[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.

[A trial of simple and rapid carbapenemase big five gene analysis using LAMP method].

Reliable detection and typing of carbapenemase is important in the treatment of infectious diseases. In this study we newly designed LAMP primer based on the latest information, and established a detection method for Carbapenemase Big five gene. For DNA extraction from strains, alkaline boiling method and commercial kit were used. The reaction temperatures of the LAMP method was VIM: 65°C, NDM: 63°C, KPC: 65°C, OXA-48-like: 65°C, IMP: 61°C. And simultaneous LAMP method was at 63°C, for 60 min. It was possible to detect up to 103 copies/ml. The reactivity of LAMP using 36 strains verified by Multiplex-PCR was VIM (4/4: number of LAMP method positive strains/number of strains evaluated), NDM (2/2), KPC (4/4), OXA-48-like (4/4), IMP (17/17). The type of carbapenemase determined by the LAMP method were all consistent with multiplex PCR. All strains were detected within 30 min. In VIM, both VIM-1-like and VIM-2-like were able to detect. In this study, although the number and variation of the strains evaluated was limited, LAMP method was clinically useful as a simple and rapid carbapenemase detection method.

[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.

Phosphorylation of plasma membrane H+-ATPase Thr881 participates in light-induced stomatal opening.

Plasma membrane (PM) H+-ATPase is crucial for light-induced stomatal opening and phosphorylation of a penultimate residue, Thr948 (pen-Thr, numbering according to Arabidopsis AHA1) is required for enzyme activation. In this study, a comprehensive phosphoproteomic analysis using guard cell protoplasts from Vicia faba shows that both red and blue light increase the phosphorylation of Thr881, of PM H+-ATPase. Light-induced stomatal opening and the blue light-induced increase in stomatal conductance are reduced in transgenic Arabidopsis plants expressing mutant AHA1-T881A in aha1-9, whereas the blue light-induced phosphorylation of pen-Thr is unaffected. Auxin and photosynthetically active radiation induce the phosphorylation of both Thr881 and pen-Thr in etiolated seedlings and leaves, respectively. The dephosphorylation of phosphorylated Thr881 and pen-Thr are mediated by type 2 C protein phosphatase clade D isoforms. Taken together, Thr881 phosphorylation, in addition of the pen-Thr phosphorylation, are important for PM H+-ATPase function during physiological responses, such as light-induced stomatal opening in Arabidopsis thaliana.

Identification of Genes Preferentially Expressed in Stomatal Guard Cells of Arabidopsis thaliana and Involvement of the Aluminum-Activated Malate Transporter 6 Vacuolar Malate Channel in Stomatal Opening.

Stomatal guard cells (GCs) are highly specialized cells that respond to various stimuli, such as blue light (BL) and abscisic acid, for the regulation of stomatal aperture. Many signaling components that are involved in the stomatal movement are preferentially expressed in GCs. In this study, we identified four new such genes in addition to an aluminum-activated malate transporter, ALMT6, and GDSL lipase, Occlusion of Stomatal Pore 1 (OSP1), based on the expression analysis using public resources, reverse transcription PCR, and promoter-driven ß-glucuronidase assays. Some null mutants of GC-specific genes evidenced altered stomatal movement. We further investigated the role played by ALMT6, a vacuolar malate channel, in stomatal opening. Epidermal strips from an ALMT6-null mutant exhibited defective stomatal opening induced by BL and fusicoccin, a strong plasma membrane H+-ATPase activator. The deficiency was enhanced when the assay buffer [Cl-] was low, suggesting that malate and/or Cl- facilitate efficient opening. The results indicate that the GC-specific genes are frequently involved in stomatal movement. Further detailed analyses of the hitherto uncharacterized GC-specific genes will provide new insights into stomatal regulation.