Saccharospirillum impatiens gen. nov., sp nov., a novel gamma-Proteobacterium isolated from hypersaline Ekho Lake (East Antarctica)

Five Gram-negative, motile, aerobic to microaerophilic spirilla were isolated from various depths of the hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica). The strains are oxidase- and catalase-positive, metabolize a variety of sugars and carboxylic acids and have an absolute requirement for sodium ions. The predominant fatty acids of the organisms are C-16: (1)omega7c, C-16:0 and C(18:1)omega7c, with C-10:1 3-OH, C-10:0 3-OH, C-12:0 3-OH, C-14:1 3-OH, C-14:0 3-OH and C-19:1 present in smaller amounts. The main polar lipids are diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmonomethylamine. The DNA base composition of the strains is 54-55 mol% G + C. 16S rRNA gene sequence comparisons show that the isolates are related to the genera Oceanospirillum, Pseudospirillum, Marinospirillum, Halomonas and Chromohalobacter in the gamma-Proteobacteria. Morphological, physiological and genotypic differences from these previously described genera support the description of a novel genus and species, Saccharospirillum impatiens gen. nov., sp. nov. The type strain is EL-105(T) (= DSM 12546(T) = CECT 5721(T)).

Suttonella ornithocola sp nov., from birds of the tit families, and emended description of the genus Suttonella

Phenotypic and phylogenetic studies were performed on three strains of Gram-negative, rod-shaped organisms recovered from dead birds of the tit families (blue tit, coal tit and long-tailed tit). Morphological, cultural and biochemical studies indicated that the organisms were related to the family Cardiobacteriaceae in the gamma-subclass of the Proteobacteria. Comparative 16S rRNA gene sequencing studies confirmed these findings and demonstrated that the bacterium represents a hitherto unknown subline within this family. The closest phylogenetic relative of the strains isolated from the birds was found to be Suttonella indologenes, although a sequence divergence of approximately 5 % demonstrated that the unknown bacterium represented a novel species. On the basis of the results of the phylogenetic analysis and phenotypic criteria, it is proposed that the bacteria recovered from the diseased birds represent a novel species, Suttonella ornithocola sp. nov., with strain B6/99/2(T) (=CCUG 49457(T)=NCTC 13337(T)) as the type strain.

Selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia

Aims/hypothesis Recent evidence suggests that a particular gut microbial community may favour occurrence of the metabolic diseases. Recently, we reported that high-fat (HF) feeding was associated with higher endotoxaemia and lower Bifidobacterium species (spp.) caecal content in mice. We therefore tested whether restoration of the quantity of caecal Bifidobacterium spp. could modulate metabolic endotoxaemia, the inflammatory tone and the development of diabetes. Methods Since bifidobacteria have been reported to reduce intestinal endotoxin levels and improve mucosal barrier function, we specifically increased the gut bifidobacterial content of HF-diet-fed mice through the use of a prebiotic (oligofructose [OFS]). Results Compared with normal chow-fed control mice, HF feeding significantly reduced intestinal Gram-negative and Gram-positive bacteria including levels of bifidobacteria, a dominant member of the intestinal microbiota, which is seen as physiologically positive. As expected, HF-OFS-fed mice had totally restored quantities of bifidobacteria. HF-feeding significantly increased endotoxaemia, which was normalised to control levels in HF-OFS-treated mice. Multiple-correlation analyses showed that endotoxaemia significantly and negatively correlated with Bifidobacterium spp., but no relationship was seen between endotoxaemia and any other bacterial group. Finally, in HF-OFS-treated-mice, Bifidobacterium spp. significantly and positively correlated with improved glucose tolerance, glucose-induced insulin secretion and normalised inflammatory tone (decreased endotoxaemia, plasma and adipose tissue proinflammatory cytokines). Conclusions/interpretation Together, these findings suggest that the gut microbiota contribute towards the pathophysiological regulation of endotoxaemia and set the tone of inflammation for occurrence of diabetes and/or obesity. Thus, it would be useful to develop specific strategies for modifying gut microbiota in favour of bifidobacteria to prevent the deleterious effect of HF-diet-induced metabolic diseases.

Large is fast, small is tight: determinants of speed and affinity in subunit capture by a periplasmic chaperone

The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh β-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function.

The O-antigen of Salmonella enterica serotype Enteritidis PT4: a significant factor in gastrointestinal colonisation of young but not newly hatched chicks

The lipopolysaccharide of Salmonella and other Gram negative pathogenic species has been implicated as a major virulence determinant and in this study we report the role of LPS of S. Enteritidis in the colonisation and persistent gastrointestinal infection of young poultry. The gene encoding the unique O-antigen ligase, waaL, was mutated by insertional inactivation in a well characterised S. Enteritidis strain, S1400/94. The waaL mutant, designated PCP, produced rough colonies on agar medium, did not agglutinate O9 antiserum, did not produce an LPS ladder on silver stained gels and was serum sensitive. PCP and a nalidixic acid marked derivative of S1400/94 (S1400/94 Nal(r)) were used to orally challenge young chicks, separately and together in competitive index experiments. At post-mortem examination of 1-day-old chicks challenged S1400/94 Nal(r) and PCP separately there were no significant differences in the numbers of S1400/94 Nal(r) and PCP bacteria in tissues sampled on days 1, 2. and 5. By day 42 after challenge S1400/94 Nal(r) bacteria were recovered in significantly higher numbers than PCP from the caecal contents (P < 0.001). In competitive index studies in the 1-day-old chick PCP colonised, invaded and persisted in lower numbers than S1400/94 Nal(r). In 4-week-old chicks challenged separately, PCP bacteria were recovered from all tissues examined in significantly lower numbers than S1400/94 Nal(r). In competitive index experiments in 4-week-old chicks, PCP was not detected at any site and at any time point. Therefore, the O-antigen of S. Enteritidis plays art important role in poultry infections although this role is less important in the newly hatched chick. Crown Copyright (C) 2004 Published by Elsevier B.V. All rights reserved.

A novel transport mechanism for MOMP in Chlamydophila pneumoniae and its putative role in immune-therapy

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE−/− mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events.

Azotobacter genomes: the genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412)

The genome of the soil-dwelling heterotrophic N2-fixing Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 (ATCC 4412) (Ac-8003) has been determined. It consists of 7 circular replicons totalling 5,192,291 bp comprising a circular chromosome of 4,591,803 bp and six plasmids pAcX50a, b, c, d, e, f of 10,435 bp, 13,852, 62,783, 69,713, 132,724, and 311,724 bp respectively. The chromosome has a G+C content of 66.27% and the six plasmids have G+C contents of 58.1, 55.3, 56.7, 59.2, 61.9, and 62.6% respectively. The methylome has also been determined and 5 methylation motifs have been identified. The genome also contains a very high number of transposase/inactivated transposase genes from at least 12 of the 17 recognised insertion sequence families. The Ac-8003 genome has been compared with that of Azotobacter vinelandii ATCC BAA-1303 (Av-DJ), a derivative of strain O, the only other member of the Azotobacteraceae determined so far which has a single chromosome of 5,365,318 bp and no plasmids. The chromosomes show significant stretches of synteny throughout but also reveal a history of many deletion/insertion events. The Ac-8003 genome encodes 4628 predicted protein-encoding genes of which 568 (12.2%) are plasmid borne. 3048 (65%) of these show > 85% identity to the 5050 protein-encoding genes identified in Av-DJ, and of these 99 are plasmid-borne. The core biosynthetic and metabolic pathways and macromolecular architectures and machineries of these organisms appear largely conserved including genes for CO-dehydrogenase, formate dehydrogenase and a soluble NiFe-hydrogenase. The genetic bases for many of the detailed phenotypic differences reported for these organisms have also been identified. Also many other potential phenotypic differences have been uncovered. Properties endowed by the plasmids are described including the presence of an entire aerobic corrin synthesis pathway in pAcX50f and the presence of genes for retro-conjugation in pAcX50c. All these findings are related to the potentially different environmental niches from which these organisms were isolated and to emerging theories about how microbes contribute to their communities.

Characterization of para-Nitrophenol-degrading bacterial communities in river water by using functional markers and stable isotope probing

Microbial degradation is a major determinant of the fate of pollutants in the environment. para-Nitrophenol (PNP) is an EPA listed priority pollutant with a wide environmental distribution, but little is known about the microorganisms that degrade it in the environment. We studied the diversity of active PNP-degrading bacterial populations in river water using a novel functional marker approach coupled with [13C6]PNP stable isotope probing (SIP). Culturing together with culture-independent terminal restriction fragment length polymorphism analysis of 16S rRNA gene amplicons identified Pseudomonas syringae to be the major driver of PNP degradation in river water microcosms. This was confirmed by SIP-pyrosequencing of amplified 16S rRNA. Similarly, functional gene analysis showed that degradation followed the Gram-negative bacterial pathway and involved pnpA from Pseudomonas spp. However, analysis of maleylacetate reductase (encoded by mar), an enzyme common to late stages of both Gram-negative and Gram-positive bacterial PNP degradation pathways, identified a diverse assemblage of bacteria associated with PNP degradation, suggesting that mar has limited use as a specific marker of PNP biodegradation. Both the pnpA and mar genes were detected in a PNP-degrading isolate, P. syringae AKHD2, which was isolated from river water. Our results suggest that PNP-degrading cultures of Pseudomonas spp. are representative of environmental PNP-degrading populations.

Synthesis and biological analysis of novel glycoside derivatives of L-AEP, as targeted antibacterial agents

To develop targeted methods for treating bacterial infections, the feasibility of using glycoside derivatives of the antibacterial compound L-R-aminoethylphosphonic acid (L-AEP) has been investigated. These derivatives are hypothesized to be taken up by bacterial cells via carbohydrate uptake mechanisms, and then hydrolysed in situ by bacterial borne glycosidase enzymes, to selectively afford L-AEP. Therefore the synthesis and analysis of ten glycoside derivatives of L-AEP, for selective targeting of specific bacteria, is reported. The ability of these derivatives to inhibit the growth of a panel of Gram-negative bacteria in two different media is discussed. β-Glycosides (12a) and (12b) that contained L-AEP linked to glucose or galactose via a carbamate linkage inhibited growth of a range of organisms with the best MICs being <0.75 mg/ml; for most species the inhibition was closely related to the hydrolysis of the equivalent chromogenic glycosides. This suggests that for (12a) and (12b), release of L-AEP was indeed dependent upon the presence of the respective glycosidase enzyme.

Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%-60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.

Antimicrobial and cytotoxic activities of medicinal plants of the Brazilian cerrado, using Brazilian cachaca as extractor liquid

Ethnopharmacological importance: Many species of plants in the Brazilian cerrado (savanna) are widely used in ethnomedicine. However, the safety and effectiveness of medicinal plants used in communities with little or no access to manufactured drugs should be evaluated. Aim of the study: Evaluate the antimicrobial and cytotoxic activities of extracts from eight plant species, obtained using Brazilian cachaca as the extractor liquid. Materials and methods: The extracts were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida parapsilosis, promastigote forms of Leishmania amazonensis, and poliovirus. In addition, cytotoxic activity was assayed in Vero cells and in human erythrocytes. Results: The plant species Curatella americana, Sclerolobium aureum, and Plathymenia reticulata showed the best activity against yeasts, especially the crude extract of C. americana and its ethyl-acetate fraction. Kielmeyera lathrophyton showed a minimum inhibitory concentration of 250 mu g/ml against S. aureus, and was inactive against Gram-negative bacteria. The extract obtained from Annona coriacea showed the best activity against the promastigote forms of Leishmania amazonensis (IC(50) = 175 mu g/ml). Only C. americana showed potential for antipoliovirus activity. The concentrations of the crude extracts that showed toxicity to VERO cells had CC(50) between 31 and 470 mu g/ml, and the lyophilized Brazilian cachaca showed a CC(50) of 307 mu g/ml. None of the extracts showed toxicity against human erythrocytes. Conclusions: Among the plant species studied. C americana proved to be effective against microorganisms, especially as an antifungal. The results will help in the search for alternative drugs to be used in pharmacotherapy, and will contribute to establish safe and effective use of phytomedicines in the treatment of infectious diseases. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Chronic Suppurative Otitis Media in Cleft Palate: Microorganism Etiology and Susceptibilities

Objective: To investigate the microbial etiology of suppurative chronic otitis media (SCOM) in patients with complete cleft lip and palate and isolated cleft palate and to determine the sensitivity of isolated microorganisms to antibiotics by drug diffusion from impregnated discs in agar and the minimum inhibitory concentration of each drug to these microorganisms by drug dilution in agar. Design/Patients: Effusion samples of SCOM obtained from 40 patients with cleft lip and palate registered at the Hospital for Rehabilitation of Craniofacial Anomalies, University of Sao Paulo, at Bauru, Brazil, were bacteriologically analyzed by cultures. The isolated bacteria were submitted to an in vitro susceptibility test to clinically used drugs. Results: Positive cultures were obtained in 100% of studied cases. Among the 57 strains observed, the most frequent were Pseudomonas aeruginosa (35%), Staphylococcus aureus (15.5%), Enterococcus faecalis (14%), and Proteus mirabilis (12%). The frequency of Gram-negative bacilli (enterobacteriaceae and nonfermentative bacilli) was 67%. Pseudomonas aeruginosa presented the highest sensitivity to ciprofloxacin, and enterobacteriaceae exhibited the highest sensitivity to gentamicin. The strains of S. aureus and E. faecalis presented the highest sensitivity to imipenem and sulfamethoxazole/trimethoprim, respectively. Conclusion: Patients with cleft lip and palate presenting with SCOM exhibited 100% positive cultures, with the highest frequency of Pseudomonas and enterobacteriaceae. With regard to the action of antibiotics, imipenem was effective against the four species of isolated microorganisms, followed by ciprofloxacin, which was effective against 75% of isolated species.

Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities

BACKGROUND: Chloroform, ethyl acetate and methanol extracts of a sample of red propolis from the state of Alagoas (northeast Brazil) were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry. Antimicrobial and antioxidant activities were also obtained. RESULTS: The propolis sample contained low content of narigenin-8-C-hexoside, this being the first report of a C-glycoside in propolis. The main constituent found was characterized as 3,4,2`,3`-tetrahydroxychalcone. Other important constituents were the chalcone isoliquiritigenin, the isoflavans (3S)-vestitol, (3S)-7-O-methylvestitol, the pterocarpan medicarpin, the phenylpropenes trans-anethol, methyl eugenol, elimicin, methoxyeugenol and cis-asarone, and the triterpenic alcohols lupeol and alpha- and beta- amyrins. The methanol extract exhibited high antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl and beta-carotene/linoleic acid assay methods, and antimicrobial activity toward Gram-positive and Gram-negative bacteria. CONCLUSION: Structures are suggested for new substances never before seen in any kind of propolis. This is the first report of 3,4,2`,3`-tetrahydroxychalcone and a flavone C-glycoside in a propolis sample. (C) 2011 Society of Chemical Industry

Insulin suppresses LPS-induced iNOS and COX-2 expression and NF-kappa B activation in alveolar macrophages

The development of septic shock is a common and frequently lethal consequence of gram-negative infection. Mediators released by lung macrophages activated by bacterial products such as lipopolysaccharide (LPS) contribute to shock symptoms. We have shown that insulin downregulates LPS-induced TNF production by alveolar macrophages (AMs). In the present study, we investigated the effect of insulin on the LPS-induced production of nitric oxide (NO) and prostaglandin (PG)-E(2), on the expression of inducible nitric oxide synthase ( iNOS) and cyclooxygenase (COX)-2, and on nuclear factor kappa B (NF-kappa B) activation in AMs. Resident AMs from male Wistar rats were stimulated with LPS (100 ng/mL) for 30 minutes. Insulin (1 mU/mL) was added 10 min before LPS. Enzymes expression, NF-kappa B p65 activation and inhibitor of kappa B (I-kappa B) a phosphorylation were assessed by immunobloting; NO by Griess reaction and PGE(2) by enzyme immunoassay (EIA). LPS induced in AMs the expression of iNOS and COX-2 proteins and production of NO and PGE(2), and, in parallel, NF-kappa B p65 activation and cytoplasmic I-kappa B alpha phosphorylation. Administration of insulin before LPS suppressed the expression of iNOS and COX-2, of NO and PGE(2) production and Nuclear NF-kappa B p65 activation. Insulin also prevented cytoplasmic I-kappa Ba phosphorylation. These results show that in AMs stimulated by LPS, insulin prevents nuclear translocation of NF-kappa B, possibly by blocking I-kappa Ba degradation, and supresses the production of NO and PGE(2), two molecules that contribute to septic shock. Copyright (C) 2008 S. Karger AG, Basel.

Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin

Introduction: Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages. Methods: Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT. Results: We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-gamma-dependent pathways and is not mediated by either interleukin-4 or interleukin-10. Conclusion: This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.