Septic shock is associated with receptor for advanced glycation end products ligation of LPS.

Septic shock is a severe systemic response to bacterial infection. Receptor for advanced glycation end products (RAGE) plays a role in immune reactions to recognize specific molecular patterns as pathogen recognition receptors. However, the interaction between LPS, the bioactive component of bacterial cell walls, and RAGE is unclear. In this study, we found direct LPS binding to RAGE by a surface plasmon resonance assay, a plate competition assay, and flow cytometry. LPS increased TNF-α secretion from peritoneal macrophages and an NF-κB promoter-driven luciferase activity through RAGE. Blood neutrophils and monocytes expressed RAGE, and TLR2 was counterregulated in RAGE(-/-) mice. After LPS injection, RAGE(+/+) mice showed a higher mortality, higher serum levels of IL-6, TNF-α, high mobility group box 1, and endothelin-1, and severe lung and liver pathologies compared with RAGE(-/-) mice without significant differences in plasma LPS level. Administration of soluble RAGE significantly reduced the LPS-induced cytokine release and tissue damage and improved the LPS-induced lethality even in RAGE(-/-) as well as RAGE(+/+) mice. The results thus suggest that RAGE can associate with LPS and that RAGE system can regulate inflammatory responses. Soluble RAGE would be a therapeutic tool for LPS-induced septic shock.

Genome sequence of a nephritogenic and highly transformable M49 strain of Streptococcus pyogenes.

The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.

Analysis of children with Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae respiratoryinfections by real-time PCR assay and serological tests.

We examined 73 children with respiratory infections for Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae using real-time PCR assay and serological tests. C. pneumoniae and M. pneumoniae infections were found in 11 (15.1%) and 6 (8.2%) cases, respectively. The sensitivities and specificities of real-time PCR versus definite diagnosis of acute infection were 63.6% and 100% for C. pneumoniae, and 100% and 100% for M. pneumoniae, respectively. C. pneumoniae PCR-negative results appeared to be due to poor growth of the organism. The sensitivity and specificity of ImmunoCard tests were 33.3% and 82.1%, respectively, indicating that the efficacy of rapid diagnosis was disputable. The present results suggest that real-time PCR is suitable for rapid diagnosis as a first screening test to determine first-line antibacterial agents to be used against these infectious diseases.

Antibacterial activity of honey from stingless honeybees (Hymenoptera; Apidae; Meliponinae).

The aim of the study was to examine antibacterial activity of the honey of stingless honeybees (Meliponinae). An agar well diffusion assay demonstrated that many honey samples of stingless honeybees inhibited the growth of test strains of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa; moreover, they exhibited non-peroxide antibacterial activity against those strains. This is the first time that non-peroxide antimicrobial activity of honey from a number of species of stingless honeybees has been demonstrated. These antibacterial activities appear to be powerful, even when compared to those of"manuka honey" from Apinae honeybees.

Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism, expression of the C-terminal half of heavy chain and its binding activity.

Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (H(C)) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the H(C), recombinant genes for the H(C) and two hybrid H(C) in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant H(C) of 111/NT to the protein receptor synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid H(C) revealed that mutation of 23 residues in carboxy terminal half of H(C) (1029-1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.

Clostridium absonum alpha-toxin: new insights into clostridial phospholipase C substrate binding and specificity.

Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene. We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme. Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa. However, the Caa enzyme had a lower activity than Cpa to the free (i.e. not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity. The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa. The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures. This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop. Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.

Phthisis bulbi caused by late congenital syphilis untreated until adulthood.

PURPOSE: To report a case of phthisis bulbi resulting from late congenital syphilis untreated until adulthood. DESIGN: Observational case report. METHODS: We report clinical and laboratory evaluations of a 43-year-old woman who presented with a palpebral ulcer of the right eye. RESULTS: The patient had a gummatous palpebral ulcer and a phthisis bulbi in the right eye and a gumma on the left eyelid. A silent interstitial keratitis of the left eye was detected. The patient had hearing loss in the right ear, her nose was missing, and her right leg had been amputated. Treponemal pallidum hemagglutination (TPHA) test was positive. Although we administered intensive oral penicillin, the clinical symptoms of the patient did not improve. CONCLUSIONS: This is a rare case of phthisis bulbi resulting from late congenital syphilis. We emphasize that treatment for late congenital syphilis must be carried out early and completely.

Incidence and bacteriology of bacteremia associated with various oral and maxillofacial surgical procedures.

OBJECTIVE: The aim of this study was to determine the incidence and bacteriology of bacteremia associated with various oral and maxillofacial surgical procedures. METHODS: A total of 237 patients who underwent oral and maxillofacial surgery were included in this study. Blood samples were obtained for bacteriological examination immediately after the essential steps of the surgical procedure had been performed. RESULTS: Bacteremia was detected in patients who underwent surgery for tumor, infection and trauma, and surgical reconstruction of jaw. In particular, decortication for osteomyelitis and tooth extraction resulted in a higher incidence of bacteremia compared with other surgical procedures. The incidence of bacteremia was not affected by oral hygiene, gingival inflammation, blood loss, and duration of surgery. Furthermore, concerning tooth extraction, there was no statistical difference in the incidence of bacteremia with respect to the number of teeth extracted and the method of extraction. Extraction of teeth with odontogenic infection (periodontitis, periapical infection, and pericoronitis) did however produce a significantly increased incidence of bacteremia compared with infection-free teeth (P < .01). Viridans streptococci were the predominant group of bacteria isolated from the bacteremias. CONCLUSION: Oral and maxillofacial surgery involving transoral incision produces bacteremia, regardless of the extent and degree of surgical invasion. In particular, surgical procedure at infected sites is more likely to result in bacteremia compared with infection-free sites.

Linkage between toxin production and purine biosynthesis in Clostridium difficile.

The production of toxins A and B by Clostridium difficile was greatly enhanced under biotin-limited conditions, in which a 140-kDa protein was expressed strongly. Gene cloning revealed that this protein was a homologue of formylglycinamidine ribonucleotide synthetase (FGAM synthetase, EC 6.3.5.3), which is known as PurL in Escherichia coli and catalyses the fourth step of the de novo purine biosynthesis pathway. This enzyme consisted of a single polypeptide, although FGAM synthetases of gram-positive bacteria usually consist of two subunits. Inhibition of the enzymic activity of C. difficile PurL by O-diazoacetyl-L-serine (azaserine) resulted in enhanced toxin B production even in biotin-sufficient conditions. In contrast, blockade of the preceding step of the PurL catalysing step by sulfamethoxazole inhibited toxin B production almost completely. These results suggest that accumulation of formylglycinamide ribonucleotide (FGAR), a substrate of FGAM synthetase, enhances toxin production by C difficile and depletion of FGAR reduces toxin production.

Clostridium difficile colonization in healthy adults: transient colonization and correlation with enterococcal colonization.

The aim of the present study was to investigate the colonization status of Clostridium difficile in healthy individuals. In total, 139 healthy adults from two study groups were examined at intervals of 3 months. Among the 18 positive subjects, the number of subjects from whom C. difficile was isolated once, twice, three times or four times was 10 (55.6%), three (16.7%), two (11.1%) and three (16.7%), respectively. In the student group, different subjects were colonized by different PCR ribotype/PFGE types. However, the same PCR ribotype/PFGE types of C. difficile were isolated from different subjects in the employee group, indicating that cross-transmission may have occurred in this group. Continuous colonization by the same PCR ribotype/PFGE type was only observed in three subjects. C. difficile-positive subjects were significantly more densely colonized by enterococci (P<0.05) than C. difficile-negative subjects: subjects that were found to be C. difficile-positive three or four times appeared to have higher concentrations of enterococci. The present results demonstrate that, although colonization by a C. difficile strain is transient in many cases, there are healthy individuals that are colonized persistently by C. difficile. They also suggest that dense colonization of the intestine by enterococci may be associated with C. difficile colonization.

Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component.

Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox). The amino acid sequences of type C and D nonTox components are almost identical. In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine. This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C- and D-16 S toxins. The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin. This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both. The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera. When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C- or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice. These results indicate that the local anti-nonTox antibodies reduce absorption of both C- and D-16 S toxins from the small intestine. The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

Association of oral yeast carriage with specific host factors and altered mouth sensation.

OBJECTIVES: The aim of this study was to determine if there was a significant association between the presence of altered mouth and taste sensations with oral carriage of yeasts and to assess the factors that influence the yeast carriage. STUDY DESIGN: The oral and dental status including unstimulated (USFR) and stimulated (SSFR) whole salivary flow rates of a total of 509 subjects was recorded. Saliva specimens were collected for microbiologic examination. Multiple logistic regression analysis was performed to identify any factors that were significantly associated with the prevalence of oral yeasts. RESULTS: Old age, clinical signs of oral dryness, denture wearing, and a reduction in USFR increased the prevalence of yeasts, whereas patient gender, levels of dentition, the sensation of dry or burning mouth, taste disorders, and SSFR were not associated with increased prevalence of oral yeasts. CONCLUSIONS: An increased prevalence of oral yeasts was not found to relate to changes in mouth sensation alone. Other factors, most notably patient age, the wearing of dentures, clinical signs of oral dryness, and salivary flow rate under rest conditions, were, however, found to be closely associated with oral yeast carriage.

Detection of cfxA and cfxA2, the beta-lactamase genes of Prevotella spp., in clinical samples from dentoalveolar infection by real-time PCR.

While most bacteria involved in dentoalveolar infection are highly susceptible to penicillin, some Prevotella strains exhibit resistance to this agent through the production of beta-lactamase. The production of beta-lactamase by Prevotella spp. is in turn associated with the expression of the genes cfxA and cfxA2. The aim of the present study was to determine the prevalence of cfxA and cfxA2 in Prevotella strains by use of real-time PCR and to assess the performance of this molecular method for the direct detection of the genes in 87 clinical samples (pus and root canal exudates) from dentoalveolar infection. Production of beta-lactamase by each isolate was determined using a nitrocefin disk. beta-Lactamase production was seen in 31% of Prevotella isolates, while all isolates of other species were beta-lactamase negative. The penicillin resistance of isolates strongly correlated with the production of beta-lactamase. Real-time PCR was found to detect the cfxA and cfxA2 genes from at least five cells per reaction mixture (5 x 10(3) CFU/ml of pus). Using real-time PCR, the presence of cfxA and cfxA2 was evident for all 48 beta-lactamase-positive Prevotella strains. In contrast, neither beta-lactamase-negative Prevotella (n = 91) or non-Prevotella (n = 31) strains were positive for the genes. In this study, 31 of the 87 samples yielded beta-lactamase-positive Prevotella results, and cfxA and cfxA2 were detected in all 31 samples. Of the 56 culture-negative samples, 8 (14%) were positive for cfxA and cfxA2 by the real-time PCR. This sensitive and specific molecular method offers a rapid clinical test for aiding in the selection of an appropriate antibiotic for treatment of dentoalveolar infection. Although penicillin remains largely effective in the treatment of dentoalveolar infection, beta-lactamase-stable antibiotics should be considered in cases in which beta-lactamase-positive Prevotella strains are involved.

Proteinase activity of prevotella species associated with oral purulent infection.

Prevotella intermedia and Prevotella nigrescens are often regarded as principal causes of acute dentoalveolar infection; however, other species within the genus are also known to be associated with such infection. The aim of this study was to determine the in vitro proteolytic activity of these different Prevotella species that have been implicated with dentoalveolar infection. A total of 234 strains were obtained from pus specimens from dentoalveolar infection and from the plaque of healthy volunteers. Prevotella loescheii, Prevotella oralis, Prevotella melaninogenica, Prevotella buccae, and Prevotella denticola were all shown to have a proteolytic activity (8.5-10.5 x 10(-8) A-units) lower than that of P. intermedia and P. nigrescens (21.1-23.5 x 10(-8) A-units). In the case of P. loescheii, P. melaninogenica, and P. intermedia, the level of proteolytic activity for clinical strains was significantly (P < 0.05) higher than that recorded for commensal strains. Proteolytic activity for all species of Prevotella examined was inhibited by N-ethylmaleimide and phenymethylsulfonyl fluoride. This study suggests that Prevotella species associated with oral purulent infection produce cysteine and serine proteinases and that in certain species of Prevotella, the strains involved in infection exhibit higher proteolytic activity when compared with strains from healthy sites.

Occurrence of the Fimbria Gene hifA in clinical isolates of nonencapsulated Haemophilus influenzae.

The adherence of Haemophilus influenzae to epithelial cells plays a crucial role in infections. However, little is known about the occurrence of fimbriae. In this study, we examined the distribution of the fimbria gene (hifA) by PCR among 167 H. influenzae strains isolated from patients with respiratory infections. Almost all (163; 98%) of the isolates were nonencapsulated strains. The carriage rate of hifA by the nonencapsulated strains was 18.4%. Electron microscopy showed that fimbriae were abundantly present on the cell surface of hifA-positive strains tested. Only four (2.4%) isolates were encapsulated, all of which were type b and did not possess hifA. The present work suggests that fimbriae may play a considerable role as adhesins in nonencapsulated H. influenzae strains.

Clostridium sardiniense Prévot 1938 and Clostridium absonum Nakamura et al. 1973 are heterotypic synonyms: evidence from phylogenetic analyses of phospholipase C and 16S rRNA sequences, and DNA relatedness.

Clostridium sardiniense Prévot 1938 and Clostridium absonum Nakamura et al. 1973 have long been considered similar in terms of their biological and biochemical properties, but their taxonomic positions have not been clarified by DNA-DNA hybridization studies or rigorous analysis of 16S rRNA genes. In the present study, DNA-DNA hybridization analysis revealed that C. absonum strains DSM 599(T), DSM 600 and KZ 1544 shared 83.0-86.3 % DNA relatedness with C. sardiniense DSM 2632(T). 16S rRNA gene sequence analysis showed that the C. absonum strains also shared high identity with C. sardiniense DSM 2632(T) (99.7, 99.3 and 99.8 % for DSM 599(T), DSM 600 and KZ 1544, respectively), implying that C. absonum and C. sardiniense are synonyms. In addition, alignment of the inferred amino acid sequences for phospholipase C (PLC) indicated 96.5 % identity between PLCs from C. sardiniense and C. absonum, but relatively low identity with other clostridial species. These results strongly suggest that the species C. sardiniense and C. absonum should be united, with the name C. sardiniense having priority.

Colonization by Clostridium difficile of neonates in a hospital, and infants and children in three day-care facilities of Kanazawa, Japan.

The intestinal-carriage rates of Clostridium difficile in neonates hospitalized in the University Hospital's Center for Perinatal and Reproductive Health and in infants and children enrolled in two day-nurseries and a kindergarten were examined. Swab samples from the floors of these facilities were also analyzed to determine the extent of environmental contamination by this organism. C. difficile was found in the stool of only one of 40 neonates during the normal 1-week stay in the hospital after delivery. The isolate from the neonate was identical to that of her mother, as determined by PCR ribotyping, pulsed-field gel electrophoresis analysis, and toxin gene type, suggesting that the C. difficile-positive neonate acquired the organism from her mother rather than from the environment. By contrast, 47 (48.0%) of the 98 infants and children, comprising 50 enrolled in two day-nurseries who were >= 3 years old and 48 enrolled in a kindergarten who were 2-5 years old, carried C. difficile. The carriage rate in infants under 2 years of age was much higher (84.4%) than in children 2 years old and older (30.3%). When analyzed according to age group, the carriage rates were 100, 75.0, 45.5, 24.0, 38.5, and 23.5% in infants and children 0, 1, 2, 3, 4, and 5 years old, respectively. The observation that several children were colonized with the same type of C. difficile strain in each day-care facility, and that the floors of day-nursery A and kindergarten C were contaminated with C. difficile strains identical to those colonizing the intestines of children enrolled in those facilities suggests that cross-infection of C. difficile among children occurs through C. difficile-carrying children or their contaminated environments.

PCR identification of the plasmid-borne enterotoxin gene (cpe) in Clostridium perfringens strains isolated from food poisoning outbreaks.

It is generally accepted that Clostridium perfringens strains associated with food poisoning carry their enterotoxin gene, cpe, on the chromosome, while C. perfringens strains isolated from non-food-borne diseases, such as antibiotic-associated diarrhea and sporadic diarrhea, carry cpe on the plasmid. However, we recently encountered a food poisoning outbreak caused by C. perfringens bearing a plasmid cpe. We therefore investigated a total of 31 clinical and non-clinical C. perfringens strains to locate the cpe gene by PCR. The cpe of nine heat-sensitive (100 degrees C for 10min) strains isolated from three outbreaks of food poisoning were located on the plasmid, while those of six heat-resistant strains from other food poisoning outbreaks were located on the chromosome. Moreover, the cpe of 5 heat-sensitive strains isolated from healthy human feces and those of 11 heat-sensitive soil strains were also located on the plasmid. These findings indicate that heat-sensitive, cpe-plasmid-borne C. perfringens strains should not be disregarded as causative agents of food poisoning.