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Na+/H+ antiporter plays an important role in maintaining cellular homeostasis by regulating osmotic pressure and intracellular pH. It plays an important role in maintaining cellular homeostasis. InHelicobacter pylori, whole genome sequencing has revealed the presence of two types of Na+/H+ antiporter. A gene (nhaA) homologous to the Na+/H+ antiporter ofEscherichia colihas been investigated and its function has been analyzed. However, another gene homologous to the Na+/H+ antiporter ofEnterococcus hirae(napA) is not yet known in detail. In this study, we investigated the function of this gene (napAinH.pylori). First, to confirm the genetic presence ofnapAin 20H. pyloriclinical isolates, PCR analysis was performed, and thenapAgene was confirmed in all strains. The amount of Na+ extrusion was measured by atomic absorption spectroscopy. The results showed that the Na+ concentration was decreased in the wild-type strain compared to thenapAmutant strain. In addition, there was a significant dose-dependent difference in CFU of Na+ concentration in thenapAmutant strain compared to the wild-type strain. We examined whether thenapAgene is related to motility using both wild-type andnapAmutant strains. As a result, in the motility agar test, the bacterial motility observed in the wild-type strain was not observed in thenapAmutant strain. However, no difference in flagellar proteins was observed by SDS-PAGE analysis. These results suggest that thenapAgene ofH. pylorimay regulate homeostasis by extruding Na+ and may also regulate motility.&nbsp

We evaluated the effect of restriction and modification genes expressed inCampylobacter jejuni, a naturally competent organism, on transformation efficiency. Fourteen strains ofC. jejuniwere tested for sensitivity to 29 restriction enzymes. Strains that showed the same susceptibility pattern by either agarose gel electrophoresis or PFGE were grouped together. Natural transformation between strains within and between groups was assessed using two chromosomal markers, one an artificially introduced gene and the other a selected point mutation. However, no correlation was found between transformation efficiency and sensitivity to the restriction enzymes tested. The activity of the restriction and modification system alone could not explain the failure of transformation to occur or the generation of stable clones in a population ofC. jejuniwith other abilities. It is likely that other factors influence horizontal gene flow in this organism. We suggested that the rule that the restriction and modification system affect horizontal gene flow, based on studies of transfection withE. coli, may not apply to the exchange of chromosomal DNA inC. jejuni.

Helicobacter pylori(H. pylori) urease generates both ammonia (NH3) and carbon dioxide (CO2) from urea. NH3helpsH. pylorito survive in the stomach in part by neutralizing gastric acid. However, the relationship between CO2andH. pyloriis not completed cleared. We examined the effect of CO2generated by urease on multiplication ofH. pyloriby using isogenicureBmutant andureBcomplemented strain fromH. pyloristrain JP26. Wild-type strain survived in the medium supplement with 1mM urea in room air, however, the urease negative strain did not. To discern whether CO2was incorporated intoH. pylori,14C in bacillus was counted after 6 hours incubation with14C urea in both acidic and neutral medium. Significant more14C uptake was detected in wild-type strain compared toureBmutant strain and this uptake in the wild-type strain was more under acidic condition compared to under neutral condition, but no difference was identified in the mutant strain. These results suggest that CO2generated by urease plays a role in multiplication ofH. pylori.

Pyelonephritis is an inflammation of the kidney tissue, calyces, and renal pelvis. It is commonly caused by bacterial infection that has spread up the urinary tract. Common bacteria are Escherichia coli and Enterococcus faecalis. Beta hemolytic streptococci also cause pyelonephritis. Beta hemolytic streptococcus such as Streptococcus agalactiae, and S. dysgalactiae subsp. equisimilis, when grown on blood-agar, produce complete hemolysis around each colony, usually cause severe infections including many cases of cellulitis, pneumonia, meningitis and sepsis. However the analysis of pyelonephritis causing beta hemolytic streptococcus has seldom been investigated. In this study, we tried to clarify the relationship between beta hemolytic streptococcus and pyelonephritis patient in Japan. We investigated beta hemolytic streptococcus isolated from pyelonephritis patient at two tertiary care hospitals in the central region of Japan by analyzing the background and antimicrobial susceptibility of beta hemolytic streptococcus. We recovered total sixteen beta hemolytic streptococcus in nine years. In pyelonephritis patients, beta hemolytic streptococcus was isolated from 9 S. agalactiae and 7 S. dysgalactiae subsp. equisimilis, respectively. The numbers of streptococci from female patients were more than those from male patients. Bacteria were not isolated from under 30 year patients. All S. dysgalactiae subsp. equisimilis were isolated from 60-year-age patients. About one fifth of beta hemolytic streptococcus had erythromycin and minocycline-resistant ability. Four beta hemolytic streptococci that were all S. agalactiae, had levofloxacin-resistant ability. Only one S. agalactiae had trimethoprim-sulfamethoxazole-resistant ability. Ampicillin was effective against all beta hemolytic streptococci. In our results, beta hemolytic streptococcus caused pyelonephritis in aged people and the quarter of these bacteria had some antibiotics resistance.

Haemophilus influenzae is an important pathogen that caused several infection diseases, such as sinusitis, otitis media, sepsis, and meningitis. This study was conducted to find out the prevalence and antimicrobial susceptibility pattern of Haemophilus influenzae isolates at general hospital in the central region of Japan from December 2015 to January 2016. Haemophilus influenzae was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by micro dilution assay according to CLSI recommendation. One hundred ninety-one Haemophilus influenzae were isolated, among which 95 (49.7%) were from male and 96 (50.3%) were from female. The age incidence of (0) years, (≤2) years, (≤5) years, and (6≤) years groups were 22(11.5%), 92(48.2%), 61(31.9%), and 16(8.4%), respectively. Positive samples were received mostly from the nasal discharge (177/92.7%), sputum (6/3.1%), tonsillar (6/3.1%), and pharynx (2/1.0%). Ceftriaxone was the most active antibiotics with 100% susceptible rates, followed by ciprofloxacin (99.5%) and minocycline (99%) in our study. Furthermore, we categorized four patterns: beta lactamase-negative ampicillin-sensitive strain (BLNAS), beta lactamase-negative ampicillin-re- sistant strain (BLNAR), beta lactamase-positive ampicillin resistant strain (BLPAR), and beta lactamase-positive amoxicillin-clavulanic acid-resistant strain (BLPACR) from those ampicillin susceptible results. The numbers of female were significant greater than those of male in BLPAR (p = 0.0336). With respect to antimicrobial susceptible pattern, there was no minocycline and piperacillin resistant strain in both BLNAS and BLNAR (p < 0.0001). Haemophilus influenzae infection spreads worldwide and inadequate use of antibiotics contributes to uptake of their new antimicrobial resistance. Continuous antimicrobial surveys are need for control the emergence and spread of antimicrobial resistance to reduce the morbidity and mortality.

Streptococcus dysgalactiae subsp. equisimilius are pathogenic bacteria which cause various infectious diseases from acute pharyngitis to streptococcal toxic shock syndrome. Although Streptococcus dysgalactiae subsp. equisimilius infection has been increasing recently, the recent characteristic investigation of respiratory tract associated Streptococcus dysgalactiae subsp. equisimilius has not been performed in Japan. In this study, we analyzed the relationship between respiratory tract and norespiratory tract associated Streptococcus dysgalactiae subsp. equisimilius by investigating the recent clinical characteristics and antimicrobial susceptible patterns of 98Streptococcus dysgalactiae subsp. equisimilius isolated during 2013-2015 in Japan. There was significant difference of gender between respiratory tract and no respiratory tract associated Streptococcus dysgalactiae subsp. equisimilius. No Streptococcus dysgalactiae subsp. equisimilius from no respiratory tract were found under 18 years-old patients. The prevalence of clindamycin non susceptible Streptococcus dysgalactiae subsp. equisimilius isolates from respiratory tract was significant greater than those from non-respiratory tract. We also analyzed the relationship between respiratory tract associated Streptococcus dysgalactiae subsp. equisimilius and Streptococcus pyogenes in 2014.There was no significant difference of gender between Streptococcus dysgalactiae subsp. equisimilius and Streptococcus pyogenes. The numbers of Streptococcus pyogenes-infected patients were significantly greater than those of Streptococcus dysgalactiae subsp. equisimilius-infected patients in children from 1 to 18 years old. From antimicrobial resistance, we found that the numbers of clarithromycin resistant Streptococcus pyogenes were greater than those of clarithromycin resistant Streptococcus dysgalactiae subsp. equisimilius among respiratory tract isolation. Our results suggest that the clinical and antimicrobial susceptible patterns are useful for screening the respiratory tract associated Streptococcus dysgalactiae subsp. equisimilius infection.

Streptococcus pyogenesis most popularpathogen caused respiratory tract infection disease. This study was conducted to find out the recent clinical characteristics of Streptococcus pyogenes isolates from respiratory tract at Japanese hospital in 2014. Streptococcus pyogenes was identified by standard laboratory procedure. Antimicrobial susceptibility testing was performed by micro dilution assay according to CLSI recommendation. Respiratory tract isolates were defined as isolation from pharynx, nasal discharge, tonsillar, and sputum. Total one hundred sixty-one Streptococcus pyogenes were isolated among which 136 were from respiratory tract and 25 were from non- respiratory tract. Respiratory tract-associated Streptococcus pyogenes was isolated among which 102 were from male and 59 were from female. The age incidence among 0 - 1 years age group was 8, among 1 - 18 years age group, 131 (p < 0.01), among 19 - 64 years age group, 17 (p < 0.01), and in 65-years it was 5. Although we investigated the relationship between season and bacterial isolation, we did not find any significant differences between respiratory tract and no respiratory tract isolation. However, our study revealed that clarithromycin was less antimicrobial effective in respiratory tract disease than in no respiratory tract disease significantly (p < 0.01). Although several antibiotics such as penicillin are still effective against Streptococcus pyogenes, incidence of Streptococcus pyogenes infection is not decreasing worldwide. Our study suggests the need for continuous epidemiological surveillance of Streptococcus pyogenes.

This study reports a case of Staphylococcus warneri dissemination in the hair of ICU/ER doctors. Eleven of 23 doctors in the first survey and 11 of 24 doctors in the second survey, which were conducted after 6 months, carried S. warneri, while hair contamination of other coagulase-negative staphylococci was only sporadic. Chromosomal DNA analysis with PFGE revealed that 7 S. warneri strains isolated in the first survey and 7 strains isolated in the second survey showed similar PFGE patterns, suggesting that a cross-infection with a S. warneri strain continued over 6 months among the ICU doctors.

Detection of Streptococcus pneumoniae colonized in the pharynx of healthy carriers currently relies on conventional culture methods of direct plating with pharyngeal swab specimens. The accurate measurement of the carriage of pneumococci, however, has not been necessarily achieved with these methods due to low density colonization and contamination of numerous oral streptococci that express α-hemolysis. A PCR-based detection method of pneumococci-specific for lytA as well as PCR serotyping of S. pneumoniae was recently developed and their effectiveness was confirmed. We modified the reaction conditions of these methods to improve the detection rate and applied them to the measurement of S. pneumoniae carried in healthy adults. Pharyngeal swab specimens obtained from 110 healthy volunteers over 40 and living in Nagoya were enriched for 5 hours with broth medium supplemented with rabbit serum and the template DNA for PCR was extracted from the mixed enriched culture. Of 110 specimens 36 (32.7%) were lytA-positive, the rate of which was much higher than the results of previous culture-based studies. The DNA template preparations were then used for PCR-based serotyping with primers specific for each of the types included in pneumococcal 23 valent vaccine (PPV23). We found that 28 out of 36 lytA-positive carriers were identified as being positive for the serotypes belonging to PPV23, although serotypes 6A and 6B were indistinguishable with the PCR method. The most frequent serotype was serotype 14, and serotypes 4, 18C, and 6A/B were also frequently identified. Five lytA-positive carriers were previously vaccinated with PPV23, and among them, 4 were positive for serotypes contained in PPV23. We recommend PCR-based identification and serotyping of S. pneumoniae in broth enrichment culture of pharyngeal swab specimens as a reliable method for the surveillance of healthy carriers with low density colonization.

In Streptococcus pyogenes, we have described the two-component signal transduction sensor and regulatory systems, CovR/S affect the antimicrobial susceptibility including penicillin G before. But the mechanism how two-component sensor protein CovS modulates penicillin G susceptibility has not been elucidated till date. This study aimed to determine how the CovS affected penicillin G susceptibility in Streptococcus pyogenes by northern blot analysis. At first, we investigated the covS mRNA expression under penicillin G induction. We found that the decrease of covS mRNA expression under Penicillin G stimulation. Next we investigated the expression of cell wall synthesis gene, pbp2a and glmM with use of covS knockout mutants from emm1 Streptococcus pyogenes strain 1529. We found that the cell-wall synthesis gene expression of the ?covS mutant strain were lower than that of the wild-type strain. Furthermore the expression of glmM mRNA gene was lower than the expression of pbp2a mRNA gene in the ?covS mutant strain. The covS-complemented strain almost restored the mRNA expression compared to covS mutant strain. The two-component sensor protein CovS affects the susceptibility to penicillin G in Streptococcus pyogenes by modulation of cell-wall synthesis.

Streptococcus pyogenes frequently causes purulent infections in humans. Biofilm formation is an important virulence property of S. pyogenes because of decreased susceptibility of bacteria to antibiotic treatment. Biofilm is composed of various types of matrix including glycocalyx which is an important exocellular matrix material related to bacterial sugar metabolism. A putative antiterminator protein, LicT (Spy0571), is one of the components of the glucose-independent β-gluco-side-specific phosphotransferase system (PTS). Although the PTS, a carbohydrate metabolic system, may play a role in biofilm formation, the relationship between LicT and biofilm formation has not yet been elucidated. Here, we evaluated whether LicT affected biofilm formation in modified chemically defined medium (CDMM) supplemented with glucose or β-glucoside:salicin. We created licT- and licT-complemented mutant strains from S. pyogenes 1529. Although the licT mutant strain tended to have higher growth rate than wild-typestrain in CDMM with glucose, it had a significant lower growth rate than the wild-type strain in CDMM with salicin. In addition, the licT mutant exhibited lower biofilm formation in CDMM containing salicin than the wild-type strain by 96 well plate analysis and confocal laser scanning microscopic analysis. Our results suggest that LicT plays an important role in biofilm formation of S. pyogenes.

Antimicrobial activity is one of the well-known biological characteristics of catechins, the main extract of green tea leaves. It is thought that catechins intercalate into the bacterial cell membrane and damage the lipid bilayer. However, the association between catechins and lipopolysaccharides, which consist of an O side chain, core oligosaccharide, and lipid A, has not been previously investigated. In this study, we evaluated the catechin sensitivity of Salmonella enterica mutants that lack the O side chain and have core oligosaccharides of different lengths. These rough mutants were more sensitive to catechins than a bacterial strain with intact lipopolysaccharide. We conclude that the O side chain and core oligosaccharide play an important role in protecting Gram-negative bacteria against the antimicrobial activity of catechins.

Klebsiella oxytoca is an opportunistic pathogen and is isolated at the second highest frequency among genus Klebsiella from hospitalized patients. According to previous reports, the major virulence factors of K. pneumoniae include capsules and several kinds of pill, whereas the virulence factors of K. oxytoca have not been well investigated. We noticed an increased frequency of K. oxytoca isolates from patients who had undergone a biliary tract operation in a general hospital from May through November, 2009. We then performed a PCR analysis of the virulence factors and an epidemiological analysis with capsular typing (serotyping) and pulsed field gel electrophoresis (PFGE) for K. oxytoca of 11 blood isolates and 10 bile isolates. As a result, serotypes of K9, K15, K26, K31, K43, K47, K55, K70, and K79 were identified in these strains, and K1 and K2 which are frequent serotypes in K. pneumoniae strains were not observed. Two blood isolates of the K55 serotype showed almost the same PFGE pattern, suggesting that these isolates were very closely related and caused cross-infection in a hospital ward. Strains of the K43 serotype were three blood isolates and 1 bile isolate, all of which showed different PFGE patterns. There were no common isolates among the blood and bile isolates. A PCR search revealed that fimH and mrkD genes which are relevant to type 1 and type 2 pili, respectively, were present in all strains, whereas kfuBC, an iron uptake gene, and cf29a were detected in only a few strains. Neither of the mucoid phenotype-related genes magA and rmpA was present in any strains. These results strongly suggest that type 1 and/or type 3 pili would have important roles in the pathogenesis of blood infection and bile infection caused by K. oxytoca.

The LytR family of cell envelope-associated transcriptional attenuators in bacteria has been brought into focus of scientific interest on the expression of various virulence factors, as well as bacterial cell envelope maintenance. However, this protein of Streptococcus pyogenes has been only described as cell surface-associated protein, and its function is completely unknown. We created lytR mutant strains from two independent S. pyogenes strains to analyze the function of LytR. The protease assay in culture supernatant showed that lytR mutant had the higher cysteine protease activity than wild-type. Two-dimensional gel electrophoresis and western blotting analysis revealed that the amount of cysteine protease, SpeB in lytR mutant was more compared with that in wild-type. The level of speB mRNA in lytR mutant also increased compared with that of wild-type. The membrane integrity and potential in lytR mutant also were decreased compared with that of wild-type. Murine infection model showed that less survival was detected in mice inoculated with lytR mutant than that with wild-type, and the size of wound lesion of mice with lytR mutant was larger than that with wild-type. Our data suggest that the lytR regulates the expression of SpeB in S. pyogenes with relation to membrane integrity.

Background: Streptococcus species account for 17%–50% of the microorganisms causing infectious endocarditis (IE), but S. pneumoniae rarely causes IE. Most clinical microbiology laboratories today depend on the optochin test to identify pneumococci. However, there have been several reports of optochin-resistant pneumococci. Case Report: A 64-year-old man suffered from sepsis and had a large vegetation on the aortic valve. The bacteria detected in the blood cultures showed α-hemolysis and were optochin-resistant, which suggested that these microorganisms were viridans streptococci. However, the bile solubility test, additional biochemical testing, and genetic analysis by polymerase chain reaction (PCR) revealed that the isolate was S. pneumoniae . Conclusions: Alpha-hemolytic streptococci showing optochin resistance should undergo additional tests before being identified as viridans streptococci, especially if the strain has been isolated from a sterile site in the body, such as the blood or cerebrospinal fluid. * IE : infectious endocarditis PCR : polymerase chain reaction AST : aspartate transaminase ALT : alanine transaminase LDH : lactate dehydrogenase HN : medical technologist lytA : autolysis gene ply : pneumolysin gene MIC : minimal inhibitory concentration CLSI : Clinical Laboratory Standards Institute

Streptococci secrete a large number of exoproteins including virulence-associated toxins and enzymes. To construct a reliable database of streptococcal exoproteins, we integrated the results that were derived from two approaches: LC-based shotgun proteomic analysis and 2-D PAGE-based proteomic analysis. We identified 74 and 82 proteins by LC-based and gel-based analysis, respectively. Forty-five proteins were identified by both methods. In addition, two proteins, one identified by both methods and the other only by LC-based shotgun analysis, were newly annotated. We therefore found the importance of combinational analysis by the two methods for the construction of a more reliable database.

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.

Recently, hospital-associated as well as community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strains showing a low level of resistance to oxacillin have emerged worldwide, and as a result, a highly sensitive method to detect MRSA has become more important. To prevent MRSA being overlooked, some selection agar media have recently been developed. We evaluated six commercially available selection agar media in regard to the detection of 35 borderline MRSA (BOMRSA) strains which were mecA-positive but showed low resistance to oxacillin. The MIC values of oxacillin differed between the broth dilution method and the agar dilution method, and 11 of the 35 BOMRSA strains were judged as sensitive by the broth dilution method and 14 of the 35 strains were judged as sensitive by the agar dilution method. Thirty-two of the 35 strains were also judged as sensitive by an oxacillin disk diffusion test. Moreover, there was no consistent pattern of resistance to the tested beta-lactams among the BOMRSA strains. Some commercially available selection media designed for the detection of MRSA contain a beta-lactam antibiotic; oxacillin, ceftizoxime, or cefoxitin, and we evaluated media containing each of these agents. The detection sensitivities of cefoxitin-based agar media, such as CHROMagar MRSA and MRSA ID, for BOMRSA were 100% at 24-h culture. On the other hand, the media containing oxacillin or ceftizoxime gave lower results at 24 h, suggesting that, possibly, BOMRSA strains may not to be able to grow on these media. These results suggest that cefoxitin-based agar media should be recommended for the first-round screening of BOMRSA.

The administration of high-dose clindamycin (CLI) along with penicillin is recommended for the treatment of streptococcal toxic shock syndrome. However, the prevalence of CLI-resistant Streptococcus pyogenes strains is increasing worldwide, and the effect of CLI on CLI-resistant S. pyogenes strains remains unknown. We aimed to evaluate the effect of CLI on the in vitro production of three major virulent exoproteins, namely, streptolysin O (Slo), NAD glycohydrolase (Nga), and streptokinase (Ska), by CLI-resistant S. pyogenes strains. After the incubation of M1 serotype CLI-resistant S. pyogenes D2TY in the presence of 1 μg/ml CLI, the amounts of Slo, Nga, and Ska and the levels of slo , nga , and ska mRNA in the supernatant were analyzed by Northern blotting and Western blotting, respectively. The results of both assays showed that the production of Slo, Nga, and Ska was higher with CLI treatment than without CLI treatment. We evaluated the role of the sensor kinase CovS, which is involved in the two-component system of S. pyogenes , in the CLI-induced production of these three exoproteins. Northern blotting analysis revealed that CLI induced the expression of covS mRNA in wild-type strain D2TY. Furthermore, both Northern blotting and Western blotting analyses showed that CLI decreased the levels of expression of Slo, Nga, and Ska in isogenic covS mutant D2TYcovS. These results suggest that CLI increases the production of three virulent exoproteins in CLI-resistant S. pyogenes strains via the action of CovS.

Helicobacter pylori infection is implicated in the pathogenesis of extradigestive diseases such as acne rosacea and idiopathic chronic urticaria and autoimmune diseases such as autoimmune gastric atrophy, rheumatoid arthritis, anti phospholipid antibody syndrome, autoimmune thyroiditis, Sjoegren syndrome, Henoch-Schoenlein purpura, and Type B insulin resistance syndrome. H. pylori eradication ameliorated the condition in some, but not all, of those with these autoimmune diseases. Recent studies primarily in Italy and Japan found that H. pylori eradication in those infected with chronic immune thrombocytopenic purpura (ITP) results in a persistent platelet count increase in over half of those treated, suggesting that although pathogenetic mechanisms underlying the relationship between H. pylori infection and autoimmune disease remain unclear, yet-unknown immunological events induced by H. pylori infection almost certainly occur in the development of autoimmune response. A majority of isolated H. pylori strains express human Lewis (Le(x) and/or Le(y) determinants and in some strains, Le(a), Le(b), sialyl-Le(x)), and H determinants in the O-chain of the surface lipopolysaccharide. Previous studies showed that this molecular mimicry helps the bacterium evade host responses while evoking autoantibody responses to Le antigens. The anti-Le(y) autoantibody is also reported to promote H. pylori adhesion to gastric epithelial cells, leading to development of gastric atrophy. Moreover, one can hypothesize that anti-Le autoreactive antibodies induced by H. pylori infection are involved in the development of autoimmune diseases, although no clinical studies showing that anti-Le immune responses are involved in the etiology of these autoimmune diseases have been conducted. Proving this hypothesis would require quantitative and qualitative analysis of autoantibodies and T cell functions to Le antigens. High frequent phase variation of Le structures in the O-polysaccharide of H. pylori may influence the immune response of patients to Le antigens.

To study comprehensive toxin profiles and the chromosomal diversity of current Japanese hospital-associated meticillin-resistant Staphylococcus aureus (HA-MRSA) strains, we conducted PCR-based identification of 28 toxin genes, and staphylococcal cassette chromosome mec (SCCmec) typing and PFGE analysis of 208 MRSA strains isolated from 100 hospitals throughout Japan. Of the tested HA-MRSA strains, 80.3 % were tst-positive. The most frequent toxin gene profile was characterized by the carriage of 13 genes, tst, sec, seg, sei, sel, sem, sen, seo, lukED, hla, hlb, hld and hlg-2. Ninety of the 208 strains had this profile, which was named pattern A. Among the 118 non-pattern A strains, 100 had similar toxin gene profiles, the concordance rates to pattern A of which were more than 80 %. Consequently, 91.3 % of the examined HA-MRSA strains carried similar toxin profiles, although PFGE patterns showed a wide variation. These strains belonged to SCCmec type II, agr II and coagulase type II. We concluded that, unlike MRSA from many other countries, most of the Japanese HA-MRSA strains belonged to, or were related to, a specific group carrying the set of 13 toxin genes, irrespective of chromosomal diversity. In addition, among the 13 toxin genes, the coexistence rates of tst, sec and sel, and those of seg, sei, sem, sen and seo, were higher than for the other toxin genes. High coexistence rates of tst, sec and sel genes suggested the presence of the pathogenicity island SaPIn1 in these strains.

We have developed a new method for a fast and precise analysis of circle-to-circle amplification (C2CA) product for specific gene detection by microchip electrophoresis. In this method, we have added a new enzymatic step to the C2CA reaction, which could be carried out isothermally at 37 degrees C. Compared to the original single-stranded DNA, the double-stranded DNA that is produced by this enzymatic reaction is more reliable for analysis by microchip electrophoresis. C2CA product was detected within 55 s with high reproducibility by this method which was successfully applied to the detection of 10-ng genomic DNA of the pathogenic bacteria Vibrio. cholerae within 110 s. Purification was found to be an indispensable step for the analysis of the C2CA product of genomic DNA samples.

Phenotypic and molecular studies have established that cereulide-producing strains of Bacillus cereus are a distinct and probably recently emerged clone within the Bacillus population. We analyzed a set of B. cereus strains, both cereulide producers and nonproducers, by multilocus sequence typing. Consistent with earlier reports, nonproducers demonstrated high heterogeneity. Most cereulide-producing strains and all flagellar antigen type H1 strains were allocated to the known sequence type of exclusively emetic B. cereus strains. Several cereulide-producing strains, however, were recovered at a new phylogenetic location, all of which were serotype H3 or H12. We hypothesize that the group of cereulide producers is diversifying progressively, probably by lateral transfer of the corresponding gene complex.

Gastric mucosal biopsy is widely used in the detection of Helicobacter pylori but is associated with a number of problems, including false-negative results due to sampling error and massive bleeding after biopsy. Given the extended period required to culture H. pylori , detection would be further improved by the use of rapid detection methods such as PCR. Here, we developed a rapid, safe, and convenient method for collecting H. pylori which combines endoscopic brushing with the loop-mediated isothermal amplification (LAMP) method. The specificity and sensitivity of LAMP were examined using nine urease-generating non- H. pylori bacterial species, Escherichia coli , Clostridium perfringens , Campylobacter jejuni , Helicobacter hepaticus , and 51 H. pylori strains. Results showed that H. pylori -specific LAMP primers amplified H. pylori DNA only and that the lowest detection limit of the LAMP reaction was 10 2 CFU. Brushing and biopsy samples taken from 200 patients with peptic ulcer at Nagoya University Hospital and a regional health care center were subjected to both LAMP and culturing. No adverse effects such as severe bleeding or penetration occurred during the procedure. By LAMP assay, 123 patients were confirmed as H. pylori positive when brushing technique samples were assayed, whereas only 100 were positive when biopsy samples were assayed. Culture assay detected H. pylori in 117 patients when it was combined with the brushing technique and in 96 when it was combined with biopsy. Combination of the endoscopic brushing technique with LAMP is considered a useful and safe system for identifying H. pylori infection.

Bacillus cereus strains from cases of severe or lethal systemic infections, including respiratory symptoms cases, were analyzed using multilocus sequence typing scheme of B. cereus MLST database. The isolates were evenly distributed between the two main clades, and 60% of them had allele profiles new to the database. Half of the collection's strains clustered in a lineage neighboring Bacillus anthracis phylogenetic origin. Strains from lethal cases with respiratory symptoms were allocated in both main clades. This is the first report of strains causing respiratory symptoms to be identified as genetically distant from B. anthracis. The phylogenetic location of the presented here strains was compared with all previously submitted to the database isolates from systemic infections, and were found to appear in the same clusters where clinical isolates from other studies had been assigned. It seems that the pathogenic strains are forming clusters on the phylogenetic tree.

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 microg of VT-2 or 1.0 microg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.

Mucosal T cells are essential to immune tolerance in the intestine, an organ constantly exposed to large amounts of dietary and bacterial Ags. We investigated whether local fibroblasts affect mucosal T cell survival, which is critical for maintenance of immune tolerance. Coculture with autologous fibroblasts significantly increased viability of mucosal T cells by inhibiting IL-2 deprivation- and Fas-mediated apoptosis, an effect that was both contact- and secreted product-dependent. Investigation of antiapoptotic factors in the fibroblast-conditioned medium (FCM) revealed the presence of IL-10 and PGE2, but not IFN-β, IL-2, or IL-15. Although recombinant IFN-β, but not PGE2, effectively prevented T cell apoptosis, neutralizing Ab studies showed that only IL-10 blockade significantly increased T cells apoptosis, whereas neutralizing IFN-β or IFN-α failed to inhibit the antiapoptotic effect of FCM. To confirm that fibroblast-derived IL-10 was responsible for preserving mucosal T cell viability, IL-10 mRNA was demonstrated in fibroblasts by Southern blotting and RT-PCR. When FCM was submitted to HPLC fractionation, only the peak matching rIL-10 contained the antiapoptotic activity, and this was eliminated by treatment with an IL-10-neutralizing Ab. Finally, when fibroblasts were transiently transfected with IL-10 antisense oligonucleotides, the conditioned medium lost its T cell antiapoptotic effect, whereas medium from fibroblasts transfected with IFN-β antisense oligonucleotides displayed the same antiapoptotic activity of medium from untransfected fibroblasts. These results indicate that local fibroblast-derived IL-10 is critically involved in the survival of mucosal T cells, underscoring the crucial importance of studying organ-specific cells and products to define the mechanisms of immune homeostasis in specialized tissue microenvironments like the intestinal mucosa.

A total of 73 clinical isolates of Streptococcus pneumoniae were measured for susceptibilities to nine fluoroquinolones, and nucleotide sequences of the quinolone resistance-determining regions (QRDRs) were determined. MIC90s of sparfloxacin, tosufloxacin, grepafloxacin, and gatifloxacin were less than 0.5 mg/L and the MIC90 of ciprofloxacin was 2 mg/L, although MIC values of some isolates to ciprofloxacin were more than 2 mg/L. We found that 60 of 73 isolates had only Ile-460-Val/ParE substitution and two isolates had an additional substitution of Ser-114-Gly/GyrA, while none of the isolates had any other substitutions in QRDRs of either ParC/E or GyrA/B. The isolates carrying Ile-460-Val/ParE substitution were more resistant to the fluoroquinolones norfloxacin and ciprofloxacin than the isolates with no amino acid substitution and the differences in MIC values were significant, suggesting that Ile-460-Val/ParE substitution in recent clinical S. pneumoniae isolates should be involved in the low-level fluoroquinolone resistance.

High-dose clindamycin (CLDM) and benzylpenicillin (PCG) are the recommended chemotherapeutic remedies for toxic shock-like syndrome caused by group A streptococci. One reason for this is that it has been shown that CLDM suppresses the expression of some exoproteins, e.g., SpeB, SpeA, and streptolysin O (Slo). We analyzed the effects of antibiotics on the production of whole exoproteins by two-dimensional gel electrophoresis. Unexpectedly, we found that the levels of several exoproteins, Slo, NAD + -glycohydrolase (Nga), M protein, and Sic, were increased by CLDM treatment, although we also confirmed previous findings that the levels of various exoproteins, including SpeB, were decreased. The increases in exoprotein levels were also detected by using other protein synthesis inhibitor antibiotics: erythromycin, kanamycin, tetracycline, chloramphenicol, and linezolid. Peptidoglycan synthesis inhibitors (such as PCG, cefazolin, and imipenem), DNA replication inhibitors (such as gatifloxacin), and an RNA polymerase inhibitor (rifampin) did not have significant effects on exoprotein production. The combination of CLDM and PCG had no advantageous effects with regard to exoprotein production compared to the effect achieved with CLDM alone. We also analyzed the transcriptional levels of slo and nga by reverse transcription-PCR and found that this change was also detected at the transcriptional level. Furthermore, the phenomenon was seen not only in strains of the M1 serotype but also in strains of the other M serotypes. Our study suggests that the clinical effectiveness of CLDM might be due to the inhibition of the production of a limited number of exoproteins.

Glycine is the simplest amino acid and is used as a metabolic product in some bacteria. However, an excess of glycine inhibits the growth of many bacteria, and it is used as a nonspecific antiseptic agent due to its low level of toxicity in animals. The effect of glycine on Helicobacter pylori is not precisely known. The present study was conducted to investigate (i) the effect of glycine on clarithromycin (CLR)-resistant and -susceptible strains of H. pylori , (ii) the effect of glycine in combination with amoxicillin (AMX), and (iii) the postantibiotic effect (PAE). The MIC at which 90% of strains are inhibited for glycine was almost 2.5 mg/ml for 31 strains of H. pylori , including CLR-resistant strains. We constructed isogenic CLR-resistant mutant strains by natural transformation and investigated the difference between clinical wild-type strains and isogenic mutants. There were no differences in the MICs between CLR-resistant and -susceptible strains or between clinical wild-type and mutant strains. The combination of AMX and glycine showed synergistic activity, with the minimum bactericidal concentration of AMX with glycine decreasing to 1/10 that of AMX alone. Glycine showed no PAE against H. pylori . These results suggest that glycine may be a useful antimicrobial agent against H. pylori not only alone but also in combination with antibacterial drugs for the treatment of H. pylori -associated diseases. Glycine may represent a component of a new type of eradication therapy for CLR-resistant H. pylori .

Background. The association between Helicobacter pylori infection and idiopathic thrombocytopenic purpura (ITP) has been reported widely. We investigated the prevalence of H. pylori infection, its virulence profile and the effectiveness of its eradication in patients with ITP. Materials and Methods. Twenty patients with ITP, 20 with peptic ulcer (10 gastric ulcer (GU), 10 duodenal ulcer (DU)) and 20 with NUD were studied. The virulence profile of the strains was assessed by genotyping for cagA, vacA, iceA, and hpyIIIR/hrgA and by assaying for IL-8 and DNA fragmentation after incubation with AGS cells. Infected patients and two uninfected ITP patients received triple therapy and platelets were counted before and 1 month, 6 months, 1 year, and 2 years after eradication therapy. Results. H. pylori infection was found in 17 ITP (85%), 20 ulcer (100%) and 13 NUD (65%) patients. Biopsies and strains were collected from five ITP, 20 ulcer and 13 NUD patients. The ITP patients had a pangastritis or corpus-predominant gastritis pattern. All H. pylori isolates, from ITP, ulcer and NUD patients, were cagA+ and vacA s1/m1, and did not differ in levels of IL-8 induction or DNA fragmentation. Fifteen ITP (88%) and 17 ulcer (85%) patients had successful eradication of H. pylori. Ten of these 15 (67%) H. pylori-eradicated ITP patients had platelet recovery. There was no significant change in platelet count in the two ITP patients in whom eradication failed or in the two originally H. pylori-uninfected ITP patients, or in the treated ulcer patients. Age at onset of ITP was the main determinant of platelet recovery: 100% of patients diagnosed after the age of 60 recovered compared with only 22% of those diagnosed before 50. Conclusions. H. pylori-infected ITP patients have a corpus-predominant pattern of gastritis but the virulence profile of their strains does not differ from that of ulcer or NUD patients. Eradication of H. pylori infection is a good therapeutic option for some patients with chronic ITP, especially for those who develop ITP in older age.

Cereulide is a cyclic dodecadepsipeptide from a pathogenic bacteria Bacillus cereus , which shows the emetic toxicity. Molecular diversity, or variety in homologation was found as a minor constituent of this cyclic peptide. Its molecular weight is 1152 but its homologs were observed as 1138 and 1166, which had 14 mass lower and higher differences from cereulide. This homologation was observed in about 10% of cereulide. It seemed to be difficult to determine the heterogeneous amino acids directly by MS/MS analysis on the intact molecules of cereulide. And hydrolysis of this cyclic peptide gave dipeptides, which were analyzed to determine their heterogeneous components by means of nano-HPLC-ESI-Q-TOF-MS and MS/MS. Among all amino- and oxy-acids, we found that O -Val and O -Leu were the keys of variation in cereulide. These findings will be significant to establish an identification method for pathogenic bacteria on the basis of biosynthetic pathways.

Porphyromonas gingivalis autolyzes in the culture media. To examine in more detail the molecular components of the autolysate, two-dimensional gel electrophoresis was performed. Many protein spots varied both in number and volume. One of these spots included Arg-gingipain (Rgp) as determined by N-terminal amino acid sequencing. Corresponding to the increase in spot volume, Rgp activity also increased during autolysis. The results of this study suggested that Rgp and other proteins in the P. gingivalis autolysate may be involved with the prolongation of periodontal disease, even after the death of P. gingivalis cells.

Background: Failure of Helicobacter pylori eradication occurs frequently despite use of multiple microbial agents. Aim: We aimed to study differences between H. pylori strains isolated before and after eradication failure. Methods: We treated 87 patients with peptic ulcer using triple therapy consisting of omeprazole plus combinations of clarithromycin, amoxicillin, or metronidazole. We studied the status of cagA, vacA, and iceA by PCR, and examined the differences in H. pylori isolates by pulsed-field gel electrophoresis and arbitrary primer polymerase chain reaction. The minimum inhibitory concentration of clarithromycin, amoxicillin, or metronidazole was determined by an agar dilution method. Results: Eradication therapy failed in 12 patients (14%); H. pylori isolates were obtained from all of these both before and after therapy. After eradication therapy, 10 patients were colonized with the same strain as before therapy, while the other two patients were colonized with different strains from those before therapy. In the former group, one isolate changed from metronidazole-sensitive to -resistant, one changed from clarithromycin- and metronidazole-sensitive to -resistant, and four were resistant to clarithromycin or metronidazole both before and after therapy. The other four isolates remained sensitive to clarithromycin and metronidazole after therapy. In the two patients who yielded apparently different isolates after therapy, they changed from clarithromycin- and metronidazole-sensitive to -resistant. Conclusion: Eradication of H. pylori by first-line therapy is an important goal in the treatment of H. pylori-positive peptic ulcer, and that appropriate antimicrobial sensitivity testing should be conducted in patients with eradication failure.

Background . Our previous study showed that histological scores of gastric mucosal inflammation and Helicobacter pylori density decreased even in patients who failed to eradicate Helicobacter pylori after antimicrobial therapy including clarithromycin. This may reflect indirect suppressive effects of lower concentrations of clarithromycin on Helicobacter pylori , as suggested in other Gram-negative rod infections. Aims . To investigate whether clarithromycin suppresses virulence factors of Helicobacter pylori at sub-minimal inhibitory concentration. Methods . Six clarithromycin-susceptible Helicobacter pylori isolates and 7 clarithromycin-resistant isolates were obtained from patients with peptic ulcer disease. These isolates were analysed for urease activity, motility, and ability to bind to gastric epithelial cells after they were incubated with or without clarithromycin at sub-minimal inhibitory concentrations. Results . Incubation of Helicobacter pylori isolates with clarithromycin at sub-minimal inhibitory concentrations reduced urease activity, motility, and binding to gastric epithelial cells in a dose-dependent manner. These findings were observed both in clarithromycin-susceptible and resistant strains. Conclusions . Suppressive effects of clerithromycin on virulence factors of Helicobacter pylori at sub-minimal inhibitory concentrations may be associated with observed attenuation of gastric inflammation and Helicobacter pylori density in patients who failed in bacterial eradication after triple therapy including clarithromycin.

The proteins in the culture supernatant (exoproteins) from Streptococcus pyogenes serotype M1 were separated by two-dimensional gel electrophoresis, and their N-terminal amino acid sequences were determined. The amino acid sequences were compared to sequences in the S. pyogenes genome database. The coding sequence showed similarity to sequences of two genes, mf2-v ( mf2 variant) and mf3, which had sequence similarity to genes encoding mitogenic factor (MF); MF has DNase activity. The recombinant genes were expressed in Escherichia coli and the proteins were synthesized. Mf2-v and Mf3 had DNase activity. The activity of Mf2-v was localized to the C-terminal half of the protein. The mf3 gene was shown to be present in most clinically isolated strains of S. pyogenes tested, and the mf2gene was detected in 20% of the isolates. The products of the mf2 and mf3 genes in clinically isolated S. pyogenes strains were thus shown to be DNases.

Neonatal toxic shock syndrome-like exanthematous disease (NTED) is a new entity of methicillin-resistant Staphylococcus aureus (MRSA) infection. Most of NTED cases reported previously in the literature were sporadic ones. In the present report, we describe an outbreak of NTED that occurred in a neonatal intensive care unit (NICU) between April, 1999 and April, 2000 in Japan. All MRSA strains isolated from 14 patients (6 NTED, 2 infections and 6 colonizations) in this outbreak belonged to the group of coagulase II and produced toxic shock syndrome toxin 1 (TSST-1). Of these, 14 strains produced staphylococcal enterotoxin C (SEC). No other superantigenic toxins were produced by these strains. The pulsed field gel electrophoresis (PFGE) patterns of genomic DNA digested with SmaI were indistinguishable each other due to no band shifting in all of the 13 strains except for strain O-21 and M56. Strain M56 was different from the dominant type in the positions of only 2 bands, whereas the pattern of strain O-21 had no similarity with the other pattern, suggesting that this outbreak was associated with the spread of a unique MRSA strain in the NICU. Two-dimensional electrophoresis (2-DE) analysis of exoproteins revealed that the patterns of these 14 strains were very indistinguishable to each other, and that these strains produced very large amounts of TSST-1 and SEC3 subtype superantigens, as measured with computer-assisted image analysis of the intensity of 2-DE spots. The 2-DE gel of O-21 showed the different pattern from the others. These results as well as the profiles of toxin production also supported the conclusion drawn from PFGE analysis. Based on these results, the involvement of TSST-1 and SEC3 in the pathogenesis of NTED is discussed.

Background: Clarithromycin (CAM) may have certain indirect effects on Helicobacter pylori (H. pylori) other than its inhibitory activity on bacterial growth, as indicated in other infections with Gram-negative micro-organisms. In the present study, we examined the effects of lower concentrations of CAM on the release of heat shock protein B (HspB), one of the major antigenic proteins from H. pylori cells, as well as the changes in humoral immune response and histological degree of antral gastritis in patients who received eradication therapy with CAM. Methods: The H. pylori strain 26695 and three CAM-resistant clinical isolates were cultured in broth with and without CAM (2–500 ng/mL). Expression of H. pylori proteins was examined by two-dimensional (2D)-electrophoresis followed by N-terminal amino acid sequencing. Changes in host immune response and histological degree of antral gastritis were monitored in patients with peptic ulcer disease who received H. pylori eradication therapy. Results: 2D electrophoresis showed 26 spots in extracellularly released proteins with different profiles from those in cytoplasmic proteins. The release of HspB increased after incubation with CAM (30–500 ng/mL) in all three H. pylori clinical isolates tested. Patients with failed H. pylori eradication after triple therapy with CAM, but not those with failed eradication after dual therapy without CAM, showed an increase in serum IgG1 and IgG2 antibodies against HspB along with a decrease in the degree of neutrophil and H. pylori colonization density in tissue sections. Conclusions: CAM may induce a humoral immune response against H. pylori and a decrease in gastric mucosal inflammation through up-regulation of the release of HspB from the bacteria in infected patients.

Expression levels of Gb3/CD77 synthase together with Gb3/CD77 antigen were analyzed using human hematopoietic tumor cell lines and normal cells. Among about 40 kinds of cells, Burkitt lymphoma cells showed the highest gene expression concomitant with the expression levels of Gb3/CD77. Unexpectedly, megakaryoblastic leukemia lines also expressed fairly high levels of mRNA of Gb3/CD77 synthase and its product. A megakaryoblastic leukemia line, MEG-01 was sensitive to verotoxins from Escherichia coli O157 and apoptosis was induced via the caspase pathway. We also demonstrated that the cell surface Gb3/CD77 expression was reduced on differentiated MEG-01 although the mRNA level of the α1,4Gal-T gene increased. In this case, the localization of Gb3/CD77 was changed from the cell surface to the cytoplasm as stained with a granular pattern, co-localizing with platelet GPIIb-IIIa, indicating that some of them were platelet precursors. Small particles outside of cells also showed similar staining patterns. These results agreed with the previous report that platelets produced in mature megakaryoblasts abundantly contained Gb3/CD77 antigen. Here, we propose the possibility that verotoxins bind immature megakaryoblasts and induce their apoptosis, leading to the arrest of platelet generation in the bone marrow. This may be one of the causes of thrombocytopenia in patients with hemolytic uremic syndrome.

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid-rich conditions. We found that the Shiga Toxin-producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K-12 strain in Luria-Bertani (LB)-2-morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini-Tn5 mutagenesis and, from 50,000 colonies, five mutants that showed a clear acetic acid-sensitive phenotype were isolated. The insertion of mini-Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O-polysaccharide, loss of both O-polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O-polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O-polysaccharide and ECA, and normal growth was restored in organic acid-rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O-polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O-polysaccharide and ECA as well as an acid resistance mechanism.

Staphylococcus aureus and Staphylococcus epidermidis are pathogenic bacteria that often cause invasive infections in humans. In this study, we characterized the composition and growth characteristics of staphylococcal biofilms under various incubation atmospheres. We assessed the effect of incubation atmosphere (aerobic, 5% CO2, anaerobic, and microaerobic) on the biofilm production capabilities of S. aureus strains isolated from healthy volunteers and from patients with catheter-related bloodstream infection. In addition, the composition of S. aureus and S. epidermidis biofilms was determined by assessment of biofilm degradation after treatment with DNase I, proteinase K, and dispersin B. The strains obtained from healthy volunteers and patients showed similar biofilm formation capabilities. Biofilms of S. aureus were rich in proteins when developed under ambient atmospheric conditions, 5% CO2, and microaerobic condition, whereas S. epidermidis biofilms contained large amounts of poly-β (1, 6)-N-acetyl-D-glucosamine when developed under ambient atmospheric conditions and microaerobic condition. The biofilm-producing capability of S. epidermidis was considerably higher than that of S. aureus under aerobic condition. Staphylococcal isolates obtained from healthy individuals and patients with catheter-related infections have similar biofilm-forming capabilities. Under microaerobic conditions, S. aureus and S. epidermidis form protein-rich and poly-β (1, 6)-N-acetyl-D-glucosamine-rich biofilms, respectively. These components may play an important role in the development of biofilms inside the body and may be the target molecules to prevent catheter-related infections caused by these organisms.

Here, we report the draft genome sequence of the Legionella pneumophila Nagoya-1 strain, serogroup 4, which was isolated from a clinical sample from a patient with legionellosis. Several virulence-associated genes, including those encoding the type IV (Dot/Icm) secretion system and effector proteins, were highly conserved.

The phenomenon of postantibiotic effect (PAE) encompasses not only the effects of bacterial growth inhibition but also the suppression of virulence factors. We tentatively designated the latter effect the postantibiotic suppression effect (PASE). The flagella of Gram-negative bacteria are involved in the development of biofilms. We measured the PASE of erythromycin (ERY) and azithromycin (AZM) on the expression of flagellin in Pseudomonas aeruginosa and Proteus mirabilis. Flagellin preparations were subjected to sodium dodecylsulfate (SDS) polyacry lamide gel electrophoresis (PAGE) analysis and the flagellin band was identified by N-terminal amino acid sequence analysis. We thus evaluated the flagellin by the intensity of the band. The mean durations of the PAE of ERY and AZM on bacterial growth were 0.9 and 2.0 h for P. mirabilis, and 0.6 and 2.7 h for P. aeruginosa, respectively. The PASE of these drugs on flagellin expression was also observed. The apparent PASEs of ERY and AZM on flagellin were up to 5 h for P. mirabilis and up to 6 h for P. aeruginosa after a single 0.5 × minimum inhibitory concentration (MIC) treatment for 5 h. Our results suggest that certain combinations of antibiotics may have prolonged suppressive effects on the expression of virulence factors in certain Gram-negative bacteria.

In the present study we showed by molecular analysis that the inhibition of motility by macrolides inProteus mirabilisandPseudomonas aeruginosawas well correlated with the loss of the expression of flagellin. Erythromycin, clarithromycin, and azithromycin at subinhibitory concentrations (sub-MICs) suppressed the expression of flagellin dose dependently. Azithromycin had the strongest inhibitory effect on the expression ofP. aeruginosaflagellin, whereas 16-membered rokitamycin had only a weak inhibitory effect. These results indicate the potential effectiveness of sub-MICs of erythromycin, clarithromycin, and azithromycin for the treatment of patients withP. mirabilisandP. aeruginosainfections.

Staphylococcus aureus and S. epidermidis are common pathogens in hospitals, and care should be taken not to disseminate these organisms among patients. We have focused on human hair as a source of bacterial contamination. We treated hair with culture solutions of S. aureus and S. epidermidis, and then performed scanning electron microscopy. Bacteria were detected on the surface of the cuticles of the hair, and the attached bacteria were not completely removed even by repeated washing with detergents. These results suggested that hair could be a source of bacterial contamination and indicated the importance of decontamination of hair.

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.

Cereulide is a principal toxin causing emetic syndrome produced by Bacillus cereus. This paper deals with biosynthetic studies on this unusual cyclic depsipeptide toxin from 13C labeled L-amino acid precursors (Val, Leu, Ala) upon cultivation in synthetic media. The analyses were made at atomic level of the constituent amino- or oxy-acids through NMR and ESI-MS/MS spectroscopic methods on cereulide and its hydrolysate dipeptides. The incorporation of the 13C atom was 95% in each O-Val, O-Leu and L-Val, while 40% in D-Ala of cereulide.

The nucleotide sequence of a 15 600-bp DNA fragment containing the staphylokinase gene ( sak NU3-1) of methicillin-resistant Staphylococcus aureus (MRSA) NU3-1 was determined. The sak gene was found within the ply gene encoding N -acetylmuramyl- L -alanine amidase and thus the ply gene should be inactivated. In the flanking region of the sak gene, the tandem repeat sequences (GAAGTGTT and GAATGGTT) were present as possible junction points between the sak and ply genes. No sequences characteristic of the presence of an IS-like element were found. Upstream from the ply gene, the kdpA , kdpB and kdpC homologues were present. Downstream from the ply gene, the tagA , tagH and tagG homologues were present. The sak gene was inserted into the same position of ply in 5/6 of sak + MRSA isolates with different genotypes. In all of these sak + isolates, Sak was detected in the culture supernatant.