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.

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.

Effect of Lonicera caerulea var. emphyllocalyx Fruit on Biofilm Formed by Porphyromonas gingivalis.

Porphyromonas gingivalis is an important pathogenic anaerobic bacterium that causes aspiration pneumonia. This bacterium frequently forms biofilms in the oral cavity and in respiratory tract-associated medical devices. Bacterial colonization that occurs in association with this biofilm formation is the main reason for incurable aspiration pneumonia. The Lonicera caerulea var. emphyllocalyx (LCE) fruit has been used in folk medicine in Hokkaido, the northern part of Japan. The aim of this study was to elucidate one of the antimicrobial mechanisms of LCE methanol extract (LCEE)-the inhibitory effect of LCEE on biofilm formation by P. gingivalis. Our results show that LCEE significantly reduced biofilm formation by three different P. gingivalis isolates in a concentration- and time-dependent manner that were quantified by the adsorption of safranin red. When LCEE was added to biofilms already formed by P. gingivalis, LCEE did not degrade the biofilm. However, treatment with LCEE significantly promoted the removal of existing biofilm by vibration compared to that of control. We also confirmed biofilm formation in LCEE-treated P. gingivalis in tracheal tubes using scanning electron microscopic (SEM) analysis. Cyanidin 3-O-glucoside (C3G), one of the components of LCE, also inhibited the formation of biofilm by P. gingivalis in a concentration-dependent manner. Our results reveal that LCEE may be an effective antibacterial substance for P. gingivalis-induced aspiration pneumonia because of its role in the suppression of bacterial biofilm formation in the oral cavity.

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.

Methanol extract of Lonicera caerulea var. emphyllocalyx fruit has antibacterial and anti-biofilm activity against Streptococcus pyogenes in vitro.

Streptococcus pyogenes causes several infectious diseases such as tonsillitis, cellulitis, and streptococcal toxic shock syndrome. As antibiotics are used for the general treatment of S. pyogenes infection, cases of treatment failure due to drug-resistant bacteria have increased. Lonicera caerulea var. emphyllocalyx (LCE) has been used as a folk medicine in northern Japan (Hokkaido). In this study, we investigated the antibacterial effect of methanol extracts of the fruit, stem, and leaf of LCE (LCEEs) against S. pyogenes using disk diffusion assay. As LCEE (fruit) had the strongest antibacterial activity among the three LCEEs, we focused on functional analysis of antibacterial effects of LCEE (fruit). LCEE (fruit) suppressed the growth of S. pyogenes in a dose-dependent manner. Morphological analysis by transmission electron microscopy demonstrated that LCEE (fruit) damaged the shape of S. pyogenes. Microplate and confocal laser microscopy analysis showed that biofilm formation was also suppressed by LCEE (fruit) in a dose-dependent manner. To further evaluate the surface structure of these biofilms, we performed hydrophobic analysis, which demonstrated that LCEE (fruit) reduced the hydrophobicity of the bacterial surface structure. Our data demonstrated that LCEE (fruit) had anti-bacterial and anti-biofilm effects on S. pyogenes in vitro, suggesting that the direct anti-bacterial effects of the LCEE (fruit) may be useful for treatment of local S. pyogenes infection.

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.

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.

Methanol extract of Lonicera caerulea var. emphyllocalyx fruit has anti-motility and anti-biofilm activity against enteropathogenic Escherichia coli.

Foodborne diseases have become a worldwide problem that threatens public health and welfare. Enteropathogenic Escherichia coli (EPEC) is one of major pathogens of moderate to severe diarrhea. The increased prevalence of EPEC strains that produce extended spectrum ß-lactamase (ESBL) has deepened the problem. The fruit of Lonicera caerulea var. emphyllocalyx (LCE) has been used as a traditional food preservative and medicine in northern temperate zones such as Hokkaido Island, Japan. In this study, we investigated the antibacterial effect of LCE fruit extract (LCEE) against EPEC. The antibacterial activities of LCEE were examined by bacterial growth, time-kill curve, soft-agar motility, electron microscopy, and 96 well-microplate biofilm assays. We also investigated the bacterial mRNA expression of biofilm-associated genes (fliC, csgA, and fimA) by quantitative real-time PCR assays. LCEE was found to suppress the growth, time-kill curve, and spread of EPEC. It also reduced the biofilm formation in a dose-dependent manner. Morphological analysis using transmission and scanning electron microscopy revealed that LCEE diminished the function of flagella resulting in reduced motility and biofilm formation. The mRNA expression of all three biofilm associated genes was downregulated under LCEE treatment. Extracts of the fruit of LCE inhibit the motility and biofilm formation of EPEC as a result of the inhibition of flagella development and function. We propose LCEE as a therapeutic candidate for the effective therapy of EPEC-associated infectious diseases.

The immunostimulatory effects and chemical characteristics of heated honey.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), honey has been used as an additive in the heat-processing of herbal medicines to enhance their immunostimulatory activities. AIM OF THE STUDY: We investigated the immunostimulatory activity of heated honey in vitro and in vivo. MATERIALS AND METHODS: For the in vitro study, we compared the differences among the inducible effects of honey subjected to various heating conditions on granulocyte colony-stimulating factor (G-CSF) secretion from the cultured enterocytes and investigated the active ingredient. For the in vivo study, we conducted a survival test of mice infected by Streptococcus pyogenes with and without oral administration of heated honey. RESULTS: We found that heating the honey induced the appearance of G-CSF secretions from the cultured enterocytes, and that this appearance depended on the heating temperature and time. No G-CSF secretions appeared when honey was not heated. Mice infected with Streptococcus pyogenes that were fed heated honey revealed prolonged survival. The active ingredient in heated honey was a high-molecular compound with about 730 kDa. When this compound was hydrolyzed, galactose, glucose, rhamnose, α-ribofuranose ß-ribofuranose 1,5':1',5-dianhydride, and 5-hydroxymethylfurfural were generated. CONCLUSIONS: Heated honey reveals immunostimulatory activity both in vitro and in vivo. These results support the scientific evidences of the TCM theory.

Effect of Hochuekkito (Buzhongyiqitang) on Nasal Cavity Colonization of Methicillin-Resistant Staphylococcus aureus in Murine Model.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) infections are largely preceded by colonization with MRSA. Hochuekkito is the formula composing 10 herbal medicines in traditional Kampo medicine to treat infirmity and to stimulate immune functions. We evaluated the efficacy of hochuekkito extract (HET) against MRSA colonization using a nasal infection murine model. Methods: We evaluated the effects of HET as follows: (1) the growth inhibition by measuring turbidity of bacterial culture in vitro, (2) the nasal colonization of MRSA by measuring bacterial counts, and (3) the splenocyte proliferation in mice orally treated with HET by the ³H-thymidine uptake assay. Results: HET significant inhibited the growth of MRSA. The colony forming unit (CFU) in the nasal fluid of HET-treated mice was significantly lower than that of HET-untreated mice. When each single crude drug-Astragali radix, Bupleuri radix, Zingiberis rhizoma, and Cimicifugae rhizome-was removed from hochuekkito formula, the effect of the formula significantly weakened. The uptake of ³H-thymidine into murine splenocytes treated with HET was significantly higher than that from untreated mice. The effects of the modified formula described above were also significantly weaker than those of the original formula. Conclusions: Hochuekkito is effective for the treatment of MRSA nasal colonization in the murine model. We suggest HET as the therapeutic candidate for effective therapy on nasal cavity colonization of MRSA in humans.

Comparison between the Effects of Oral and Intramuscular Administration of Shin'iseihaito (Xinyiqingfeitang) in a Streptococcus pyogenes-Induced Murine Sinusitis Model.

Streptococcus pyogenes (S. pyogenes) is a species of Gram-positive coccoid bacteria having many virulence factors. Its capsule and exotoxins can cause upper respiratory tract infections such as sinusitis. The general treatment for S. pyogenes-induced sinusitis is administration of antibiotics such as penicillin and macrolides; however, a serious problem associated with these antibiotics is their attenuated effect. Shin'iseihaito (Xinyiqingfeitang), a formula of Japanese traditional Kampo medicine and traditional Chinese medicine, has been used for the treatment of sinusitis. In general, formulas of Japanese traditional Kampo medicine are orally administered. This is in contrast to certain formulas of traditional Chinese medicine, which are being recently administered intramuscularly or intravenously. Regarding these traditional Chinese medicine formulas, the injection methodology is reported to be more effective than oral intake. In this study, we compared the efficacy between orally and intramuscularly administered Shin'iseihaito against S. pyogenes-induced sinusitis. We evaluated the antibacterial effect of Shin'iseihaito extract (SSHT) against S. pyogenes by K-B disk diffusion assay. Furthermore, we investigated the nasal colonization of S. pyogenes, determined cytokine (TNF-α, IL-1ß, and IL-6) levels, and conducted a splenocyte proliferative assay in a murine sinusitis model. SSHT displayed direct anti-S. pyogenes activity. Intramuscular administration of SSHT decreased the nasal colonization of S. pyogenes compared with oral administration. Thymidine uptake analysis revealed that the proliferation of splenocytes from S. pyogenes-infected mice under intramuscular SSHT treatment was upregulated compared to that of splenocytes from S. pyogenes-infected mice under oral SSHT treatment. We also found that TNF-α, IL-1ß, and IL-6 levels in the nasal discharge from intramuscularly treated S. pyogenes-infected mice were lower than those from orally treated mice. Our findings suggest that intramuscular administration of Shin'iseihaito may be useful for the treatment of murine S. pyogenes-induced sinusitis.

Effect of Shin'iseihaito (Xinyiqingfeitang) on Acute Streptococcus pneumoniae Murine Sinusitis via Macrophage Activation.

Streptococcus pneumoniae (S. pneumoniae) causes sinusitis. The general treatment of S. pneumonia sinusitis is by using antibiotics; however, one of their serious problems is the attenuation of their effect. Shin'iseihaito (Xinyiqingfeitang), a formula of Japanese traditional Kampo medicine, has been used for the treatment of sinusitis in Japan. In this study, we investigated the efficacy of Shin'iseihaito against S. pneumoniae-caused sinusitis in mice. Oral administration of Shin'iseihaito extract (SSHT) decreased the nasal colonization of S. pneumoniae in both prophylactic and therapeutic treatments, respectively, and the former was more effective than the latter. Histopathological analysis revealed that the epithelial tissue from S. pneumoniae-infected nose under SSHT treatment recovered the tissue destruction in comparison to infected nose. We also confirmed this result by scanning electron microscopic analysis. Murine peritoneal macrophages from SSHT-treated mice had significant phagocytic activity in comparison to those from untreated group. We also found that tumor necrosis factor-α, interleukin-1ß, interleukin-6, and monocyte chemotactic protein-1 levels and the migration of macrophages from S. pneumoniae-infected mice with the treatment with SSHT were increased compared to those from untreated group. Our data suggest that Shin'iseihaito may be useful for the treatment of S. pneumoniae-induced sinusitis.

Shin'iseihaito (Xinyiqingfeitang) Suppresses the Biofilm Formation of Streptococcus pneumoniae In Vitro.

Streptococcus pneumoniae (S. pneumoniae) is the important pathogen that causes otolaryngeal diseases such as sinusitis. S. pneumoniae frequently forms the biofilm to prevent severe circumstances such as antimicrobial agents. Shin'iseihaito (xinyiqingfeitang) is a formula of Japanese traditional Kampo medicine that has 9 crude drugs and provides the medicinal usage for sinusitis. The objective of the present study is to reveal the mechanism of antibiofilm activity by Shin'iseihaito extract (SSHT). SSHT significantly inhibited the formation of biofilm from S. pneumoniae ATCC 49619 in dose- and time-dependent manners. SSHT also significantly suppressed the biofilm formation by other five different cps types of S. pneumoniae clinical isolates. We found that the extracts of 8 out of 9 components in Shin'iseihaito had the inhibitory effects of biofilm formation, and the extract of the root of Scutellaria baicalensis had the strongest effect among the ingredients of Shin'iseihaito. We found that the capsule of SSHT-treated S. pneumoniae was significantly thinner than that of the untreated group and that SSHT reduced the hydrophobicity of bacterial cell surface. Our results suggest that Shin'iseihaito may be a useful agent for the treatment of S. pneumoniae-induced sinusitis because of the inhibition of biofilm formation of S. pneumoniae.

Effect of Shin'iseihaito on murine allergic reaction induced by nasal sensitization.

Shin'iseihaito (Magnolia Flower Lung-Clearing Decoction; xin yí qing fèi tang), a formula of traditional Japanese kampo medicine ( rì ben hàn yi) and traditional Chinese medicine (TCM; zhong yi), has been used for the treatment of chronic sinusitis. The objective of this study was to evaluate the anti-allergic effect of shin'iseihaito on murine allergic reaction induced by nasal sensitization using ovalbumin (OVA) as an antigen. Extract of shin'iseihaito (SSHT) could reduce the eosinophil, serum IgE and interleukin (IL)-4 levels, while increased the interferon (IFN)-γ levels in allergic mouse. Furthermore, allergic-murine serum treated with SSHT could not activate passive cutaneous anaphylaxis (PCA) reaction in murine model. Thus, our study showed that SSHT may possess anti-allergic activity. We suggested that SSHT may contribute to inhibit the exacerbation of allergic reaction induced by nasal sensitization.

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.

The cell envelope-associated protein, LytR, regulates the cysteine protease SpeB in Streptococcus pyogenes.

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.

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.

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.