Culture-based bacterial evaluation of the appendix lumen in patients with and without acute appendicitis.

PURPOSE: Controversy exists over whether bacterial flora within the appendix differs between patients with and without appendicitis. To examine these potential differences, we cultured the appendiceal luminal microbiota of patients with and without acute appendicitis, and identified the bacterial species therein. METHODS: Fifty-seven patients with acute appendicitis and 37 patients without acute appendicitis who underwent curative resection of colorectal cancer and prophylactic appendectomies (control group) were included. Appendicitis patients were classified into the phlegmonous group or the gangrenous appendicitis group histopathologically. There was no patient with perforated appendicitis. Aerobic isolates were identified using standard identification schemata, and anaerobic isolates were identified according to the Japanese guidelines. RESULTS: There were no significant differences among the three groups in the median number aerobe species present per patient. However, the median number anaerobe species in the gangrenous appendicitis group was significantly higher than that of the control group and the phlegmonous appendicitis group. In addition, the incidence of patients with Bacillus species, Fusobacterium nucleatum, and Bilophila wadsworthia increased as the disease progressed from phlegmonous to gangrenous appendicitis. CONCLUSION: The present results suggest that increased diversity of anaerobes and the translocation of Bacillus species, F. nucleatum, and B. wadsworthia are associated with the progression of acute appendicitis.

Recurrent bacteremia with different strains of Streptococcus pyogenes in an immunocompromised child.

We report an immunocompromised child who experienced two episodes of bacteremia due to Streptococcus pyogenes. Random amplification of polymorphic DNA profiles, emm genotypes, superantigen profiles, antimicrobial susceptibility, and resistance-related genes were investigated, and the results showed different profiles between the two isolates. This is the first report describing recurrent bacteremia caused by different strains of S. pyogenes.

Fast three-dimensional terahertz computed tomography using real-time line projection of intense terahertz pulse.

We demonstrated fast three-dimensional transmission terahertz computed tomography by using real-time line projection of intense terahertz beam generated by optical rectification in lithium niobate crystal. After emphasizing the advantage of intense terahertz pulse generation for two-dimensional spatio-temporal terahertz imaging, peak-to-peak amplitudes of pulsed terahertz electric field have been used to obtain a series of projection images at different rotation angles. Then a standard reconstruction algorithm has been employed to perform final three-dimensional reconstruction. Test samples including a medicine capsule have been investigated with a total acquisition time to only 6 minutes.

Analysis of the pathological lesions of the lung in a mouse model of cutaneous infection with Streptococcus pyogenes.

Invasive diseases such as toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) are re-emerging infectious diseases. The mechanism of pathogenesis is not completely understood although the virulence of this organism has been analyzed using animal model systems, particularly using mice. The analysis of the progression of infection, however, is difficult. Computed tomography (CT) scanning is an extremely powerful technique that we applied to the mouse model of cutaneous infection with S. pyogenes. Two or three days after subcutaneous administration of bacteria, high density reticular areas were detected in the lung by CT. Histopathological examination of the lung was performed to examine the results of CT. Increased numbers of cytokeratin-positive epithelial cells, probably alveolar type II epithelial cells, were detected but no remarkable increase of inflammatory cell infiltrates was observed. Our results show that the pathological lesions of the lung in this model, wherein relatively few numbers of neutrophils were in the alveoli, are well correlated with the lung of a part of streptococcal toxic shock syndrome patients. Therefore, CT may be useful in assessing the progression of S. pyogenes infection, particularly in the pathological lesions of the lung in this model.

Shin'iseihaito (Xinyiqingfeitang) extract ameliorates ovalbumin-induced murine allergic rhinitis by regulating cytokines.

Shin'iseihaito (Xinyiqingfeitang) is a formula of traditional Japanese Kampo medicine and traditional Chinese medicine (TCM) and for chronic sinusitis. However, the precise action mechanism has been unknown. We examined the effect of shin'iseihaito extract (SSHT) on murine allergic rhinitis model using ovalbumin (OVA). We decocted the mixture of 9 crude drugs in water to prepare SSHT. SSHT (20 times amount of human dose) was orally administered to mice treated with OVA. After mice were sacrificed on day 28, immunoglobulin (Ig) E, interleukin (IL)-4, IL-13, interferon (IFN)-γ, and thymic stromal lymphopoietin (TSLP) levels in nasal lavage fluid samples were measured by enzyme-linked immunosorbent assay (ELISA). The pathological tissue sections from the nasal epithelial mucosa were histopathologically investigated by optical and scanning electron microscopies. We also investigated the effects of modified SSHTs prepared by removing one crude drug from shin'iseihaito to clarify the active ingredients. SSHT suppressed IgE, IL-4, IL-13, and TSLP levels, while increased the IFN-γ levels in OVA-induced allergic mice. Sensitization with OVA resulted in an increase in eosinophilia and goblet cells in murine nasal cavity tissue in comparison with those in untreated group, however, those were significantly reduced by the treatment with SSHT. The extracts of 8 crude drug's mixtures except for the removal of Gypsum fibrosum (GF) from shin'iseihaito counteracted on the suppressive effects of SSHT on IgE, IL-4, IL-13, and TSLP levels in nasal lavage fluid. Our result demonstrated that SSHT may contribute to inhibit the exacerbation of OVA-induced murine allergic rhinitis by regulating cytokines, and the components except for GF contributed anti-allergic effect of shin'iseihaito.

Analysis of two-component sensor proteins involved in the response to acid stimuli in Streptococcus pyogenes.

The virulence of Streptococcus pyogenes depends on proteins that are produced by this bacterium. The production of virulence proteins depends on environmental factors, and two-component regulatory systems are considered to be involved in sensing these factors. One of the environmental factors is acid stimuli. We established knockout strains in all speculated two-component regulatory sensor proteins of the M1 clinical strain of S. pyogenes and examined their relevance to acid stimuli. The parental strain and its derived knockout strains were cultured in a medium adjusted to pH 7.6 or 6.0, and their growth in broth was compared. The spy1622 sensor knockout strain showed significant growth reduction compared with the parental strain in broth at pH 6.0, suggesting that the Spy1622 two-component sensor protein is involved in sensing acid stimuli. To further examine the role of the Spy1622 two-component sensor protein in virulence, blood bactericidal assays and mouse infection model experiments were performed. We found that the spy1622 knockout strain was less virulent than the parental strain, which suggests that the Spy1622 two-component sensor protein could play an important role in virulence.

Real-time line projection for fast terahertz spectral computed tomography.

We demonstrated fast terahertz spectral computed tomography by using real-time line projection of a terahertz beam. Two types of cross-sectional images of continuously rotating samples have been measured in only a few seconds. From temporal data, a peak-to-peak sinogram and cross sections have been reconstructed using a filtered backprojection algorithm. Using fast Fourier transform from temporal data, spectral cross sections of the sample have been obtained.

Variation in M protein production among Streptococcus pyogenes strains according to emm genotype.

M protein is an important virulence determinant in Streptococcus pyogenes, but the amounts of M protein in various strains of the species remain to be elucidated. To assess the amount of M protein in strains of each emm genotype, dot blot analysis was performed on 141 clinically isolated strains. Among the cell membrane-associated proteins, M protein was present in greater quantities in the emm1, 3, and 6 strains than in the other emm strains. In addition three strains, one each of the emm1, 3, and 6 types, showed prolific M protein production (M protein-high producers). These three emm genotypes are frequently isolated in clinical practice. Sequencing of the csrRS gene, one of the two-component signal transduction systems implicated in virulence, was performed on 25 strains bearing different amounts of M protein. CsrS mutations, in contrast to CsrR protein, were detected in 11 strains. The M protein-high producer strain of emm1 type carried two amino acid substitutions, whereas the other three emm1 strains carried only one substitution each. The M protein-high producer expressed its emm gene more strongly than the corresponding M protein-low producer did according to TaqMan RT-PCR. These observations suggest that the accumulation of amino acid substitutions in CsrS protein may contribute, at least in part, to the large amount of M protein production seen in several emm genotypes.

Prevalence of a streptococcal inhibitor of a complement-mediated cell lysis-like gene (sicG) in Streptococcus dysgalactiae subsp. equisimilis.

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.

[Antimicrobial susceptibility and serotype distribution in perinatal group B Streptococcus isolates--a 1999-2009 multicenter study].

We determined temporary changes in group B Streptococcus antimicrobial susceptibility and serotype distribution from perinatal strains. We examined invasive microbiological isolates from neonates with early-onset group B streptococcal disease (n = 14), and colonized isolates from those born uneventfully (n = 55) and from the genital tracts of pregnant and puerperal women (n = 198), collected between 1999 and 2009. All isolates were susceptible to penicillin. No significant differences were seen in susceptibility of 12 antimicrobial agents examined between invasive and colonized isolates. MIC50, MIC90, and resistance did not differ between stage I (1999-2005) and II (2006-2009) isolates. Serotype distribution significantly differed, however, serotypes III and Ia predominated among invasive isolates, while serotypes Ib and VI were common among their colonized counterparts. These findings suggest that to date, penicillin remains effective in intrapartum prophylactic use in colonized pregnant women.

Clindamycin-induced CovS-mediated regulation of the production of virulent exoproteins streptolysin O, NAD glycohydrolase, and streptokinase in Streptococcus pyogenes.

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 microg/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.

Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes.

We investigated culture supernatant proteins from the M1 serotype of Streptococcus pyogenes by two-dimensional gel electrophoresis and peptide mass mapping analysis, and characterized the single protein spots. Among them, we analysed the Spy0747 protein. This protein is homologous to the SsnA protein, a cell-wall-located DNase expressed in Streptococcus suis serotype 2. We designated the Spy0747 protein as SpnA. SpnA protein was also detected in the insoluble fraction of whole-cell lysates using shotgun proteomic analysis, suggesting that SpnA is also located in the cell wall. SpnA was expressed as a glutathione S-transferase-fusion protein in Escherichia coli. We confirmed that the recombinant protein had DNase activity that was dependent on Ca(2+) and Mg(2+), like SsnA. Blood bactericidal assays and mouse infection model experiments showed that the spnA knockout strain was less virulent than the parental strain, thus suggesting that SpnA could play an important role in virulence. Using PCR, we found that the spnA gene was present in all clinical S. pyogenes strains we examined. Our results, together with a previous report identifying Spy0747 as a surface-associated protein, suggest that SpnA is an important cell-wall-located DNase that is generally produced in S. pyogenes and is involved in virulence.

NADase as a target molecule of in vivo suppression of the toxicity in the invasive M-1 group A Streptococcal isolates.

BACKGROUND: NAD-glycohydrolase (NADase) secreted by M-1 group A streptococcal (GAS) isolates are suspected as one of the virulence factors to cause severe invasive disease including streptococcal toxic shock-like syndrome (STSS). M-1 GAS strains were divided into three groups based on NADase activity: high activity, low activity and no activity in our previous report. RESULTS: The representative high activity isolates taken from STSS patients showed higher virulence compared with isolates from the low activity group, when used to infect mice. The knockout mutant of the nga gene, which encodes NADase also showed reduced virulence in a mouse infection study. The cloned nga gene was able to significantly complement the lost virulence. In addition, the solution containing purified recombinant IFS, which is an inhibitor of NADase, partially rescued mice infected with S. pyogenes. CONCLUSIONS: These results indicate that NADase is important for the virulence of S. pyogenes in vivo and is the potential target to suppress the virulence.

Characterization of Streptococcus pyogenes isolated from balanoposthitis patients presumably transmitted by penile-oral sexual intercourse.

Streptococcus pyogenes is indigenous to the human pharynx and causes acute pharyngitis. Balanoposthitis is an inflammatory disease of the glans and the foreskin. However, balanoposthitis caused by S. pyogenes is not widely recognized as a sexually transmitted disease. In addition, bacteriological features of the isolates causing balanoposthitis are unclear. The four S. pyogenes strains isolated from adult balanoposthitis were examined. We performed emm typing, T antigen typing, RAPD assay, PCR assay for the streptococcal pyrogenic exotoxin-related genes and antibiotic-resistant genes, and antibiotic susceptibility assay. All four strains were suspected to be transmitted by penile-oral sexual intercourse, were found to be different by genetic analysis, and also harbored some antibiotic-resistant factors. We propose that S. pyogenes should be considered as a causative agent of sexually transmitted disease. The drug resistant S. pyogenes must be taken into account when balanoposthitis patients are treated with antibiotic.

Effects of Lonicera japonica Flower Bud Extract on Citrobacter rodentium-Induced Digestive Tract Infection.

Background: Although antibiotic therapy is currently a gold standard for bacterial infections, it is not used for severe diseases like enterohemorrhagic Escherichia coli, in which the Shiga toxin is overproduced by antibiotic action. The Lonicera japonica flower bud (LJF) is an herbal component used against purulent diseases in traditional Japanese and Chinese medicine. We investigated the effects of LJF extract (LJFE) on Citrobacter rodentium-induced digestive tract infection in a mouse model. Methods:Citrobacter rodentium and LJFE were orally administered to C57BL/6 mice. The survival rate and bacterial colonization in the large intestine, mesenteric lymph node, and blood of mice were evaluated. Cytokines secreted from intraperitoneal macrophages of LJFE-treated mice were measured using ELISA. Moreover, the phagocytic activity of intraperitoneal macrophages against Citrobacter rodentium was compared between LJFE- or chlorogenic acid (CGA)-treated mice. Results: LJFE significantly increased the survival rate and decreased Citrobacter rodentium colonization in mice. Moreover, the values of tumor necrosis factor-α, interleukin-1ß, and interferon-γ secreted from macrophages were increased following LJFE treatment. While macrophages of LJFE-treated mice showed a significant phagocytic activity, macrophages of CGA-treated mice only showed a phagocytic tendency. Conclusions: LJF may be useful for treating Citrobacter rodentium-induced digestive tract infection.

Suppressive Effects of Hainosan (Painongsan) against Biofilm Production by Streptococcus mutans.

Streptococcus mutans, a bacterium that causes dental plaques, forms a biofilm on tooth surfaces. This biofilm can cause gingivitis by stimulating the gingival margin. However, there is no established treatment for biofilm removal. Hainosan (Painongsan), a traditional Japanese Kampo formula, has been used to treat gingivitis. Therefore, we investigated the biofilm suppressive effects of the hainosan extract (HNS) and its components on S. mutans. We conducted scanning electron microscopy and confocal laser microscopy analyses to clarify the anti-biofilm activities of HNS and its crude drugs. We also performed a quantitative RT-PCR assay to assess the biofilm-related gene expression. HNS showed a significant dose-dependent suppressive effect on biofilm formation. Both the scanning electron microscopy and confocal laser microscopy analyses also revealed the significant inhibitory effects of the extract on biofilm formation. Transmission electron microscopy analysis showed that HNS disrupted the surface of the bacterial wall. Furthermore, HNS reduced the hydrophobicity of the bacteria, and suppressed the mRNA expression of ß-glucosyltransferase (gtfB), glucosyltransferase-SI (gtfC), and fructosyltransferase (ftf). Among the constituents of hainosan, the extract of the root of Platycodon grandiflorum (PG) showed the strongest biofilm suppression effect. Platycodin D, one of the constituent natural compounds of PG, inhibited S. mutans-associated biofilm. These findings indicate that hainosan eliminates dental plaques by suppressing biofilm formation by S. mutans.

Potential role for matrix metalloproteinase-3 in gastric ulcer healing.

BACKGROUND/AIMS: Matrix metalloproteinases (MMPs) are endopeptidases which perform important functions in extracellular matrix remodeling, cell proliferation, and inflammatory processes. Here, we compared MMP-3 levels with those of tissue inhibitor of metalloproteinases (TIMP)-1 and several inflammatory cytokines in gastric ulcer (GU) patients. METHODS: This study enrolled 50 patients with GU and 6 with functional dyspepsia (FD). Samples of gastric mucosa from the antrum and the ulcer site were harvested from GU patients and of antral mucosa alone from FD patients during upper gastrointestinal endoscopy. Mucosal biopsy tissues were cultured for 24 h, and the culture supernatant was measured for levels of MMP-3, TIMP-1, IL-1beta, IL-6, and IL-8. RESULTS: All GU patients were positive for Helicobacter pylori, while all FD patients were negative. Antral levels of TIMP-1, IL-1beta, IL-6, and IL-8 were significantly higher in GU than FD patients. Further, MMP-3 levels were significantly higher in GU patients at the ulcer site than in the antrum, and had a significantly positive correlation with TIMP-1, IL-1beta, IL-6, and IL-8. CONCLUSION: MMP-3 levels were significantly higher at the ulcer site than in the antrum, suggesting that MMP-3 may perform an important function in gastric ulcer healing.

Relationship between nutritional therapy and surgery in Crohn's disease.

BACKGROUND/AIMS: Crohn's disease is often refractory and some patients require repeated surgical treatment. Nutritional therapy with an elemental diet has been reported effective in improving nutritional state and suppressing inflammation, and might be expected to assist in minimizing the need for surgery. We evaluated the relationship between an elemental diet and the period that patients spent without intestinal resection. METHODOLOGY: A total of 153 patients with Crohn's disease who visited our hospital from July, 1999 to July, 2005 were enrolled. The relationship between the caloric content of an elemental diet and surgery as an endpoint was examined using Cox regression analysis. Cumulative non-operation rates were calculated by the Kaplan-Meier method. Statistical significance was determined using the log-rank test. RESULTS: Among patients with jejunoileal involvement, patients receiving an elemental diet providing 900 kcal or more per day showed a statistically significant improvement in cumulative non-operation rate. Among those without jejunoileal involvement, in contrast, the cumulative non-operation rate did not differ among those receiving an elemental diet of less or more than 900 kcal per day. CONCLUSIONS: The use of an elemental diet of 900 kcal per day may be effective in avoiding surgery in patients with jejunoileal lesions. This diet may be useful in improving long-term convalescence in these patients.

Effect of Lonicera caerulea var. emphyllocalyx Extracts on Murine Streptococcus pyogenes Infection by Modulating Immune System.

Streptococcus pyogenes (S. pyogenes) causes several infectious diseases such as tonsillitis, cellulitis, and streptococcal toxic shock syndrome. The general treatment of S. pyogenes infection is by using ß-lactam antibiotics; however, the cases of treatment failure were increasing as serious problems. Lonicera caerulea var. emphyllocalyx (LCE) has been used in the folk medicine in the northern part of Japan, the northern part of China, Korea, and Russia. In this study, we investigated the efficacy of three parts (fruit, stem, and leaf) of Lonicera caerulea var. emphyllocalyx extract (LCEEs) against murine S. pyogenes infection. Oral administration of LCEEs increased the mortality in murine model, and the extracts of its stems and leaves were more effective than the fruit extract significantly. Murine splenocytes and mesenteric lymph nodal cells treated with LCEEs suppressed the excess production of inflammatory cytokine such as TNF-α in comparison to those from untreated cells. LCEEs stimulated the differentiation of pluripotent hematopoietic stem cells in those murine lymph nodal cells. It also activated the proliferative response of murine lymph nodal cells. We also found that the stem and leaf extracts seemed to be more effective than the fruit extract in those phenomena. The concentration of lignins in LCEE prepared from the stems was larger than that from leaves, and that was larger than that from the fruits. Our data suggest that LCE, especially the stems and the leaves, may be useful for the treatment of S. pyogenes infection.

In Vitro Effect of the Traditional Medicine Hainosan (Painongsan) on Porphyromonas gingivalis.

Background: Hainosan (Painongsan) is a traditional Japanese and Chinese medicine that is used to treat several purulent diseases, including gingivitis and periodontitis. This formulation contains three crude drug components: The dried immature fruit of Citrus aurantium (Aurantii Fructus Immaturus), the dried root of Paeonia lactiflora (Paeoniae Radix), and the dried root of Platycodon grandiflorum (Platycodi Radix). Here we evaluated the in vitro antibacterial effects of hainosan extract (HNS) and extracts of its three components against Porphyromonas gingivalis, one of the pathogenic bacteria that causes periodontitis. Methods: The antibacterial activities of HNS and its components were examined by counting the number of colony-forming units (CFUs) and through transmission electron microscopy. Results: We found that HNS had direct antibacterial activity against three P. gingivalis isolates (JCM12257, JCM8525, and JCM19600), with HNS-treated cells being significantly smaller than those of untreated bacteria. Extracts of Platycodi Radix and Paeoniae Radix significantly suppressed the growth of P. gingivalis in a dose-dependent manner, with Platycodi Radix extract having the greatest antibacterial effect. In addition, P. gingivalis that were treated with Platycodi Radix extract were significantly larger than those treated with Aurantii Fructus Immaturus or Paeoniae Radix extracts. Further analysis showed that platycodin D, which is one of the ingredients of Platycodi Radix, reduced bacterial growth. Conclusions: Platycodi Radix is the active component in Hainosan and may represent a useful agent for the treatment of P. gingivalis-induced gingivitis and periodontitis.