Molecular Analysis for Potential Hospital-Acquired Infection Caused by Aspergillus Tubingensis Through the Environment.

The identification of Aspergillus species has been performed mainly by morphological classification. In recent years, however, the revelation of the existence of cryptic species has required genetic analysis for accurate identification. The purpose of this study was to investigate five Aspergillus section Nigri strains isolated from a patient and the environment in a university hospital. Species identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identified all five black Aspergillus strains as Aspergillus niger. However, calmodulin gene sequence analysis revealed that all five strains were cryptic species, four of which, including the clinical strain, were Aspergillus tubingensis. Hospital-acquired infection of the patient with the A. tubingensis strain introduced from the environment was suspected, but sequencing of six genes from four A. tubingensis strains revealed no environmental strain that completely matched the patient strain. The amount of in vitro biofilm formation of the four examples of the A. tubingensis strain was comparable to that of Aspergillus fumigatus. An extracellular matrix was observed by electron microscopy of the biofilm of the clinical strain. This study suggests that various types of biofilm-forming A. tubingensis exist in the hospital environment and that appropriate environmental management is required.

Characteristics of Biofilms Formed by C. parapsilosis Causing an Outbreak in a Neonatal Intensive Care Unit.

BACKGROUND: We dealt with the occurrence of an outbreak of Candida parapsilosis in a neonatal intensive care unit (NICU) in September 2020. There have been several reports of C. parapsilosis outbreaks in NICUs. In this study we describe our investigation into both the transmission route and the biofilm of C. parapsilosis. METHODS: C. parapsilosis strains were detected in three inpatients and in two environmental cultures in our NICU. One environmental culture was isolated from the incubator used by a fungemia patient, and another was isolated from the humidifier of an incubator that had been used by a nonfungemia patient. To prove their identities, we tested them by micro satellite analysis. We used two methods, dry weight measurements and observation by electron microscopy, to confirm biofilm. RESULTS: Microsatellite analysis showed the five C. parapsilosis cultures were of the same strain. Dry weight measurements and electron microscopy showed C. parapsilosis formed biofilms that amounted to clumps of fungal cells. CONCLUSIONS: We concluded that the outbreak happened due to horizontal transfer through the humidifier of the incubator and that the C. parapsilosis had produced biofilm, which promoted an invasive and infectious outbreak. Additionally, biofilm is closely associated with pathogenicity.

Reducing the urine collection rate could prevent hospital-acquired horizontal transmission of multidrug-resistant Pseudomonasaeruginosa.

INTRODUCTION: Multidrug-resistant Pseudomonas aeruginosa (MDRP) is a waterborne pathogen that occasionally causes hospital-acquired infection in immunocompromised or critically ill patients. Urine is frequently collected to evaluate renal function or to perform hormonal examinations, but the procedure involves risk due to the possibility of healthcare workers with contaminated hands. Our objective was to evaluate the association between the urine collection and hospital-acquired horizontal transmission of MDRP. METHODS: We monitored the urine collection rate from 2011 to 2017, as part of ongoing efforts to reduce the need to collect urine. The urine collection rate and the frequency of isolation of MDRP, Methicillin resistant S. aureus (MRSA) and extended spectrum ß-lactamases (ESBL)-producing E. coli were analyzed during the same period. PFGE and MLST were also performed to analyze the identity of 5 MDRP strains detected on the same ward in 2014-2015. RESULTS: The urine collection rate was dramatically decreased from 4.8% in 2011 to less than 0.5% in 2017, because the isolation rate of MDRP was significantly positively associated (RR = 1.72, 95%CI:1.03-2.85) with the urine collection rate. Isolations of MRSA and ESBL-producing E. coli showed no significant. Molecular typing showed the PFGE patterns of 3 of 5 MDRP strains were closely related as did MLST (ST17), and the remaining 2 MDRP strains had different PFGE and MLST patterns (ST14, ST655). Our data implicated the urine collection as one of the causes of hospital-acquired MDRP infections. CONCLUSIONS: We concluded that a reducing the urine collection rate could contribute to preventing hospital-acquired horizontal transmission of MDRP.

Infection Control for a Carbapenem-Resistant Enterobacteriaceae Outbreak in an Advanced Emergency Medical Services Center.

BACKGROUND: A carbapenem-resistant Enterobacteriaceae (CRE) outbreak occurred in an advanced emergency medical service center [hereafter referred to as the intensive care unit (ICU)] between 2016 and 2017. AIM: Our objective was to evaluate the infection control measures for CRE outbreaks. METHODS: CRE strains were detected in 16 inpatients located at multiple sites. Environmental cultures were performed and CRE strains were detected in 3 of 38 sites tested. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and detection of ß-lactamase genes were performed against 25 CRE strains. FINDINGS: Molecular typing showed the PFGE patterns of two of four Klebsiella pneumoniae strains were closely related and the same MLST (ST2388), and four of five Enterobacter cloacae strains were closely related and same MLST (ST252). Twenty-three of 25 CRE strains harbored the IMP-1 ß-lactamase gene and 15 of 23 CRE strains possessed IncFIIA replicon regions. Despite interventions by the infection control team, new inpatients with the CRE strain continued to appear. Therefore, the ICU was partially closed and the inpatients with CRE were isolated, and the ICU staff was divided into two groups between inpatients with CRE and non-CRE strains to avoid cross-contamination. Although the occurrence of new cases dissipated quickly after the partial closure, a few months were required to eradicate the CRE outbreak. CONCLUSION: Our data suggest that the various and combined measures that were used for infection control were essential in stopping this CRE outbreak. In particular, partial closure to isolate the ICU and division of the ICU staff were effective.

Disruption of Aspergillus fumigatus biofilm by Streptococcus pneumoniae: Mycelial fragmentation by hydrogen peroxide.

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.

Molecular epidemiology, antimicrobial susceptibility, and characterization of fluoroquinolone non-susceptible Streptococcus pyogenes in Japan.

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.

Biofilm-Forming by Carbapenem Resistant Enterobacteriaceae May Contribute to the Blood Stream Infection.

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.

Typhoid Vaccination among Japanese Travelers to South Asia and the Factors Associated with Compliance.

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.

Hepatitis B Vaccination Status among Japanese Travelers.

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.

Post-Travel Consultations in a Regional Hub City Hospital, Japan.

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.

A mutational shift from domain III to II in the internal ribosome entry site of hepatitis C virus after interferon-ribavirin therapy.

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.

Two hydrophobic segments of the RTN1 family determine the ER localization and retention.

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.

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis.

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 RTN gene 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 two-hybrid 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.

Promoter/origin structure of the complementary strand of hepatitis C virus genome.

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.

Molecular cloning and characterization of the mouse reticulon 3 cDNA.

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.

Kinetic analysis of C-terminally truncated RNA-dependent RNA polymerase of hepatitis C virus.

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+).