant bacteria such as Carbapenem-resistant enterobacterales will also be transmitted to humans through contact with companion animals.

[Reliability of the Identification Result of Score Value ≧2.000 with the MALDI Biotyper: What Kind of Polymicrobial Species Are Included].

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.

[On the identification accuracy of MALDI-TOF MS influence of pretreatment methods, types of medium, coating skills, and preservation methods].

In bacterial identification by MALDI-TOF MS, there are many reports of usefulness concerning direct identification from blood culture and identification of bacteria which cannot be identified with automatic analysis equipment. On the other hand, there are very few studies that investigate how various conditions influence on identification accuracy, such as the type of medium used for bacterial isolation and pure culture, the pretreatment methods, the difference in coating technique, and preservation methods. Therefore, we examined 10 strains of 2 drug-resistant bacteria species and 9 strains of 1 unnormal bacterium species. As a result, no significant differences were found in accuracy of identifying all strains of the target bacteria incubated for 24 hours and changing the types of medium, the pretreatment methods, and the coating techniques. In particular, methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum ß-lactamase (ESBL) producing Escherichia coli showed little change in the score value and the mass spectrum that assayed every 24 hours during the preservation period in all of the medium. In the case of Vibrio vulnificus, however, identification accuracy was decreased by the specific medium and storage conditions. It is suggested as this factor that the growth state of bacteria may have influenced the identification accuracy.

[On the performance evaluation of VersaTREK introduced in blood culture].

We evaluated performance of Versa TREK, blood culture system used in our hospital. Compared with BacT/ALERT 3D, the detection time of bacteria in the VersaTREK was shorter in most of strains. Compared with BacT/ALERT Virtuo, there was little difference in the detection time of bacteria. In addition, VersaTREK was able to detect Helicobacter cinaedi which could not be detected by other equipment, and H. cinaedi was detected in clinical specimens within 2 days. There were 147 bottles judged to be false positives at our facility, of which 7,290 were 2,0% of the total. Ninety one points eight percentage of the cause was due to the change in the temperature inside the device, 3.4% was due to incorrect procedure. So, it is considered that false positives are further decreased by appropriate management of the installation and sample collection.

[Evaluation of novel nucleic acid detection kit for Mycoplasma pneumoniae].

For diagnosis of Mycoplasma pneumoniae infection, highly sensitive and rapid diagnosis is important. Because antibiotics are limited for the treatment of M. pneumoniae infection. In this study, we evaluated new rapid nucleic acid detection kit for M. pneumoniae. This kit does not require excessive pretreatment of specimens and molecular diagnosis of M. pneumoniae is possible within 40 min. Using 120 nasopharyngeal specimens, we compared this kit with a commercially available molecular diagnostic reagent (LAMP). 51 of 120 cases were M. pneumoniae positive, and the results of both assays were all consistent. In addition, sequencing of 23S rRNA gene was performed on 51 cases positive for M. pneumoniae. As a result, macrolide resistance mutation (2063A>G) was observed in 19 cases (37.3%). The gene mutations estimated by this kit coincided completely with the sequencing. In conclusion, new rapid nucleic acid detection kit could detect M. pneumoniae with the same sensitivity as other molecular diagnostics, in a simple process.

[Development and Evaluation of a New Selective Culture Medium, KBM Anaero RS-GNR, for Detection of Anaerobic Gram Negative Rods].

The laboratory culture methods for isolating drug-resistant pathogens has been the gold standard in medical microbiology, and play pivotal roles in the overall management of infectious diseases. Recently, several reports have emphasized the development of antibiotics-resistance among anaerobic gram-negative rods, especially Genus Bacteroides and Prevotella. Therefore, a selective culture method to detect these pathogens is needed. We developed here the new selective culture medium, termed "KBM Anaero RS-GNR," for detecting anaerobic Gram-negative rods. Growth capability and selectivity of the agar medium were assessed by using the pure culture suspensions of more than 100 bacterial strains as well as the 13 samples experimentally contaminated with these bacterial strains. This new medium, "KBM Anaero RS-GNR," successfully showed the selective isolation of anaerobic Gram-negative rods. Compared with commercially available medium, "PV Brucella HK Agar, " which is also designed to detect anaerobic Gram-negative rods, there was no significant difference of the overall detection efficiency between two media. However, "KBM Anaero RS-GNR" showed superior to selectivity for anaerobic Gram-negative rods, especially from the samples contaminated with Candida species. Thus, the culture method using KBM Anaero RS-GNR is relevant for isolation of anaerobic Gram-negative rods especially from clinical specimens.

[Utility of the Rapid Staining with the Use of Microwave for Detection of Genus Mycobacterium].

Recently, many laboratories use fluorescence microscopy for rapid screening of clinical specimens for detection of Genus Mycobacterium. The success of the stain depends on the staining temperature at which the fluorescent dye could uniformly penetrate the cell wall through waxy lipid barrier of the mycobacterial organism. Therefore, this process requires a precise heating control. In this study, to control the temperature during fluorescent auramine- rhodamine staining, we explored the potential use of microwave. The efficiency of microwave irradiation during the staining process was evaluated by using a Mycobacterium avium-containing sputum of which the smear slide was irradiated with several different conditions in combination of time and wattage. As a result, 1) the liquid temperature of the stain correlated well with wattage of microwave irradiation. 2) The tubercle bacilli were easily visualized as brilliant fluorescent bacilli in an orange color when it was set at the best condition of 600 W and 10 sec irradiation. 3) The sensitivity of microscopy with this staining method (MW method) was higher than those of conventional staining methods such as Ziehl-Neelsen staining and standard auramine-rhodamine staining, demonstrating that MW method can be applicable to the sputum slides which contained a few bacilli. Thus, we established the new staining method that is rapid and easy to perform in clinical laboratories. Since the MW method has not yet been utilized in order to conduct fluorescence microscopy for sputum smears, advancement on this method will make a vast change in testing of acid fast bacilli.