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Clostridioides difficile is the most common anaerobic bacterium that causes healthcare-associated infections, and prompt diagnosis and infection control are important because it causes C. difficile infection (CDI). In this evaluation, the C. difficile nucleic acid detection reagent, Smart Gene CD Toxin B (Mizuho Medy Co., Ltd., hereinafter referred to as the "evaluation reagent") was evaluated for its clinical performance in comparison with real-time PCR and toxigenic culture (TC). Measurement of evaluation reagents and real-time PCR were performed on 157 residual stool specimens from suspected CDI patients. For TC, stool culture was performed, and colonies in which C. difficile was identified by a mass spectrometer (MALDI Biotyper) were checked for toxin production using a rapid antigen diagnostic kit. The results of the evaluation reagents showed a high concordance rate; 100% sensitivity (81/81) and 100% specificity (76/76) with real-time PCR, 89.8% sensitivity (79/88), and 97.1% specificity (67/69) with TC. The evaluation reagent enables a simple nucleic acid amplification test (NAAT) in a short time and is thought to be useful in CDI treatment, which requires rapid diagnosis and infection control.

With the rapid spread of multidrug-resistant bacteria, carbapenem-resistant Enterobacteriaceae (CRE) has been reported worldwide as a major concern because of limited treatment options. Carbapenem resistance is mainly due to carbapenem-ase, a carbapenem-degrading enzyme, which is mainly encoded on a plasmid to spread across bacterial species. However, there have been only small-scale attempts to determine the similarities or accommodations of the plasmids disseminating regionwide. We analysed the 230 CRE isolates carrying bla IMP from 43 medical facilities in the northern Osaka area focusing on the plasmids, the main carriers of the drug resistance genes. Combination of whole genome sequencing and Southern blotting revealed the predominant dissemination of bla IMP-6 by the pKPI-6 plasmid among genetically distinct isolates, as well as the emergences of derivatives that acquired various advantages. We iden-tified heteroresistance likely causing stealth transmissions, which was generated by the transcriptional regu-lation of bla IMP-6 , stabilization of bla IMP-6 through chromosomal integration, enhanced carbapenem resistance through plasmid multimerization, or broadened antimicrobial resistance due to a single point mutation in bla IMP-6 . In this article, I dis-cussed the mechanisms of regional spread of CRE and enhancement of carbapenem resistance providing the insights to prevent their disseminations.

This review summarizes current knowledge regarding the pathological mechanism of Streptococcus pneumoniae, a major cause of pneumonia, sepsis, and meningitis, with focus on our previously presented studies.To identify pneumococcal adhesins or invasins on cell surfaces, we investigated several proteins with an LPXTG anchoring motif and identified one showing interaction with human fibronectin, which was designated PfbA. Next, the mechanism of pneumococcal evasion form host immunity system in blood was examined and pneumococcal α-Enolase was found to function as a neutrophil extracellular trap induction factor. Although S. pneumoniae organisms are partially killed by iron ion-induced free radicals, they have an ability to invade red blood cells and then evade antibiotics, neutrophil phagocytosis, and H 2 O 2 killing. In addition, our findings have indicated that zinc metalloprotease ZmpC suppresses pneumococcal virulence by inhibiting bacterial invasion of the central nervous system. Since evolutionarily conserved virulence factors are potential candidate therapeutic targets, we performed molecular evolutionary analyses, which revealed that cbpJ had the highest rate of codons under negative selection to total number of codons among genes encoding choline-binding proteins. Our experimental analysis results indicated that CbpJ functions as a virulence factor in pneumococcal pneumonia by contributing to evasion of neutrophil killing.Use of a molecular biological approach based on bacterial genome sequences, clinical disease states, and molecular evolutionary analysis is an effective strategy for revealing virulence factors and important therapeutic targets.

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 10 3 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.

Bangladesh is considered as a high-risk country for emerging infectious diseases because of its high population density, poverty, and unhygienic conditions. Although control efforts have primarily been focused on major infectious diseases such as diarrheal diseases, tuberculosis, malaria, and HIV infection, the prevalence and impact of many local or minor infectious diseases are still unclarified in this country. In this review, we present our recent experience and outcomes of collaborative research on puerperal infection (PI), which is a poorly defined infectious disease in Bangladesh. PI is the most common complication during the perinatal period in developing countries. We investigated the incidence of individual species of aerobic bacteria causing PIs and their drug resistance, and the genetic traits of isolates during the two-year period (2010-2012). The common species of isolates from patients with PIs were Escherichia coli, Enterococcus faecalis, Staphylococcus haemolyticus, Proteus mirabilis, Staphylococcus aureus, and Klebsiella pneumoniae. A remarkable finding was the high rates of resistance to cephalosporins among Gram-negative bacteria harboring extended-spectrum beta-lactamase genes, which were associated with carbapenem resistance in a few isolates. This study defined the importance of control of antimicrobial resistance in Bangladesh, and provided suggestions for the future direction of collaborative research on infectious diseases in Bangladesh.

In 2013, two outbreaks of enterohemorrhagic Escherichia coli (EHEC) occurred in Saitama city. According to reports from each of the medical institutions that detected the EHEC isolates, the isolates seemed to differ in their production of Vero Toxin (VT / Shiga Toxin: Stx) since one isolate produced only Stx1 and the other produced both Stx1 and Stx2. However, a patient survey conducted by a public health center revealed that common foodstuffs had been consumed in both outbreaks. Because, the two EHEC isolates were newly detected from two people in one patient's family, we analyzed the phenotypic and genetic relationships among four isolates in total. All the isolates were serotyped as O157: H-, and both stx1 and stx2 were detected. Subsequently, all four isolates were shown to have the same pulsed-field gel electrophoresis (PFGE) banding pattern. The findings suggested that these isolates belonged to the same strain group. Among these cases, the isolates had stx2c which is one of the stx2 subtypes. Reportedly, some cases with the Stx2 subtype can not be detected using conventional tests for toxin. In addition, Stx2 can be overlooked as a result of this limitation of Stx-production tests. Both epidemiological research by public health centers and genetic analysis by prefectural and municipal public health institutes (PHIs) are very important for clarifying possible relationships among outbreaks, as in the present cases. Moreover, collaborations and networks among medical institutions, PHIs and public health centers should be further strengthened to prevent the spread of infections.

Chronic infection with Helicobacter pylori is involved in a variety of clinical outcomes including gastric cancer. In the present study, we focused on the infection strategies of H. pylori associated with establishment of chronic infection. As a result, the following four findings revealed. 1) alpha-ketoglutarate oxidoreductase (KOR) is an essential survival enzyme for energy metabolism in the coccoid form of H. pylori, and inactivation of the KOR activity exerted a potent bactericidal action against H. pylori by preventing induction of the coccoid form. 2) SodB expression is derepressed by amino acids mutation of ferric uptake regulator (Fur), which is associated with the development of Metronidazole resistance. 3) FecA1 is an important determinant of the host-colonization ability through Fe(2+) supply to SodB, suggesting that FecA1 may be a possible target for the development of a novel bactericidal drug. 4) Intracellular CagA oncoprotein is degraded by autophagy and therefore short lived. However, in the CD44v9-expressing gastric cells, CagA specifically accumulated through the repression of autophagy induction.

Reports of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have recently increased in Japan. To determine the status of MRSA infections in our hospital, we investigated their Staphylococcal cassette chromosome mec (SCCmec) types and prevalence of Panton-Valentine leukocidin (PVL). In addition, we investigated the relation between their SCCmec and antimicrobial susceptibility. The 191 strains were isolated from January to July in 2011 and were classified as SCCmec type I (2, 1.0％), type II (136, 71.2％), type IV (36, 18.8％), type V (4, 2.1％) and type VIII (2, 1.0％). Eleven isolates (5.8％) were designated as nontypable. No isolates were PVL-positive in this study. The SCCmec type IV strains were more susceptible to imipenem (MIC90, 0.25 μg/ml) than SCCmec type II strains (MIC90, >16 μg/ml). This difference was also observed between SCCmec type IV and SCCmec type II in susceptibility levels to clarithromycin, clindamycin, minocycline, and levofloxacin, but not to gentamicin. In particular, SCCmec type IV strains were susceptible to imipenem and minocycline. The result indicates these susceptibility is useful to discriminate CA-MRSA from Hospital-associated MRSA (HA-MRSA).

Purpose: We aimed to investigate the prevalence and possible transmission routes of rifampicin (RFP) mono-resistant Mycobacterium tuberculosis strains., Methods: Drug susceptibility testing was used to identify 15 RFP-resistant strains out of 4633 M. tuberculosis isolates. Sequencing of the rpoB gene and VNTR analysis were performed to further confirm the genetic classification., Results: Resistance-conferring mutations in the RFP resistance-determining region (RRDR) of the rpoB gene were found in 14 of the 15 strains with phenotypic RFP mono-resistance. VNTR analysis revealed 2 clusters of 5 identical strains each., Conclusions: Although the community prevalence of RFP mono-resistant M. tuberculosis is low, the results of VNTR analysis suggested that rather than being recently transmitted, these strains may have been widely transmitted as latent infections in the population.

Since the discovery of antibiotics, the battle between humans and drug resistant bacteria has never stopped. Bacteria have developed various ways to resist the toxic effects of antibiotics and other drugs. Multidrug efflux pumps are integral membrane proteins that utilize cellular energy to extrude antibiotics or biocides actively out of the cell. In this symposium, I first introduce the post-genomic approach to analyze all putative drug efflux genes. Next, I discuss the regulation of drug efflux pumps responding to environmental signals. I also introduce the physiological roles of drug efflux pumps in virulence, which is an ongoing research area. Multidrug efflux pumps have greater clinical relevance than has previously been thought, because there is now accumulating evidence that certain classes of efflux pumps not only confer resistance to drugs used in therapy but also have a role in bacterial pathogenicity.

The Gram-positive bacterium Bacillus subtilis forms spores when conditions are unsuitable for growth. The spores are encased in a multilayered shell that includes a cortex and a spore coat, and remain viable for long periods in the harsh environment. In the present article, recent progress in our understanding of the outer structure of B. subtilis spores is reviewed in the Japanese language. Although spore coat assembly involves the deposition of at least 70 distinct protein species, the positions of most of such proteins have not been experimentally determined. To this end, the diameters of the protein layers and spores were measured using fluorescence microscopy and then the positions of proteins in the spore coat of B. subtilis spores were estimated. The locations of 16 proteins were determined using this method. One protein was assigned to the cortex, nine to the inner coat, and four to the outer coat. Further, two proteins, CgeA and CotZ, were assigned to a previously unidentified outermost layer. McKenney et al. have also identified the outermost layer using a similar method; the layer was termed the "crust". Immunofluorescence microscopy revealed that the crust is indeed the most external layer of B. subtilis spores. Mutational analysis indicated that all genes in the cotVWXYZ cluster were involved in spore crust synthesis and that CotY and CotZ played critical roles in crust formation.

To determine the Karp-type Orientia tsutsugamushi subtype in northern Japan, i.e., Yamagata, Niigata, and Akita Prefectures, we analyzed the partial nucleotide sequence of the 56-kDa protein-encoding gene of 30 isolates from scrub typhus cases. Based on sequencing results, we classified isolates into two groups of 27 and 3 isolates. Nucleotide sequences of 27 isolates were homologous to the yeo-joo strain, classified as a JP-1 subtype. The three isolates were each homologous to a stain of CMM1, KNP1, or KNP2, classified as JP-2 subtypes. Phylogenetic tree analysis showed the 27 isolates forming a cluster with the yeo-joo strain and the three isolates with the CMM, KNP1, and KNP2 strains and therefore belonging to these subtypes. The Karp-type O. tsutsugamushi JP-2 subtype predominates in Japan, the JP-1 subtype probably the predominates in the area investigated. O. tsutsugamushi JP-1 subtype strains must therefore be isolated from subjects in this area and comprehensively studied.

A 70-year-old man with diabetes mellitus seen for fever, right chest pain, and right-lung field consolidation on chest X-ray was found in thoracoabdominal computed tomography (CT) to have variable-sized nodules in both lung fields and multiple low-density hepatic areas. On physical examination, his pulse was 145 beats per minute and blood pressure 92/68mmHg, indicating a preshock state. Laboratory tests showed elevated WBC of 15,200/microL, serum-C-reactive protein (CRP) of 34.4 mg/dL, and a decreased platelet count of 16,000/microL. Suspecting liver abscesses complicated by a septic pulmonary embolism, we immediately conducted percutaneous transhepatic abscess drainage (PTAD). Liver abscess blood culture and drainage fluid grew the Klebsiella pneumoniae hypermucoviscosity phenotype, carrying the rmpA gene. Although the man had been in critical condition on admission, broad-spectrum antibiotics and PTAD treatment improved his clinical condition to where he could be discharged without problem.

Unlabelled: Gene mutation of Mycobacterium leprae was studied on bacilli-positive multibacillary leprosy patients since 2000 in Japan., Subjects: LL 31 cases, BL 7 cases., Results: gene mutation of folP was found in 19/36 cases (52.8%), that of rpoB in 13/33 cases (39.4%), that of gyrA in 6/31 cases (16.8%). Five cases showed both mutations of folP and rpoB, and one case showed those of fol/P and gyrA. Mutations of folP, rpoB andgyrA all were found in 4/36 cases (10.3%). High incidence of resistance to DDS or rifampicin was observed, and incidence of resistance to of loxacin was considerably high. These results reveal existence of multidrug-resistant bacilli in Japanese leprosy patients. Treatment of these cases was arranged according to the result of gene mutation analysis and satisfactory effects were obtained in 34/38 cases. One case did not response to newer treatment. Three cases refused use of improved treatment and their disease activities are not controlled. This study proved that gene mutation analysis is a rapid and accurate method to know the drug resistance against DDS, rifampicin and new quinolones, and important in chemotherapy of leprosy.

Nine clones of non-pathogenic streptococci were isolated from the pharynges of seven healthy subjects, and grown on sheep blood agar plates with a hemolysis or gamma hemolysis, then cultured in LB broth for 16 hrs. Purified streptolysin O (SLO) purchased from Sigma Chemical Co. (Sigma-SLO), SLO antigen as a latex agglutination reagent from A company (A-SLO) and supernatants from four culture media were electrophoresed on 12% SDS-polyacrylamide gel and transferred to PVDF membranes. Immunological analyses of antibodies against SLO in healthy sera and proteins in culture medium demonstrated that healthy sera contained an antibody recognizing Sigma-SLO, A-SLO and a protein of the same size as SLO (SLO-like protein) in culture medium. These findings suggest that healthy subjects develop an antibody directed against SLO-like protein produced by non-pathogenic streptococci, and that this antibody cross-reacts with Sigma-SLO and A-SLO. Using DNA from Streptococcus pyogenes and non-pathogenic streptococci, the SLO gene and SLO-like protein gene were analyzed by direct sequencing with oligonucleotide primers designed to cover no. 74 to approximately 1900 of the SLO gene. There were three different bases resulting in amino acid substitution between the SLO gene and SLO-like protein gene, namely 101Lys (AAA) of SLO to Asn (AAT), 175Met (ATG) to Arg (AGG) and 185Asp (GAT) to Asn (AAT). Remaining 560 residues of 563 amino acids constituting SLO-like protein were homologous to SLO. Non-pathogenic streptococci on the pharynges of healthy subjects produce an SLO-like protein composed of amino acids similar to those of SLO, which induces an antibody against this SLO-like protein in serum. It is likely that an antibody against SLO-like protein in healthy sera cross-reacts with SLO and causes a pseudo-positive reaction on ASO measurement by the latex agglutination method using SLO antigen.

Purpose: Evaluation of rifampicin-resistance by the line probe assay, for rifampicin-susceptible Mycobacterium tuberculosis strains which were classified as rifampicin-resistant by the phenotypic drug susceptibility testings., Materials and Methods: A total of 15 clinical isolates from NHO Kinki-chuo Chest Medical Center consisting of 6 rifampicin-resistant strains by the line probe assay despite susceptible result by the drug susceptibility testings, and 9 clinical isolates which showed the fluctuating results on repeated drug susceptibility testings. After we conducted 3 drug susceptibility testings and the line probe assay, we have examined the sequence analysis for confirming mutations in the rpoB gene., Results: All strains were determined rifampicin-susceptible or intermediate by the drug susceptibility testings with Minimum Inhibitory Concentration (MIC) which ranged from 0.25 to 4 microg/ml by BrothMIC MTB-1, whereas these isolates indicated rifampicin-resistance by the line probe assay and revealed mutations in the hot-spot region (69 bp) by the sequence analysis., Conclusion: We verified that the line probe assay might be useful for the correct determination of drug susceptibility, especially about the low-level rifampicin-resistant M. tuberculosis strains.

Objective: To clarify the genetic microheterogeneity of Mycobacterium lentiflavum and identify the predominant genotype., Materials and Methods: Clinical isolates of M. lentiflavum used in this study were obtained from sixteen patients of lung diseases. In order to assess their intraspecies variability, four gene fragments, from the 16S rDNA (1471 bp), 16S-23S ITS (282 bp), rpoB (306 bp), and hsp65 (401 bp), were sequenced., Results: Intraspecies variabilities were found in all of the four targeting fragments. As multilocus sequence typing with these four targets, 16 clinical isolates were divided into 3 genotypes, i.e., MLST2, MLST3, and MLST4. Among them, MLST2 to which 12 clinical isolates belonged, was a predominant genotype. Three strains belonged to MLST3 and the remaining one strain belonged to MLST4. Drug susceptibility study indicated that there was no clear relation between sequence types and drug susceptibility., Conclusion: Multilocus sequence typing could aid in characterization and in better understanding of the epidemiology of M. lentiflavum.

We studied distribution of thermostable direct hemolysin (TDH)- and TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in coastal sea water, sediment, and shellfish and related retail shellfish contamination in Shimane Prefecture, Japan, between 2002 and 2004. V. parahaemolyticus was isolated from > 80% of sea water, sediment, and shellfish. The detection of TDH gene (tdh) and TRH gene (trh)-positive V parahaemolyticus in sea water was 11%, in sediment 16%, and in shellfish 26%. The number of genes and gene-related in seawater was 23 MPN/L, in sediment 29 MPN/100 g, and in shellfish 460 MPN/10 g. TDH- and TRH-producing V. parahaemolyticus detected in seawater was 5%, in sediment 11% and in shellfish 14%. The continuous distribution of TDH-producing O2:K28, O4:K88, O4:K37, and O4:KUT organisms on the western coast and TRH2-producing O5:k30, O5:K43, O10:K19, O10:KUT, O11:K40, O11:KUT, and OUT:KUT organisms on the Oki Island coast suggested the settlement of these organisms in these coastal environments. From 7 (12%) of 59 retail short-necked clam samples, we isolated TDH-producing O 1:KUT, O3:K6 (2 strains from 2 samples imported from Korea), O4:K12, OUT:K8, and TRH2-producing OUT:K40 and OUT:K51 organisms. These findings suggested that TDH- and TRH-producing V. parahaemolyticus are widely distributed along the coast of this prefecture and are transported by contaminated retail shellfish from other areas.

Purpose: To classify a specific Mycobacterium among various mycobacteria utilizing sequencing of rpoB gene. To classify mycobacteria not identified by DNA-DNA hybridization (DDH) using sequencing of rpoB and 16S rRNA gene., Objects and Methods: Classification of 106 Mycobacteria strains, one Nocardia strain, one Rhodococcus strain, four Gordona strains was made by using partial sequencing of rpoB and 16S rRNA (RIDOM). Thereafter, 38 mycobacteria clinical strains not identified by DDH were classified utilizing the DNA sequencing data., Results: Pairs of M. kansasii and M. gastri, M. abscessus and M. chelonae, M.fortuitum (ATCC49404) and M. polcinum, M. peregrinum and M. septicum, M. farucinogense and M. senegalense and M. fortuitum (ATCC49403), Rhodococcus, Nocardia and Gordona strains were classified using sequencing of rpoB gene. Even though sequencing of rpoB and 16S rRNA gene was utilized, it was impossible to classify M. tuberculosis complex, M. avium family, M. marinum and M. ulcerans, and M. fortitum subsp. fortuitum and M. fortuitum subsp. acetamidolyticus. The 38 mycobacteria clinical strains not identified by DDH were successfully classified using sequencing of both rpoB and 16S rRNA. These sequencing analyses showed that M. heckeshornense, M. branderi, M. intermedium, M. shimoidei, M. wolinskyi, M. malmoense and M. lentiflavum could be identified. Thirty six clinical isolates (94.7%) and 32 clinical isolates (84.2%) were identified by rpoB sequencing and 16S rRNA sequencing (RIDOM), respectively., Conclusion: The classification ratio of mycobacteria including Nocardia, Rhodococcus and Gordona is 69.6% for sequencing of 16S rRNA and 89.3% for sequencing of rpoB gene. Sequencing of rpoB is useful for classification of mycobacteria due to its genetic diversity, but has some limitation in its application. In order to classify mycobacteria more accurately, it is important to combine sequencing of rpoB and 16S rRNA and biochemical/biological tests.

To develop the rapid and simple genomic diagnostic method, we analyzed the partial dnaA sequence of 27 mycobacterial species. The partial dnaA sequence could distinguish M. kansasii and M. gastri. Based on this region and RLEP sequence of M. leprae, we established the loop-mediated isothermal amplification method (LAMP) to detect each species. The LAMP method for M. kansasii and M. gastri, could detect 500 copies. Five copies of M. leprae genomic DNA could be detect in 30min. To simplify the sample processing, the LAMP assay was performed with FTA filter paper. M. leprae bacilli were applied on filter paper that lyses bacilli and bound DNA, eliminating sample centrifugation and extraction procedures. Assays of number standards showed reproducible detection rate 50 bacilli of M. leprae. Thus, The LAMP assay combined with FTA card has the advantages of rapid and simple detection and provides a practical, economical, and specific method for the diagnosis of M. leprae and NTM infection.

The minimum inhibitory concentrations (MICs) to rifampicin (RFP) for Mycobacterium tuberculosis complex distribute bipartitely, the most susceptible, wild isolates showing < or = 0.03 microg/ml of MICs and the resistant isolates are >2.0 microg/ml. As the results, a very few number of isolates showing the MICs between 0.06 microg/ml to 2.0 microg/ml are still interpreted as "indeterminate" by BrothMIC MTB. In this communication, we determined genetic mutations in rpoB gene of the isolates of M. tuberculosis complex those MICs to RFP resulted in "indeterminate" interpretations. Through the direct base-sequencing, genetic mutation (s) in rpoB gene associated with amino acid substitution(s) were found in 21 of 27 clinical isolates of M. tuberculosis complex. All the isolates with 0.06 microg/ml of MICs were confirmed as being a wild type, whereas those with > or =0.125 microg/ml of MICs have a variety of genetic mutations. There found one exception, that is, a strain with 0.5 microg/ml of MIC revealed no mutation in rpoB gene. In addition to direct base-sequencing, a line probe assay, LiPA . Rif TB (Innogenetics N.V., Zwijnaarde, Belgium) was comparatively evaluated. The results obtained were highly correlated, LiPA . Rif TB giving comparable readings for 26 (96.3S) of 27 isolates tested. With these results, it can be concluded that the interpretive breakpoints for RFP MICs determined by BrothMIC MTB should be revised as follows: susceptible, < or =0.06 microg/ml, and resistant, > or =0.125 microg/ml. Secondly, LiPA . Rif TB gave an accurate and rapid interpretation for RFP resistance, but it may be concerned for the occasional false-susceptible readings.