A long-term study of sharps injuries among health care workers in Japan.

BACKGROUND: The risk of transmission of occupational blood-borne infection is a serious problem for health care workers (HCWs) in Japan. Although the Japanese version of Exposure Prevention Information Network (EPINet) was introduced in 1997, no published data in the clinical setting have been available yet. OBJECTIVE: To examine the epidemiology of occupational sharps injuries of HCWs in a university hospital using EPINet and to analyze the trends and changes in epidemiologic characteristics of needlestick injuries in a detailed situation. METHODS: The HCWs were requested to report sharps injury incidents to the Infection Control Nurse when the incidents occurred. Those who were involved in the incidents were required to personally complete an EPINET form. RESULTS: A total of 259 cases of sharps injuries occurred during the 7-year period. Registered nurses accounted for 72.2% of the cases, constituting the largest group of the HCWs. The incidents occurred most frequently in the hospital wards. Thirty-three cases (55.9%) of the injuries with syringe-needle units occurred "after use before disposal," whereas 34 cases (73.9%) of the injuries with suture needles occurred "during use of device." More than half of the injuries with a winged steel needle occurred despite the protective mechanism. DISCUSSION: There was no apparent difference in the characteristics of the subjects compared with other reports. The circumstances of the injuries varied with the kinds of instruments. This fact may provide useful information for planning measures to sharps injuries. CONCLUSIONS: With the problem of underreporting aside, a detailed study, such as ours, comprising by job category and by kind of instrument or the like would provide more useful and effective information in terms of sharps injury prevention.

Two cases of sucrose-fermenting Vibrio vulnificus infection in which 16S rDNA sequencing was useful for diagnosis.

Vibrio vulnificus is a Gram-negative bacterium which is associated with severe infections in humans. We experienced two cases of sucrose-fermenting V. vulnificus infection. The causative agents in both cases were unidentifiable by conventional identification systems because of their unique characteristics, and sequencing of 16S rDNA was found to be useful for diagnosis.

Factors determining prognosis in streptococcal toxic shock-like syndrome: results of a nationwide investigation in Japan.

Since the first report of streptococcal toxic shock-like syndrome (TSLS) in Japan, the numbers of reported patients have been increasing. However, clinical manifestations remain somewhat unclear, and factors potentially defining prognosis remain to be identified. We conducted a retrospective nationwide postal survey of major Japanese hospitals concerning clinical manifestations of invasive streptococcal infections including necrotizing fasciitis and TSLS. We evaluated 30 patients who died and 36 survivors. The overall mortality rate was 45%. Physical and laboratory findings on admission were compared statistically between fatal cases and surviving patients. Most laboratory results from the patients who died showed greater abnormality than results from the survivors. Patients who died had significantly fewer leukocytes and platelets, although their C-reactive protein concentrations were similar to those in survivors. Creatinine was significantly higher, and temperature and blood pressure were significantly lower, in patients who died. Patients with invasive streptococcal infections should be managed aggressively when the above features are present.

Matrix metalloproteinases in the pathogenesis of asthma and COPD: implications for therapy.

While asthma is an inflammatory disorder of the airways involving mediators released from mast cells and eosinophils, inflammation alone is insufficient to explain the chronic nature of the disease. Recent progress in the understanding of disease pathogenesis has revealed that airway remodeling, which is at least in part due to an excess of extracellular matrix (ECM) deposition in the airway wall, plays a significant role in airflow obstruction. Matrix metalloproteinases (MMPs) have been suggested to be the major proteolytic enzymes to induce airway remodeling in asthma and COPD. It has been widely accepted that different inflammatory processes are involved in asthma and COPD with different inflammatory cells, mediators, and responses to treatments. Despite these different processes, airflow obstruction and airway remodeling characterize these two diseases. MMP-2 and -9 have been reported to be involved in the pathogenesis of airway remodeling in both diseases and MMP-12, in addition to these MMPs, in the pathogenesis of COPD. In this review, we discuss the current views on the role of MMPs in the pathogenesis of bronchial asthma and COPD. Anti-MMP therapy could theoretically be useful to prevent airway remodeling in asthma and COPD. However, to date no clinical data are available regarding the efficacy of anti-MMP therapies in the treatment of patients with asthma and COPD.

A case of nosocomial Legionella pneumophila pneumonia.

We report a case of Legionella pneumophila pneumonia in a patient with interstitial lung disease. Intensive environmental investigations revealed that a system of all-day-running bathwater was the source of infection. In this case, the concentration of L. pneumophila in the hospital bathwater was low. We therefore emphasize that even a low concentration of L. pneumophila in environmental water can cause serious infections to immunocompromised patients in a hospital.

Seroprevalence of Fusobacterium varium in ulcerative colitis patients in Japan.

The etiology of ulcerative colitis (UC) is unknown, while an exacerbating factor of this disease is associated with infectious agents. Recently, Fusobacterium varium has been found in the mucosa of a significant number of patients with UC. The aim of this study was to estimate the prevalence of F. varium infection based on serology, evaluate the relationship between F. varium seropositivity and UC, and determine the clinical characteristics of infected UC individuals. Seropositive patients were determined by immunoblotting with F. varium ATCC 8501 antigen. We also identified cross-reactive protein spots by peptide mass mapping analysis. These protein spots showed putative caseinolytic protease protein, putative translation elongation factor G, and putative enolase. Immunoblotting with F. varium antigen revealed signals with sera from 45 (40.2%) of the 112 UC patients and 20 (15.6%) of the 128 healthy controls, respectively (P<0.01). In terms of disease activity, seropositive UC patients were more likely to have clinically severe disease than seronegative UC patients. Disease location in seropositive patients was more extensive than the seronegative patients. In conclusion, F. varium is a predominant infection in the UC population and is a potential pathogen of UC.

A new phylogenetic cluster of cereulide-producing Bacillus cereus strains.

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.

Analysis of twin-arginine translocation pathway homologue in Staphylococcus aureus.

Staphylococcus aureus releases a large number of exoproteins, including membrane-active proteins and toxins with superantigenic activity involved in pathogenicity. However, the export pathways of exoproteins in S. aureus have not been reported. We analyzed the function of the staphylococcal twin-arginine translocation (Tat) pathway homologue, the presence of which was recently discovered according to the genome database. The amino-acid sequences of the Tat homologues of S. aureus do not have a high similarity with those of Escherichia coli and other bacteria. Constructed tatC-deficient mutants from distinct parent strains showed the same patterns of exoproteins compared with those of parent strains on two-dementional gel electrophoresis, and the amounts of secreted staphylococcal enterotoxins and toxic shock syndrome toxin-1, of which signal peptides have some features often seen in signal sequences of Tat-dependent proteins, did not change with Western blotting analyses. Therefore, it seems that the Tat pathway does not play a major role in the secretion system of S. aureus, but other export pathways may play an important role in toxin secretion. This is the first experimental report showing the influence of the Tat pathway on the secretion of S. aureus.

Growth phase-dependent effect of clindamycin on production of exoproteins by Streptococcus pyogenes.

The administration of high-dose clindamycin plus benzylpenicillin has been recommended for the treatment of streptococcal toxic shock-like syndrome caused by Streptococcus pyogenes, and clindamycin has been found to be more effective than beta-lactams in retrospective analyses of human cases. Although therapeutic doses of clindamycin have also been shown to be effective against experimental infections and clindamycin has great efficacy against the production of bacterial exoproteins, we recently reported that the level of production of some exoproteins was unchanged or even increased by a subinhibitory dose of clindamycin when it is added upon the initiation of bacterial culture and the treated cultures were analyzed by two-dimensional gel electrophoresis. In this study we further examined the effect of clindamycin on the production of exoproteins by adding it to Streptococcus pyogenes cultures during various growth phases. We found that the levels of production of some proteins, NAD+ glycohydrolase, streptolysin O, and streptococcal inhibitor of complement, were increased when clindamycin was added at early-log-phase growth, which was the result that was seen when clindamycin was added at the beginning of culture. However, clindamycin inhibited the production of most types of proteins when it was administered to Streptococcus pyogenes cultures at mid-log-phase growth. In csrS- or mga-knockout bacterial strains, the increase in exoproteins seen in parental strains was considerably inhibited. Our study indicates that the in vitro effect of clindamycin on the production of exoproteins greatly depends on the growth phase of bacteria and some regulatory factors of Streptococcus pyogenes that are involved in this phenomenon.

Use of a combination of brushing technique and the loop-mediated isothermal amplification method as a novel, rapid, and safe system for detection of Helicobacter pylori.

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.

Phylogenetic analysis of Bacillus cereus isolates from severe systemic infections using multilocus sequence typing scheme.

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.

Prevalence of Ile-460-Val/ParE substitution in clinical Streptococcus pneumoniae isolates that were less susceptible to fluoroquinolones.

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.

Effect of antibiotics on group A Streptococcus exoprotein production analyzed by two-dimensional gel electrophoresis.

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.

Close correlation of streptococcal DNase B (sdaB) alleles with emm genotypes in Streptococcus pyogenes.

DNase B is a major nuclease and a possible virulence factor in Streptococcus pyogenes. The allelic diversity of streptococcal DNase B (sdaB) gene was investigated in 83 strains with 14 emm genotypes. Of the 15 alleles identified, 11 alleles carried only synonymous nucleotide substitutions. On the other hand, 4 alleles had a non-synonymous substitution other than synonymous substitutions, resulting in the substitution of a single amino acid. The distribution of each allele was generally emm genotype-specific. Only sdaB7 was found in both emm2 and emm4. The promoter region was highly conserved and DNase B protein was similarly expressed in all alleles.

Cloning and characterization of two novel DNases from Streptococcus pyogenes.

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.

Cloning and characterization of the deoxyribonuclease sd alpha gene from Streptococcus pyogenes.

The pathogenesis of Streptococcus pyogenes infection, especially toxic shock-like syndrome (TSLS), is still not fully understood; however, the exoproteins have been considered to play a role. We analyzed the culture supernatant proteins (exoproteins) from a TSLS-related isolate belonging to M3 serotype S. pyogenes by two-dimensional gel electrophoresis and characterized a single protein spot by using BLAST database. We cloned the gene of this protein and named it sd alpha, which was similar to the deoxyribonuclease (DNase) sdc of S. equisimilis. We showed that the recombinant protein from the sd alpha gene had DNase activity. By polymerase chain reaction, we found that the sd alpha gene was present in most clinically isolated S. pyogenes including TSLS-related isolates. We thus conclude that Sd alpha is a new DNase of S. pyogenes.

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7.

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.

Two-dimensional gel electrophoresis analysis of the abundance of virulent exoproteins of group A streptococcus caused by environmental changes.

Group A streptococci regulate the expression of virulence factors in response to environmental change. In order to investigate this mechanism, the growth of group A streptococci and the abundance of virulent exoprotein production in culture supernatant were analyzed by two-dimensional gel electrophoresis (2-D electrophoresis) under several culture conditions. Judging from alterations in their growth, group A streptococci were affected by various environmental stresses. Under high O(2) and low CO(2 )concentrations, streptococcal pyrogenic exotoxin B (SpeB) and streptococcal pyrogenic exotoxin F (SpeF) significantly decreased, and the streptococcal inhibitor of complement (Sic) increased. At 30 degrees C, increases in endo-beta- N-acetylglucosaminidase (EndoS) and alpha-amylase were also detected, while at 41 degrees C EndoS became undetectable and SpeB and SpeF decreased. Sic, SpeF and mitogenic factor 3 (Mf3) decreased when cells were cultured in higher NaCl concentrations, and EndoS disappeared following culture of the cells in high glucose concentration. An increase in acid phosphatase and a decrease in several other proteins were detected when the cells were cultivated in high iron concentrations. These results suggest that group A streptococci have a versatile adaptation system that responds to several environmental stresses by altering the level of exoprotein production.