Japanese encephalitis (JE) is a seasonal epidemic disease with a 50-year recorded history in China. Its characteristics can be summarized as follows: (i) it is a seasonal epidemic disease; approximately 90% of cases are recorded in July, August, and September each year. The peak of JE onset is 1 month earlier in South China than in the north of the country; (ii) the disease is highly sporadic. It is rare for more than two cases to appear simultaneously in one family; (iii) most affected children are under 15 years old; (iv) the disease is widely distributed in all areas of the nation except Qinghai Province, Xinjiang Uygur Autonomous, and Tibet. Due to widespread application of the JE vaccine, the number of JE cases has decreased significantly nationwide, from 174,932 cases of morbidity in 1971 to 5,097 cases in 2005.

The fungal pathogen Candida albicans is an opportunistic fungal pathogen that causes oral and vaginal mucosal infections as well as systemic disease. The ability of C. albicans to adhere to host surfaces is positively correlated with its pathogenicity. We prepared a polyclonal anti-Candida albicans antibody in chicken egg yolk (anti-C. albicans IgY) and investigated its in vitro effectiveness in preventing C. albicans adherence and biofilm formation. Anti-C. albicans IgY significantly reduced the adherence of C. albicans SC5314 to human oral epithelial cells in a dose-dependent manner. The same effect was also observed in other Candida spp. including C. albicans serotype A and B. Further, the IgY inhibited biofilm formation of C. albicans in medium without serum, but the inhibition was slightly restored in medium conditioned with 10% serum. The data indicate that anti-C. albicans IgY cross-reacted with various Candida spp. and may have a protective effect against oral candidiasis and reduce the dissemination of Candida spp. This effect may be due to the blocking of the binding of Candida spp. to the host cells. However, the blocking did not play a role when Candida formed a germ tube in the presence of serum. Therefore, anti-C. albicans IgY may be considered as a prophylactic immunotherapy or possibly an adjunctive antifungal therapy under limited conditions.

Dengue is the one of the most prevalent arthropod-borne viral diseases. Dengue virus circulates between humans and mosquitoes, and causes a wide range of disease in humans. To elucidate the link between the cell tropism of dengue virus and its pathogenesis, peripheral blood cells of infected patients were analyzed by flow cytometry. The dengue virus antigen was detected in peripheral CD19⁺ cells (B cells) in one dengue hemorrhagic fever patient. Two dengue type-2 virus isolates were recovered from this patient using mosquito cell line C6/36 and human hematopoietic cell line K562, and designated VNHCM18-C/02 and VNHCM18-K/02, respectively. VNHCM18-K/02 exhibited strong binding ability and high infectivity to a B-lymphocyte cell line (RPMI8226) but showed poor growth in C6/36 cells, while VNHCM18-C/02 more efficiently and dominantly grew in C6/36 cells but did not efficiently bind to nor infect the B-cell line. Three amino acid differences were detected; one in an envelope protein (E-62) and two in nonstructural proteins. The distinct cell-binding to RPMI8226 was attributed to the difference between the two isolates in envelope protein E-62. Thus, we isolated two dengue type-2 virus variants with different cell-tropisms from the same patient, suggesting possible co-circulation in the patient.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 induces the formation of filamentous, actin-rich, pedestal-shaped structures beneath bacterial cells that have attached to intestinal epithelial cells. Pedestal formation requires the translocation of EHEC O157:H7 type III effectors. One of these type III effectors, EspFu, consists of an N-terminal signal sequence, which is necessary for the translocation of EspFu into the host cell through a type III secretion system, and almost identical proline-rich repeats (PRRs), which control actin rearrangement and increase the efficiency of actin assembly in the host cell. In this study, we report that insulin receptor tyrosine kinase substrate p53 (IRSp53) in the host cell acts as a binding partner for EspFu. Co-immunoprecipitation and fluorescence microscopy showed specific interactions between EspFu and IRSp53 as well as their co-localization in epithelial cells. Additionally, we demonstrated that the association between EspFu and IRSp53 induces dynamic membrane remodeling in epithelial cells.

Campylobacter jejuni has the potential to thrive at 37ºC (e.g., in the human intestinal tract) and 42ºC (e.g., in the poultry intestinal tract). We aimed to determine the protein expression profiles of C. jejuni cultured at 37ºC and 42ºC in vitro by two-dimensional electrophoresis (2DE). The differentially expressed spots/proteins between C. jejuni cultured at 37ºC and 42ºC were defined when their expression differed by twofold. The differently expressed spots detected from C. jejuni cultured both on agar and in broth at 37ºC and 42ºC were subjected to protein identification by MALDI TOF/TOF. Overall, 15 and 20 differentially expressed proteins were defined for C. jejuni cultured at the two temperatures on agar and in broth, respectively. All of the identified differentially expressed proteins could be clustered as proteins involving the metabolism, regulator system, periplasmic proteins and the major antigens of C. jejuni. In conclusion, there are subsets of proteins that are optimally expressed at 37ºC, which may contribute to the host adaptation and/or the pathogenicity in the human intestinal tract.

Eleven Salmonella Choleraesuis and seven Salmonella Hadar strains isolated from various clinical humand samples were investigated by plasmid profile analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and pulsed-field gel electrophoresis (PFGE) in order to obtain information at a molecular level on the epidemiology of S. Choleraesuis and S. Hadar, which are significantly present in Turkey. Plasmid profile analysis showed that 10 (90.9%) of 11 S. Choleraesuis isolates harbored one to two plasmids with sizes of 2.0, 5.0 or 6.5 kb; and 5 (71.4%) of 7 S. Hadar isolates harbored one to three plasmids ranging from 2.5 to 70 kb. ERIC-PCR was performed using ERIC-2 primers; since isolates within each serotype showed similar band models, we concluded that ERIC-PCR is not suitable for differentiating isolates within the same serotype and for grouping into clusters. In PFGE using the AvrII enzyme, S. Choleraesuis isolates formed three clusters, and S. Hadar isolates formed three clusters; using the XbaI enzyme, S. Choleraesuis isolates formed two clusters, and S. Hadar isolates formed four clusters. These results showed that plasmid profile analysis and PFGE are reliable and discriminative methods that would complement antibiograms, and could contribute to the investigation of outbreak epidemiology. This is the first report on S. Choleraesuis and S. Hadar isolates from Turkey investigated by plasmid profile analysis, ERIC-PCR and PFGE methods.

The purpose of the current study was to investigate the presence and molecular type(s) of extended-spectrum β-lactamases (ESBLs) in Salmonella spp. isolates obtained from patients with diarrhea in hospitals of Tehran, Iran. Over a period of 17 months, 129 Salmonella spp. were isolated from fecal samples and tested for susceptibility using the Kirby-Bauer disk diffusion method; then, screening for ESBL-producing isolates and determination of their minimum inhibitory concentrations were carried out using the combined disk method and standard agar dilution method, respectively. The presence and type of ESBL-encoding genes were determined by PCR and sequence analysis. The isolates were all identified as Salmonella enterica of different serovars. The highest resistance in the collected Salmonella isolates was to nalidixic acid (45.7%), followed by tetracycline (43.4%), trimethoprim-sulfamethoxazole (36.4%), ampicillin (15.5%), and chloramphenicol (14.7%). All the isolates were susceptible to ciprofloxacin, gentamicin, and cefoxitin. Three S. enterica isolates were resistant to ampicillin, piperacillin, ceftazidime, cefotaxime, ceftriaxone, cefpodoxime, cephalothin, and aztreonam. PCR and DNA sequencing revealed that two of the three isolates harbored both a bla_CTX-M-15 and a bla_TEM gene while the third one carried only a bla_CTX-M-15 gene. This is the first study providing structural data for a CTX-M-type β-lactamase produced by Salmonella isolates recovered in Iran.

Pulsed-field gel electrophoresis (PFGE) is the most common genotyping method used for the typing of a number of bacterial species. Generally, investigators use their own custom-developed protocol, but a standardized PFGE protocol would allow the comparison of typing results between laboratories and the tracing of strains around the country. In the present study, we optimized a PFGE protocol for subtyping of Acinetobacter baumannii, Escherichia coli and Klebsiella spp., which are commonly isolated from nosocomial infections in many hospitals. Reproducibility of our PFGE procedure was studied three times at 2- to 3-week intervals. Epidemiological concordance of the optimized PFGE procedure was tested on seven isolates of A. baumannii from a previous outbreak and seven A. baumannii isolates randomly selected among the clinical isolates. The optimized PFGE procedure was evaluated on a total of 174 clinical isolates including 62 A. baumannii, 50 E. coli, and 62 Klebsiella spp. The inter-laboratory reproducibility of the optimized protocol was tested at four laboratories. The optimized procedure is completed in 28 h after culturing. It is likely to be cost-effective, due to the reduction in the time, reagent volume and enzyme concentration needed. The procedure showed high concordance with epidemiological data. There were no non-typeable isolates among the tested bacteria. It is reproducible and versatile. This protocol can be used to identify outbreaks and monitor the spreading rate of nosocomial infections caused by the tested bacterial isolates. Furthermore, due to its high intra- and inter-laboratory reproducibility, the protocol has the potential to be useful for comparing PFGE fingerprinting profiles of the isolates from different settings.

By proving the bactericidal effects of a low-concentration titanium dioxide (TiO₂) particle mixture against Staphylococcus aureus, we hope to ultimately apply a mixture of this type as part of a clinical treatment regimen. A bacterial suspension of S. aureus 1×10⁵ CFU/ml was added dropwise to a TiO₂ particle mixture (19 ppm TiO₂) and irradiated by ultraviolet (UV) light. The colony-forming units were counted and the bacterial survival rate was calculated. In the control sample, the bacterial survival rate was 83.3% even after 120 min. In the TiO₂ mixture + UV sample, the bacteria count dropped sharply, reaching 17.3% of the baseline value at 30 min and 0.4% at 60 min. TiO₂ particles dispersed in water mixtures are known to elicit highly efficient UV absorption and greater bonding to bacteria. A reaction of the TiO₂ with another oxidizer altered the aqueous pH and accelerated the photocatalytic chemical reaction. The TiO₂ particle mixture showed high antibacterial action against S. aureus even at a low concentration.

Pyogenic liver abscess (PLA) formation is thought to originate from the transmission of infection via three major routes including the biliary tract, portal vein and hepatic artery. However, about 50% of PLA cases are considered to be cryptogenic. Here we report an unusual autopsy case of PLA associated with periodontopathic bacterial infection. A 59-year-old female suddenly developed cardiopulmonary arrest and died. Despite macroscopic and microscopic examinations, the infectious routes and source of infection were unidentified, and the case appeared to be cryptogenic. Since this patient had suffered severe periodontitis for a long period of time, we investigated the involvement of periodontal infection in PLA formation by performing immunohistochemical analyses. We identified several periodontopathic bacterial species in the PLA of this patient, including Fusobacterium nucleatum, Treponema denticola, Prevotella intermedia and Porphyromonas gingivalis. Thus, we demonstrate here that periodontal infection is a potential source of infection in the formation of PLA.

We investigated the linezolid susceptibility of Mycobacterium tuberculosis strains isolated from a tertiary care hospital in Izmir. A total of 67 M. tuberculosis strains (33 multidrug-resistant [MDR] and 34 non-MDR) were isolated and identified by the Tuberculosis Laboratory, Department of Microbiology and Clinical Microbiology, Faculty of Medicine, Ege University. The activity of linezolid was studied by the standard agar proportion method. For all of the strains, the MIC range was 0.06-1 mg/L, and the MIC₅₀ and MIC₉₀ values were 0.5 mg/L. No differences were observed between the MDR and non-MDR isolates. In general, linezolid was found to be effective for both the non-MDR and MDR M. tuberculosis strains.

A novel genotyping method for methicillin-resistant Staphylococcus aureus (MRSA), the phage open-reading frames typing (POT) method, was evaluated using 92 MRSA isolates collected from blood cultures between 1991 and 2003 at Nagoya University Hospital. These strains were divided into 64 distinct POT types, classified into 21 genotypes by pulsed-field gel electrophoresis (PFGE) using SmaI, and analyzed with the DICE coefficient of 80% in dendrogram analysis, with 48 subtypes analyzed with the DICE coefficient of 100%. The discriminatory indices of these three methods were 0.988, 0.719, and 0.953, respectively. The first and second prevalent PFGE subtypes A1 and A2, which comprised 16 and 13 isolates recovered serially during the study period, were both divided into 11 distinct POT types. Six isolates belonging to PFGE subtype A1 were indistinguishable with POT. The six isolates were probably involved in an outbreak. Phenotypic analysis suggested that these isolates were the siblings of the New York/Japan clone which are prevalent in many Japanese hospitals. In conclusion, in the strain population studied, POT is a more rapid and discriminatory method than PFGE, and is a useful epidemiological tool for evaluating the available clinical information.

An association between diabetes mellitus (DM) and tuberculosis (TB) has been implied for a long time. We previously reported that KDP type 1 diabetic rats and GK type 2 diabetic rats are highly susceptible to Mycobacterium tuberculosis infection. As a next step, we conducted a retrospective analysis of 2,141 patients with pulmonary TB newly diagnosed during the period from 2008 to 2009 to evaluate the influence of DM on the drug response rate and the long-term relapse rate of TB. There were 203 DM patients with TB (type 1 DM, 7 [3.4%]; type 2 DM, 196 [96.6%]). The TB relapse rate (2 years after discharge) was higher in DM patients than in non-diabetic patients (20% versus 5.3%). The frequency of multidrug-resistant-TB among DM patients with TB was higher than that among TB patients (17.7% versus 8.4%, P < 0.01). These results suggest that the period of TB treatment should be prolonged, and that in the meantime the blood glucose level should be maintained within a reference value range.

It is important to conserve the effectiveness of antifungal agents because the options for currently available agents are limited. Although echinocandins, which have been developed in recent decades, are highly active against a broad spectrum of fungi, one concern is their reduced activity against Candida albicans at high drug concentrations, which is known as the paradoxical effect. To date, resistance related to the paradoxical effect has not been reported in clinical situations, but some in vivo data suggest that the paradoxical effect potentiates the emergence of resistance. It is valuable to investigate the underlying mechanisms of as well as strategies against this paradoxical resistance. Previous reports imply that the paradoxical effect might be related to stress responses. In this study, we report that radicicol, a heat shock protein 90 (Hsp90) inhibitor, reduces the paradoxical effect of micafungin. We also confirm that radicicol reduces the tolerance to voriconazole, one of the new azoles, which is consistent with a previous report. Our results may therefore imply that common stress responses might exist in the paradoxical resistance to micafungin and also the tolerance to voriconazole, and may suggest that inhibiting Hsp90-related stress responses could help to avoid potential resistances.

Recently, the recombination event of norovirus (NoV) has been reported with high frequency, suggesting that RNA recombination is a major driving force in NoV evolution. To assess the incidence of NoV recombination in a residential area, we conducted a molecular biological survey of NoVs existing in sewage water in Toyama Prefecture, Japan. Although GII/4 was predominantly detected in sewage water that was associated with a high frequency of outbreaks caused by this genotype, other genotypes, including two types of recombinant strain, were identified during the survey period. One of the recombinants is the WUG1 type, which was first detected in Saitama Prefecture in 2000. The other recombinant is a novel type derived from two parent strains of genogroup II, GII/7 for the RNA-dependent RNA polymerase and GII/13 for the capsid. This suggests that certain NoVs circulating in the area are occasionally changing their genetic properties by recombination events.

The incidence of influenza in the Naha city area in the southernmost part of Japan was surveyed in 2007 and 2008. Patients who had influenza-like symptoms and visited one of four general hospitals in Naha City, Okinawa, Japan were included in this study. The nasal or throat swab samples were applied to the rapid test for detecting influenza A and B virus antigens. The positive rate of influenza A and/or B virus antigen was 26.2% (8,480/32,380). Most cases (82.9%) were influenza A. In 2007, influenza A cases were detected during the entire year, and an epidemic peak was also noted in July, while no outbreak occurred in the summer of 2008. The surveillance of the rapid influenza virus antigen test seemed to provide reliable epidemiological data. This finding warrants further study in this region, including study of the influences of climate and socio-behavior patterns of the residents in the region on influenza epidemics.

Infection with Japanese encephalitis virus (JEV), a mosquito-borne, neurotropic flavivirus, may cause acute encephalitis in humans. Recombinant Salmonella typhimurium BRD509 was constructed to display domain III of the envelope (E) protein of JEV (JEDIII) on its surface with the N-terminal domain of ice nucleation protein (INPN) as the display motif. Bacterial cell surface display was confirmed by Western blot analysis and immunohistochemical staining. Binding of recombinant INPN-JEDIII and JEDIII proteins to three mammalian cell lines was compared using a cell-binding ELISA; the human neuroblastoma cell line SK-N-SH, which had a low level of binding, was selected for further studies. The display of JEDIII on the surface of BRD509 did not significantly influence its invasiveness was confirmed by measuring released bacterial antigen using whole-cell ELISA. The relative expression of an apoptosis-related gene and total DNA damage were assessed to investigate the effects of infection on SK-N-SH cells. Compared to BRD509, infection with the recombinant bacterium reduced cell damage, suggesting that JEDIII may limit apoptosis during the early stages of JEV infection. Our studies demonstrated that it is feasible to study the pathogenesis of JEV using the approach described.