Antimicrobial and anti-inflammatory activities of leaf extract of Valeriana wallichii DC.

Valeriana wallichii DC (Valerianaceae) is one of the most widely used traditional remedies for various complications associated with nervous system and digestion. No antimicrobial and anti-inflammatory studies have so far been carried out on the aerial parts of the plant. The present work was focused to evaluate the antimicrobial (antifungal and antibacterial) and anti-inflammatory properties of V. wallichii using reported methods. Chloroform fraction (VW-2) and hexane fraction (VW-3) exhibited significant activity against S. aureus and B. subtilus, respectively. The chloroform fraction (VW-2) showed significant activity against S. aureus with 0.27 mg/ml MIC, where 0.31 mg/ml MIC was deduced for VW-3 fraction against B. subtilus. VW-3 fraction was also found to be the most potent inhibitor of M. canis, showing 70% inhibition with an MIC value of 0.19 mg/ml. Considerable inhibitory activity was also observed for VW-2 and water fraction (VW-6) against M. canis and A. flavus. A remarkable anti-inflammatory like activity was observed for the crude extract at a dose of 200 mg/kg at all observed durations. Other doses of the sample also showed excellent activity. Looking to these results it may be concluded that V. wallichii may be a potential source for activity guided isolation of natural products with antimicrobial and anti-inflammatory-like properties.

Colonization of segmented filamentous bacteria and its interaction with the luminal IgA level in conventional mice.

Segmented filamentous bacteria (SFB) colonize in the ileum. They promote the development of intraepithelial lymphocytes and immunoglobulin A (IgA)-producing cells in the small intestine. In SFB-monoassociated mice, changes in SFB colonization of the small intestine were related to the level of IgA derived from maternal milk during the suckling period and self-produced in the small intestine after weaning. In this study, we investigated whether or not maternal and neonatal IgA influence the colonization of SFB in conventional mice from 18 to 105 days old. The pups were forcedly weaned at 20 days old. SFB could be detected in the distal small intestine after day 22, and their number rapidly reached a maximum on day 28. Thereafter, they gradually declined to one-fourth of the maximum level. The lowest concentrations of IgA in the small intestinal and cecal contents were detected on day 22. Thereafter, they increased as the age of the mice increased. The expression of the polymeric immunoglobulin receptor gene in the distal small intestine increased after weaning. These results suggested that the colonization of SFB in the pre-weaning and post-weaning periods might be prevented with IgA derived from maternal milk and self-produced IgA, respectively.

A strain of Lactobacillus plantarum affects segmented filamentous bacteria in the intestine of immunosuppressed mice.

Segmented filamentous bacteria (SFB) are present in the gastrointestinal tract of mice from weaning until the maturation of the immune system. Probiotic bacteria also have an effect on host immunity. To study the relationships established between these bacteria, samples from a mouse model fed with Lactobacillus plantarum under different immunological conditions were analysed. SFB populations were measured by a newly designed group-specific quantitative PCR assay. The results confirmed the presence of the probiotic in the intestine and an expansion of SFB in the ileum of immunocompromised mice, which was abolished upon administration of L. plantarum, an effect not described to date.

Citrinin, a mycotoxin from Penicillium citrinum, plays a role in inducing motility of Paenibacillus polymyxa.

Paenibacillus polymyxa, a Gram-positive low-G+C spore-forming soil bacterium, belongs to the plant growth-promoting rhizobacteria. The swarming motility of P. polymyxa strain E681 was greatly induced by a secondary metabolite, citrinin, produced by Penicillium citrinum KCTC6549 in a dose-dependent manner at concentrations of 2.5-15.0 microg mL(-1) on tryptic soy agar plates containing 1.0% (w/v) agar. Flagellum staining showed that citrinin activated the production of flagella by P. polymyxa. This result was supported by reverse transcriptase-PCR analysis of gene expression, which showed increased transcriptional levels of sigD and hag homologues of P. polymyxa E681 in the presence of citrinin. The results presented here show that a mycotoxin, citrinin, has a newly identified function of inducing bacterial motility by transcriptional activation of related genes. This finding contributes to our understanding of the interactions between bacteria and fungal strains in nature.

Differential expression of nifH and anfH genes in Paenibacillus durus analysed by reverse transcriptase-PCR and denaturing gradient gel electrophoresis.

AIMS: The aim of this study was to develop an approach based on a reverse transcriptase (RT)-PCR/denaturing gradient gel electrophoresis (DGGE) for the detection of the functional genes nifH and anfH in Paenibacillus durus. METHODS AND RESULTS: Two sets of primers were employed to study the expression of the nitrogen fixation genes in a pure-culture system of P. durus grown in media with increasing concentrations of ammonium (NH(4)(+)), tungsten (W) or molybdenum (Mo). The results obtained indicate that the expression of nitrogenase genes from P. durus can take place in the presence of relatively high levels of fixed nitrogen. It was also observed that the addition of 20 micromol l(-1) molybdenum and 2 mmol l(-1) tungstate did not interfere in the mRNA levels of nifH and anfH genes. CONCLUSIONS: Our results demonstrate the presence and transcription of nifH and anfH in P. durus under a variety of growth conditions. A specific set of primers was designed for the detection of the alternative system for nitrogen fixation in P. durus. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-PCR/DGGE system enables the rapid gathering of incremental data about the regulation of conventional and alternative nitrogenase genes in P. durus strains.

Evaluation of culture media for Paenibacillus larvae applied to studies of antimicrobial activity.

This study was conducted to compare different liquid culture media for Paenibacillus larvae growth in order to find the best one to be used in studies on activity of antimicrobial substances, such as essential oils. P. larvae presented poor growth in usual broths such as Mueller-Hinton, commonly employed in antimicrobial activity assays. Growth in liquid media was evaluated using Paenibacillus larvae strains isolated from hives located in different geographical zones. The MYT medium (Mueller-Hinton broth, yeast extract and thiamine) was selected out of the eight liquid media analyzed, as it proved to be the most adequate due to its higher absorbance at 620 nm. The following mean values were obtained from the four P. larvae strains: 0.227 +/- 0.016 for the Cobo strain, 0.279 +/- 0.015 for La Plata strain, 0.758 +/- 0.020 for Mechongué strain and 0.244 +/- 0.0079 for Sierra de los Padres strain, respectively.

[Optimization of the growth of Paenibacillus larvae in semi-selective media]. / Optimización del desarrollo de Paenibacillus larvae en medios semiselectivos.

The sensitivity of media MYPGP, MYPGP(NALPIA) A (6 microg/ml nalidixic acid and 10 microg/ml pipemidic acid) and MYPGP(NALPIA) B (9 microg/ml nalidixic acid and 20 microg/ml pipemidic acid) for the recovery of viable spores of Paenibacillus larvae from honey, was evaluated by using different incubation times and different spore concentrations. No significant differences between incubation times, spore concentration or culture media were found. In the case of the recovery of vegetative cells from PBS at different incubation times and different dilutions no significant differences were found between the incubation times or the dilutions tested, while significant differences were found in the three media when compared with one another, MYPGP(NALPIA)B providing the lowest recovery of vegetative cells. Considering these results, we propose the use of MYPGP(NALPIA)B to recover spores of P. larvae from honey, specially for honeys with heterogeneous populations of bacterial spores; when culturing vegetative cells, MYPGP or MYPGP(NALPIA)A must be used to obtain good growth.

Protease from Sporosarcina sp. RRLJ 1.

Protease was isolated from Sporosarcina RRLJ1 which was collected from acid tea (Camellia sinensis) plantations. It showed potential for production of the enzyme for commercial purposes. The study revealed that optimum pH for growth of the organism was 6.5-7 and supplement of casein (1%) in the medium was required for production of protease. Enzyme production and enzyme activity was maximum in 72 hr old broth culture. Maximum activity of the enzyme was found at pH 6.5.

[Features of solid materials' colonization by pure and mixed cultures of methanotrophs]. / Osobennosti kolonizatsii tverdykh materialov chistymi i smeshannymi kul'turami metanotrofov.

The process of colonization of hydrophilic (glass) and hydrophobic (polysterene) carriers by pure cultures of methanotrophs Methylocystis parvus UCM B-3490T, Methylococcus capsulatus UCM B-3030, as well as by their cultures mixed with Bacillus megaterium UCM B 5723T and Pseudomonas putida VKPM B-4188 under the conditions efficient for methanotrophic bacteria. M. parvus demonstrated the highest intensity of this process on the above carriers owing to high hydrophobic cell surface. Both methanotrophs colonized the glass surface more quickly with formation of microcolonies on carriers after 6 days of incubation in pure and mixed cultures with B. megaterium. The number of bacilli on these carriers quickly decreased. In the mixed cultures with P. putida the glass and polysterene colonization intensity decreased, while the amount of pseudomonas on carriers increased.

Gracilibacillus ureilyticus sp. nov., a halotolerant bacterium from a saline-alkaline soil.

A Gram-stain-positive, halotolerant, neutrophilic, rod-shaped bacterium, strain MF38(T), was isolated from a saline-alkaline soil in China and subjected to a polyphasic taxonomic characterization. The isolate grew in the presence of 0-15 % (w/v) NaCl and at pH 6.5-8.5; optimum growth was observed with 3.0 % (w/v) NaCl and at pH 7.0. Chemotaxonomic analysis showed menaquinone MK-7 as the predominant respiratory quinone and anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(15 : 0), C(17 : 0) and C(16 : 0) as major fatty acids. The genomic DNA G+C content was 35.3 mol%. 16S rRNA gene sequence similarities of strain MF38(T) with type strains of described Gracilibacillus species ranged from 95.3 to 97.7 %. Strain MF38(T) exhibited the closest phylogenetic affinity to the type strain of Gracilibacillus dipsosauri, with 97.7 % 16S rRNA gene sequence similarity. The DNA-DNA reassociation between strain MF38(T) and G. dipsosauri DSM 11125(T) was 45 %. On the basis of phenotypic and genotypic data, strain MF38(T) represents a novel species of the genus Gracilibacillus, for which the name Gracilibacillus ureilyticus sp. nov. (type strain MF38(T) =CGMCC 1.7727(T) =JCM 15711(T)) is proposed.

Evaluation of different growth media for the recovery of the species of Alicyclobacillus.

AIMS: Five different isolation media, namely potato dextrose agar (PDA), orange serum agar (OSA), K agar, yeast-starch-glucose agar and Bacillus acidocaldarius medium were evaluated for the recovery of Alicyclobacillus spp. from inoculated diluted and undiluted fruit-juice concentrates. METHODS AND RESULTS: Plates of PDA (pH 3.7), spread with vegetative cells (3.9 x 10(6) CFU ml(-1)) of Alicyclobacillus acidoterrestris from single-strength pear juice, recovered 2.9 x 10(6 )CFU ml(-1) after 5 days at 50 degrees C (74% recovery). The recovery of endospores from single-strength pear juice, after a heat treatment at 80 degrees C for 10 min, was higher on spread plates of OSA (pH 5.5) at 50 degrees C for 5 days (97% recovery). CONCLUSIONS: PDA (pH 3.7) and OSA (pH 5.5) at 50 degrees C for 3-5 days recovered the highest numbers of vegative cells and endospores of Alicyclobacillus spp. from sterilized fruit juices and concentrates. SIGNIFICANCE AND IMPACT OF THE STUDY: The most appropriate synthetic media for the recovery of Alicyclobacillus species from inoculated fruit juices and concentrates are shown.

Anoxybacillus rupiensis sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria).

Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55-58 degrees C, and pH at 6.0-6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270(T) with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA-DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270(T), represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127(T) = NBIMCC 8387(T)). The 16S rRNA gene sequence data of a strain R270(T) have been deposited in the EMBL databases under the accession number AJ879076.

Operation of the CO dehydrogenase/acetyl coenzyme A pathway in both acetate oxidation and acetate formation by the syntrophically acetate-oxidizing bacterium Thermacetogenium phaeum.

Thermacetogenium phaeum is a homoacetogenic bacterium that can grow on various substrates, such as pyruvate, methanol, or H2/CO2. It can also grow on acetate if cocultured with the hydrogen-consuming methanogenic partner Methanothermobacter thermautotrophicus. Enzyme activities of the CO dehydrogenase/acetyl coenzyme A (CoA) pathway (CO dehydrogenase, formate dehydrogenase, formyl tetrahydrofolate synthase, methylene tetrahydrofolate dehydrogenase) were detected in cell extracts of pure cultures and of syntrophic cocultures. Mixed cell suspensions of T. phaeum and M. thermautotrophicus oxidized acetate rapidly and produced acetate after addition of H2/CO2 after a short time lag. CO dehydrogenase activity staining after native polyacrylamide gel electrophoresis exhibited three oxygen-labile bands which were identical in pure culture and coculture. Protein profiles of T. phaeum cells after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the strain exhibited basically the same protein patterns in both pure and syntrophic culture. These results indicate that T. phaeum operates the CO dehydrogenase/acetyl-CoA pathway reversibly both in acetate oxidation and in reductive acetogenesis by using the same biochemical apparatus, although it has to couple this pathway to ATP synthesis in different ways.

Emended description of Pasteuria nishizawae.

The description of the Gram-positive, obligately parasitic, mycelial and endospore-forming bacterium, Pasteuria nishizawae, is emended to include additional observations on the life cycle, host specificity and endospore morphology. The nucleotide sequence of the 16S rRNA gene is also provided.

Degradation of cyanophycin by Sedimentibacter hongkongensis strain KI and Citrobacter amalonaticus strain G Isolated from an anaerobic bacterial consortium.

Using a combination of various enrichment techniques, the strictly anaerobic, gram-positive, endospore-forming bacterium Sedimentibacter hongkongensis strain KI as revealed by 16S rRNA analysis and the gram-negative enterobacterium Citrobacter amalonaticus strain G as revealed by physiological tests were isolated from an anaerobic cyanophycin (CGP)-degrading bacterial consortium. S. hongkongensis strain KI is the first anaerobic bacterium with the ability to hydrolyze CGP to beta-Asp-Arg and beta-Asp-Lys dipeptides, as revealed by electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography analysis. However, these primary accumulated hydrolysis products were only partially used by S. hongkongensis strain KI, and significant growth on CGP did not occur. On the other hand, C. amalonaticus strain G did not degrade CGP but grew on the beta-linked iso-dipeptides formed in vitro by enzymatic CGP degradation or in vivo by metabolic activity of S. hongkongensis strain KI. Dipeptide utilization occurred at the highest rate if both strains were used in cocultivation experiments with CGP, indicating that cooperation between different bacteria occurs in anaerobic natural environments for complete CGP turnover. The amino acids obtained from the cleavage of dipeptides were fermented to ethanol, acetic acid, and succinic acid, as revealed by gas chromatographic analysis and by spectrophotometric enzyme assays.

Nocturnal production of endospores in natural populations of epulopiscium-like surgeonfish symbionts.

Prior studies have described a morphologically diverse group of intestinal microorganisms associated with surgeonfish. Despite their diversity of form, 16S rRNA gene surveys and fluorescent in situ hybridizations indicate that these bacteria are low-G+C gram-positive bacteria related to Epulopiscium spp. Many of these bacteria exhibit an unusual mode of reproduction, developing multiple offspring intracellularly. Previous reports have suggested that some Epulopiscium-like symbionts produce dormant or phase-bright intracellular offspring. Close relatives of Epulopiscium, such as Metabacterium polyspora and Clostridium lentocellum, are endospore-forming bacteria, which raises the possibility that the phase-bright offspring are endospores. Structural evidence and the presence of dipicolinic acid demonstrate that phase-bright offspring of Epulopiscium-like bacteria are true endospores. In addition, endospores are formed as part of the normal daily life cycle of these bacteria. In the populations studied, mature endospores were seen only at night and the majority of cells in a given population produced one or two endospores per mother cell. Phylogenetic analyses confirmed the close relationship between the endospore-forming surgeonfish symbionts characterized here and previously described Epulopiscium spp. The broad distribution of endospore formation among the Epulopiscium phylogenetic group raises the possibility that sporulation is a characteristic of the group. We speculate that spore formation in Epulopiscium-like symbionts may be important for dispersal and may also enhance survival in the changing conditions of the fish intestinal tract.

Soehngenia saccharolytica gen. nov., sp. nov. and Clostridium amygdalinum sp. nov., two novel anaerobic, benzaldehyde-converting bacteria.

Two anaerobic, benzaldehyde-converting bacteria were isolated from an anaerobic upflow anaerobic sludge bed (UASB)-reactor treating potato starch waste water. Strain BOR-Y(T) converted benzaldehyde to benzoate and benzylalcohol in approximately equimolar concentrations. Benzaldehyde conversion did not support growth. Strain BOR-Y(T) was Gram-positive and rod-shaped, and its cells were slightly thickened in the middle. The strain was a mesophilic spore-former that grew between 15 and 40 degrees C, with optimum growth at 30-37 degrees C. The optimum pH for growth was pH 7.0. Strain BOR-Y(T) grew on a wide range of carbohydrates and some other carbon sources including yeast extract, cysteine and serine. The G+C content of its DNA was 42 mol%. According to physiological characteristics and 16S rRNA gene sequence analysis, confirmed by DNA-DNA hybridization with its phylogenetic neighbours, strain BOR-Y(T) belongs to a novel genus of cluster XII of the clostridia, namely Soehngenia; the name Soehngenia saccharolytica is proposed for the type species (type strain BOR-Y(T)=DSM 12858(T)=ATCC BAA-502(T)). Strain BR-10(T) reduced benzaldehyde to benzylalcohol. This conversion was coupled to growth. In a medium containing yeast extract, the presence of benzaldehyde resulted in the accumulation of more than twofold more cells. Strain BR-10(T) was a Gram-positive organism that was characterized by oval- or rod-shaped cells with oval ends, which occurred singly, in pairs or sometimes in chains. The strain was moderately thermophilic and grew between 20 and 60 degrees C, with optimum growth at 45 degrees C. The optimum pH for growth was between pH 7.0 and 7.5. Strain BR-10(T) grew on a wide range of carbon sources including carbohydrates, yeast extract, casein and some amino acids. The G+C content of its DNA was 32 mol%. As determined by 16S rRNA gene sequence analysis, strain BR-10(T) represents a novel species of cluster XIVa of the clostridia; the name Clostridium amygdalinum is proposed for this novel species (type strain BR-10(T)=DSM 12857(T)=ATCC BAA-501(T)).

Characterization of endospore-forming bacteria associated with entomopathogenic nematodes, Heterorhabditis spp., and description of Paenibacillus nematophilus sp. nov.

Endospore-forming bacteria were isolated from insect-pathogenic nematodes, Heterorhabditis spp., from three diverse geographical locations. Spindle-shaped sporangia of these bacteria adhere to the free-living infective stage of the nematode, which carries them to new insect hosts, where the bacterium reproduces. These isolates were characterized based on phenotypic and chemotaxonomic properties and 16S rRNA gene sequences. Analysis of the 16S rRNA gene placed the isolates within the genus Paenibacillus. The isolates shared higher sequence similarities with each other (95.1-100%) than they did with any other named species within the genus (89.2-94%). Paenibacillus macquariensis, Paenibacillus azoreducens, Paenibacillus amylolyticus and Paenibacillus durus were among the species with highest sequence similarity to these isolates. The isolates shared a high degree of phenotypic similarity and were easily distinguished from closely related members of the genus. Anteiso-C15:0 and C16:0 were among the major fatty acid types and the DNA G + C content was approximately 44 mol% in all isolates. DNA-DNA similarity studies revealed genomic heterogeneity among the isolates, such that they are likely to represent more than one species. Two of the isolates (both from a Heterorhabditis megidis isolate from Estonia) are phenotypically distinguishable from the others and are proposed as a single species, Paenibacillus nematophilus sp. nov. The type strain for this novel species is NEM1aT (=DSM 13559T =NCIMB 13845T). The other isolates, although closely related to the proposed species, are likely to represent at least one, but most likely two, novel species.

Characteristics of Sulfobacillus acidophilus sp. nov. and other moderately thermophilic mineral-sulphide-oxidizing bacteria.

Several isolates of Gram-positive, acidophilic, moderately thermophilic, ferrous-iron- and mineral-sulphide-oxidizing bacteria were examined to establish unequivocally the characteristics of Sulfobacillus-like bacteria. Two species were evident: Sulfobacillus thermosulfidooxidans with 48-50 mol% G+C and Sulfobacillus acidophilus sp. nov. with 55-57 mol% G+C. Both species grew autotrophically and mixotrophically on ferrous iron, on elemental sulphur in the presence of yeast extract, and heterotrophically on yeast extract. Autotrophic growth on sulphur was consistently obtained only with S. acidophilus.

Role of Alicyclobacillus acidoterrestris in the development of a disinfectant taint in shelf-stable fruit juice.

AIMS: This study was undertaken to identify the bacterium and metabolic products contributing to a disinfectant taint in shelf-stable fruit juice and to determine some of the growth conditions for the organism. METHODS AND RESULTS: Microbiological examination of tainted and untainted fruit juice drinks detected low numbers of acid-dependent, thermotolerant, spore-forming bacteria in the tainted juices only. The presence of omega-cyclohexyl fatty acids was confirmed in two of the isolates by cell membrane fatty acid analysis. The isolates were subsequently identified as Alicyclobacillus acidoterrestris by partial 16S rDNA sequencing. Studies on the isolates showed growth at pH 2.5-6.0 and 19.5-58 degrees C. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify 2,6-dibromophenol (2,6-DBP) and 2,6-dichlorophenol (2,6-DCP) in the tainted juice. Challenge studies in a mixed fruit drink inoculated with the two isolates and the type strain of A. acidoterrestris, incubated at 44-46 degrees C for 4 d, showed the production of both metabolites, which were confirmed and quantified by GC/MS. CONCLUSIONS: The results show that A. acidoterrestris can produce 2,6-DBP and 2,6-DCP in shelf-stable juices. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report detailing experimental methodology showing that A. acidoterrestris can produce 2,6-DCP in foods. Control of storage temperatures (to < 20 degrees C) immediately after processing may provide an effective control measure for the fruit juice industry to prevent spoilage by A. acidoterrestris.