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Biofilm is a complex structure consisting of microorganisms such as bacteria, fungi and an extracellular matrix (ECM). Biofilms are involved in most microbial infections and show persistent resistance to antibiotic treatment and immune response. Both Aspergillus fumigatus and Streptococcus pneumoniae are colonizers that can form biofilms in the respiratory tract. These pathogens have been simultaneously isolated from the same patient, but their interaction is poorly understood. We observed morphological changes in single- and mixed-species biofilms prepared for confocal laser scanning microscopy and scanning electron microscopy (SEM). Pneumococci suppressed the development of a fungal biofilm, and it even disrupted a preformed fungal biofilm. When a preformed fungal biofilm was treated with pneumococci, the mycelial network was fragmented, and only bacteria could develop. SEM revealed that the fragmented mycelium was further disrupted into fine filaments as treatment time progressed, and that the ECM of the preformed fungal biofilm had disappeared. The pneumococcal culture supernatant contained mycelial fragmentation activity that was heat-sensitive. The culture supernatant of a mutant pneumococcal strain deficient in pneumolysin (Δply) also exhibited the mycelial fragmentation activity. Enolase and lactate oxidase, which are involved in glycolysis and hydrogen peroxide production, were identified in the culture supernatant of the Δply mutant. Neither the wild type nor the mutant strain could fragment the mycelium in the presence of catalase. These data suggest that hydrogen peroxide could fragment the mycelium and would terminate the co-existence of A. fumigatus and S. pneumoniae in biofilm.

Streptococcus pyogenes (Group A streptococci: GAS) are known to cause a wide variety of human illnesses, some of which can be life-threatening. Usually, penicillin is the first-choice agent for the treatment of GAS infections. For patients with penicillin or beta-lactam antibiotics allergies, macrolide drugs are recommended as an alternative therapy. However, an increased prevalence of macrolide-resistant GAS (MRGAS) has been reported in many countries. Furthermore, fluoroquinolone non-susceptible GAS has been reported. The present study was focused on determining the features of fluoroquinolone non-susceptible strains collected from children with pharyngotonsillitis in the southwestern areas of Japan. To reveal the characteristics of fluoroquinolone non-susceptible GAS, we investigated the MIC, T-serotype, emm typing, and PFGE of 298 GAS strains isolated in the Fukuoka southwest area of Japan between 2011 and 2013. We determined that fluoroquinolone non-susceptibility shows a MIC to tosufloxacin of ≧1 μg/ml. We identified 33 (11.1%) fluoroquinolone non-susceptible GAS strains. In these strains, 6 T-serotypes and 9 emm/MLST patterns were detected. The predominant combinations were emm6/ST382 (14 strains, 42.4%) and emm89/ST101 (5 strains, 15.2%). PFGE classified 10 pulsotypes, and each was quite different. These results showed that fluoroquinolone non-susceptible GAS strains have a variety of origins. The usage of fluoroquinolone drugs could have a negative effect on the antimicrobial drug sensitivity of GAS in Japan. Considering such a situation, continuous monitoring of quinolone non-susceptible GAS is necessary.

Objective In 2010, candid advice concerning the low rate of typhoid vaccination among Japanese travelers was received from Nepal. Recently, progressive Japanese travel clinics have encouraged Japanese travelers to be vaccinated against typhoid fever in conjunction with officially approved vaccines, such as hepatitis A vaccine. We herein report the status of typhoid vaccinations for Japanese travelers to the most endemic area (South Asia) and describe the factors associated with compliance. Methods In the travel clinic at Kurume University Hospital, we used the following criteria to retrospectively extract the records of new pre-travel Japanese clients between January 2011 and March 2015: hepatitis A vaccine administered, traveling to South Asian countries, and ≥2 years of age. We first summarized the participants and then divided them into typhoid-vaccinated and typhoid non-vaccinated groups for a comparative analysis. Results This study included 160 clients. A majority (70.0%) of these clients traveled for business. The duration of trips was long (≥1 month) (75.0%), and India was a popular destination (90.6%). A comparative study between the vaccinated group (n=122) and the non-vaccinated group (n=38) revealed that the two factors most positively associated with typhoid vaccination were business trips [adjusted odds ratio (aOR) 3.59, 95% confidence interval (CI) 1.42-9.06] and coverage by a company/organization payment plan (aOR 7.14, 95% CI 2.67-20.3). Conclusion The trend toward typhoid vaccination among Japanese travelers to South Asia with pre-travel consultation is correlated with business trips and coverage by a company/organization payment plan. If problems concerning the cost of vaccines were resolved, more travelers would request typhoid vaccination.

Bloodstream infection (BSI) due to carbapenem-resistant Enterobacteriaceae (CRE) has a high mortality rate and is a serious threat worldwide. Ten CRE strains (eight Enterobacter cloacae, one Klebsiella pneumoniae and one Citrobacter freundii) were isolated from the blood of nine patients, a percentage of whom had been treated with indwelling devices. The steps taken to establish cause included minimum inhibitory concentration (MIC) tests, a pulsed-field gel electrophoresis (PFGE), biofilm study, a multiplex PCR for resistant genes of carbapenemases and extended-spectrum beta-lactamases (ESBLs), and plasmid incompatibility typing. All strains showed a tendency toward resistance to multiple antibiotics, including carbapenems. Frequently isolated genes of ESBLs and carbapenemases include blaTEM-1 (four strains), blaSHV-12 (four strains) and blaIMP-1 (six strains). A molecular analysis by PFGE was used to divide the XbaI-digested genomic DNAs of 10 CRE strains into eight patterns, and the analysis showed that three E. cloacae strains detected from two patients were either identical or closely related. The biofilm production of all CRE strains was examined using a microtiter biofilm assay, and biofilm growth in continuous flow chambers was observed via the use of a confocal laser scanning microscope. Our study indicates that biofilm formation on indwelling devices may pose a risk of BSI due to CRE.

This study clarified the characteristics of travelers who received hepatitis B vaccinations. Subjects were 233 Japanese travelers who visited our clinic prior to travel. We summarized the characteristics of the clients and performed two comparative studies: first, we compared a hepatitis B-vaccinated group with an unvaccinated group; second, we compared a group that had completed the hepatitis B vaccine series with a group that did not complete the series. The hepatitis B vaccine was administered to 152 clients. Factors positively associated with the hepatitis B vaccination (after adjusting for age and sex) included the following: travel for business or travel as an accompanying family member; travel to Asia; travel for a duration of a month or more; and, inclusion of the vaccine in a company or organization's payment plan. Meanwhile, factors negatively associated with the vaccination were travel for leisure or education, and travel to North America or Africa. Among 89 record-confirmed cases, only 53 completed 3 doses. The completion rate was negatively associated with the scheduled duration of travel if it was from a month to less than a year (after adjusting for age and sex). The present study provides a basis for promoting vaccination compliance more vigorously among Japanese adults.

Objective To clarify the characteristics of post-travel consultation services in Japan, particularly in the provinces, we analyzed our post-travel patients in the travel clinic of Kurume University Hospital located in Kurume City (a regional hub City in southwestern Japan). Methods Sixty post-travel patients visited our clinic between April 2008 and October 2014 and participated in this study: 55 were Japanese and five were foreign. We summarized and compared the characteristics of the patients after dividing the Japanese participants into long-term travelers (>14 days) and short-term travelers (≤14 days). The foreign travelers were described in a separate analysis. Results Of the 55 Japanese travelers, the mean age (± standard deviation) was 37.3 ± 16.3 years, and 36 patients (65%) were men. Southeast Asia was the major destination (30/55, 55%), and business was stated as the major reason for travel (16/55, 29%). Post-exposure rabies prophylaxis (16/55, 29%) was the most common purpose for the consultations. There were 34 participants (62%) who were classified as short-term travelers. Fewer of the short-term travelers stated receiving pre-travel consultations compared with long-term travelers (11% vs. 79%, p=0.0002). The five foreign travelers included one dengue fever patient and two malaria patients. Conclusion Most post-travel Japanese patients visited our clinic were short-term travelers who had not received any pre-travel consultation. One of the most common complaints, post-exposure rabies prophylaxis, could have been avoided to some extent by appropriate pre-travel consultations. The results of this study suggest that pre-travel consultations should therefore be encouraged for both long- and short-term travelers.

Reticulon (RTN) proteins are localized to the endoplasmic reticulum (ER), and are related to intracellular membrane trafficking, apoptosis, inhibiting axonal regeneration, and Alzheimer's disease. The RTN proteins are produced without an N-terminal signal peptide. Their C-terminal domain contains two long hydrophobic segments. We analyzed the ER localization signal of human RTN1-A. Mutant proteins lacking the first (39 residues) or second (36 residues) hydrophobic segment showed ER localization. On the other hand, the mutant lacking both hydrophobic segments was cytosolic. Enhanced green fluorescent protein (EGFP) tagged with the first or second hydrophobic segment of RTN1-A was localized to the ER. These results suggest that each hydrophobic segment determines the ER localization. In addition, EGFP tagged with the truncated form of the first hydrophobic segment exhibited the localization to the Golgi rather than the ER. This suggests that the length of the hydrophobic segment contributes to the ER retention of RTN1-A.

Summary Background A vancomycin-intermediate Staphylococcus aureus (VISA) (vancomycin minimum inhibitory concentration: 4mg/L) outbreak occurred in an advanced emergency medical service centre [hereafter referred to as the intensive care unit (ICU)] between 2013 and 2014. Aim Our objective was to evaluate the infection control measures that were successful. Methods Seventeen VISA strains were isolated from the sputum of 15 inpatients and the skin of two inpatients. Fourteen VISA strains were recognized as colonization. However, three VISA strains were isolated from the sputum of three inpatients with pneumonia. Environmental cultures were performed and VISA strains were detected in five of 65 sites. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) was performed on 21 VISA strains. Findings Molecular typing including PFGE and MLST showed that the patterns of 19 VISA strains were identical and those of the other two VISA strains were possibly related. This meant that a horizontal transmission of VISA strains had occurred in the ICU. In August 2013, the infection control team began interventions. However, new inpatients with VISA strains continued to appear. Therefore, in October 2013, the ICU was partially closed in order to try to prevent further horizontal transmission, and existing inpatients with the VISA strain were isolated. Although new cases quickly dissipated after the partial closure, it took approximately five months to eradicate the VISA outbreak. Conclusion Our data suggest that despite the employment of various other infection control measures, partial closure of the ICU was essential in terminating this VISA outbreak.

The biochemical properties of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) truncated with C-terminal 21 amino acids and expressed in insect cells were analyzed. The enzyme carried copy-back and de novo RNA synthesis activity but not terminal nucleotidyl transferase activity. k(pol) and K(m) for de novo RNA synthesis were calculated as 10.0 pmol/microg/h and 2.5 microM under 0.5 mM GTP and 2.0 pmol/microg/h and 3.5 microM under 50 microM GTP, respectively. Those for copy-back RNA synthesis were similar under both conditions (k(pol), 1.8 pmol/microg/h; K(m), 3.0 microM). De novo RNA synthesis was activated by 0.5 mM GTP. However, the ratio of GTP to three other NTPs was important for activation. Our HCV RdRp showed high activity for the complementary sequence of the HCV internal ribosomal entry site and a synergistic effect of Mg(2+) to Mn(2+).

An epidemic of aseptic meningitis caused by human echovirus 9 (E-9) occurred in the summer of 1997 in northern Kyushu, Japan. Sequences of genome position 2504-3358, which encoded a part of VP1, of the nine isolated viruses were determined. An RGD motif and B-C loop were found in all. They were almost identical and closely related to the virulent strain Barty.

Background Influenza virus RNA polymerase is a multifunctional enzyme that catalyses both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood, although the enzyme is known to be required for cRNA vRNA synthesis. The protease related activity of PA has been discussed ever since protease-inducing activity was demonstrated in transfection experiments. Results PA protein was highly purified from insect cells infected with the recombinant baculovirus carrying PA cDNA, and a novel chymotrypsin-type serine protease activity was identified with the synthetic peptide, Suc-LLVY-MCA, in the PA protein. [3H]DFP was crosslinked with PA and a mutational analysis revealed that serine624 was as an active site for the protease activity. Conclusions These results constitute the demonstration of protease activity in PA subunit of the influenza virus RNA polymerase complexes.

Influenza A viruses (H3N2) isolated in 1998 in Nagasaki, Japan, carried a mutation (384R --> G) in one of the anchor amino acids of the HLA-B27-restricted cytotoxic T lymphocyte (CTL) epitope of NP (383-391). Phylogenetic analysis revealed that these viruses have been isolated only in Japan to date and belong to the unique lineages.

Influenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNA-cleaving DNA enzymes from 10-23 catalytic motif to target PB2-AUG initiation codon and measured their RNA-cleaving activity in vitro. Although the RNA-cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection.

Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Deltatom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.

Protein insertion into mitochondrial outer membrane (OM) vesicles isolated from Neurospora crassa has recently been reported. The N. crassa OM vesicles retained the features of the intact mitochondria concerning the dependency of insertion on the receptor protein [A. Mayer et al. (1993) J. Cell Biol. 121, 1233-1243]. In this study, OM vesicles were purified from bovine adrenal cortex mitochondria, and unilamellar proteoliposomes were reconstituted from OM vesicles using heptyl beta-thioglucoside. Both OM vesicles and the reconstituted outer membrane vesicles (ROM) were able to import porin, but unable to import the precursor of adrenodoxin, which translocates across both the outer and inner membranes of intact mitochondria. Porin insertion into both OM vesicles and ROM was inhibited in the presence of purified recombinant adrenodoxin precursor and also by ATP depletion, and was dependent on the trypsin-sensitive membrane surface factor, suggesting that the purified OM vesicles as well as ROM retained the properties of the intact OM concerning porin insertion. The protein import machinery of OM seems to be functional for the outer membrane protein without the participation of the inner membrane. The successful reconstitution of the protein import activity from solubilized OM will pave the way for further biochemical characterization of the protein import machinery of OM.

The precursor of bovine adrenodoxin (pAd), a mitochondrial protein, was expressed in Escherichia coli. The cloned cDNA of pAd was ligated to an expression vector pET-3d, and silent mutations were introduced into the N-terminal portion of the cDNA in order to increase the expression. The precursor was highly expressed (approximately 20% of the total cell protein) as the inclusion body, and contained an iron-sulfur center as judged from its optical absorption spectra. The inclusion body was solubilized with 7 M urea and pAd was purified in the presence of urea. The purified pAd was efficiently imported into isolated bovine adrenal cortex mitochondria and processed to the mature form. The import reaction required ATP inside the mitochondria in addition to the inner membrane potential, and was strongly inhibited by trypsin treatment of the mitochondria, as in the case of the in vitro translated precursor. It was, however, not dependent on the unfolding activity of the cytosolic factor with extramitochondrial ATP.

We focused on the relationship between variation in the IRES of hepatitis C virus (HCV) genotype 1b and clinical outcome, since the internal ribosome entry site (IRES) has a comparatively low heterogeneity and it might be easy to find unique substitutions. Patients infected with HCV were selected using strict criteria, and unique mutations in the IRES were extracted by the subtraction of common mutations. We found that most mutations accumulated in domain III (dIII) of IRES in sustained virological responders (SVRs) and non-SVRs before therapy. However, these mutations were exclusively observed in domain II (dII) in non-SVR at 2 weeks after the start of therapy.

An outbreak of acute keratoconjunctivitis involving 27 patients occurred in the Department of Ophthalmology, Kurume University Hospital. Adenoviral DNA was detected in four inpatients, one outpatient and one healthcare worker. Sequence-based typing of adenoviral DNA indicated serotype 3 from one inpatient, the rest being serotype 37. At a later stage of the outbreak adenoviral DNA types 37 and/or 3 were also detected from almost all environmental instruments and commonly used eye drops, despite thorough disinfection of the environment and enforcement of various infection control measures. The detection rate of adenoviral DNA in environmental swabs was 81%. A further second disinfection of the environment reduced the detection rate of adenoviral DNA to 38%. The outbreak ceased after closing the ophthalmology ward and outpatient consulting room, accompanied by enhanced cleaning of environmental instruments and the introduction of disposable eye drops for individual patients.

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTNgene produces 1–3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast twohybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis. 2002 Elsevier Science (USA). All rights reserved.

Hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp). Sequences in the 3' termini of both the plus and minus strand of HCV genomic RNA harbor the activity of a replication origin and a transcription promoter. There are unique stem-loop structures in both termini of the viral RNA. We found that the complementary strand of the internal ribosome-binding site (IRES) showed strong template activity in vitro. The complementary strand RNA of the HCV genome works as a template for mRNA and viral genomic RNA. We analyzed the promoter/origin structure of the complementary sequence of IRES and found that the first and second stem-loops worked as negative and positive elements in RNA synthesis, respectively. The complementary strand of the second stem-loop of IRES was an important element also for binding to HCV RdRp.

cDNA of mouse reticulon 3 (mRTN3) was cloned. The cloned cDNA is 1745 bp long and contains an open reading frame of 237 amino acids for a 30 kDa protein. The gene was mapped at band B of chromosome 19 by FISH. Two altematively spliced transcripts, 3.4 and 2.3 kb, of mRTN3 were found by Northem blot analysis. Both transcripts were expressed in many tissues and embryos and the highest expression of the 3.4 kb-transcript was observed in the brain, especially in neurons. The expression of 30 kDa-mRTN3 protein was also greatest in the brain. Both N and C-termini of mRTN3 faced the cytosol, indicating that they may recruit other proteins to the endoplasmic reticulum.

In Japan, the use of amantadine for treatment of influenza A virus infection was not accepted until November 1998, although it was widely used for treatment of Parkinsonism. Since then, we have monitored the emergence of amantadine-resistant viruses and isolated two viruses from patients on long-term treatment with amantadine.

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH2-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)–induced translation arrest and photo–cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH2-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

Mitochondrial import stimulation factor (MSF) unfolds wheat germ lysate synthesized aggregated mitochondrial precursor proteins and stimulates their mitochondrial import in an ATP dependent manner. Here we analysed the function of MSF mainly by utilizing chemically pure adrenodoxin precursor (pAd). MSF bound to the unfolded pAd and prevented it from losing import competence and also restored the import competence of the aggregated pAd dependent on ATP hydrolysis. The import incompetent aggregated mitochondrial precursors induced the ATPase activity of MSF and the activity was strongly inhibited by isolated mitochondrial outer membrane (OM) but not by trypsin treated outer membrane (tOM). The precursor induced ATPase activity of N-ethylmaleimide (NEM)-treated MSF was not inhibited by OM. In this context, the MSF-precursor complex specifically bound to OM and binding was abolished both by the treatment of OM with trypsin and by the treatment of MSF with NEM. These results show that MSF is a novel cytoplasmic chaperone protein with a mitochondrial precursor-targeting function.