Bacterial community associated with ensilage process of wilted guinea grass

Aims: To determine the effects of wilting, storage period and bacterial inoculant on the bacterial community and ensiling fermentation of guinea grass silage. Methods and Results: Fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. There was more lactic acid than acetic acid in all silages, but the lactic acid to acetic acid ratio decreased with storage time. This shift from lactic to acetic acid was not prevented even with a combination of wilting and bacterial inoculant. The DGGE analyses suggest that facultatively heterofermentative lactic acid bacteria (Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus pentosus) were involved in the shift to acetic acid fermentation. Conclusions: Lactic acid can dominate the fermentation in tropical grass silage with sufficient wilting prior to ensiling. Prolonged storage may lead to high levels of acetic acid without distinctive changes in the bacterial community. Significance and Impact of the Study: The bacterial community looks stable compared to fermentation products over the course of long storage periods in tropical grass silage. Acetic acid fermentation in tropical grass silage can be a result of the changes in bacterial metabolism rather than community structure.     

A study into the role of L-aspartic acid on the metabolism of L-malic acid and D-glucose by Oenococcus oeni

AIMS: The purpose of this work was to study the effect of L-aspartic acid concentration on bacterial growth, D-glucose fermentation and L-malic acid consumption of Oenococcus oeni NCFB 1707. METHODS AND RESULTS: Bacterial cultures were performed in synthetic media. Bacterial growth, D-glucose fermentation and L-malic acid consumption were reduced when L-aspartic acid concentration became excessive. This inhibitory effect of high concentrations of L-aspartic acid on bacterial growth was also observed with several Oenococcus oeni strains, except O. oeni BL01. The L-aspartic acid inhibitory effect on bacterial growth could be reduced by increasing the concentration of L-glutamic acid. L-glutamic acid transport was found to be competitively inhibited by L-aspartic acid. In addition, an excessive amount of L-aspartic acid modified D-glucose metabolism, with an overproduction of acetic acid and reduced ethanol production. CONCLUSION: Since L-glutamic acid is an essential amino acid for the bacterial strain used, the L-aspartic acid inhibitory effect on bacterial growth could be linked to its involvement in an antagonistic interaction with L-glutamic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: Such antagonistic interactions between amino acids in O. oeni strains could be another explanation for the difficulties of inducing malolactic fermentation in wines.     

Classification of Bacteria Based on the Biases of Terminal Amino Acid Residues

The frequencies of amino acid residues are known to be biased at both terminal regions of amino acid sequences deduced from bacterial genomic DNA. To investigate whether or not the features of biases of amino acid residues at the terminal regions are related to the bacterial phylogeny, we calculated the normalized amino acid compositions at both terminal regions, and used these compositions to classify 144 bacteria by hierarchical clustering analysis. Our results showed that most of these bacteria were classified into taxonomic classes by the hierarchical clustering analysis that was based on the normalized amino acid compositions at the N-terminal region. Therefore, we concluded that the features of biases of the N-terminal amino acid residues were related to the bacterial phylogeny.     

Bacterial fatty acid metabolism in modern antibiotic discovery

Bacterial fatty acid synthesis is essential for many pathogens and different from the mammalian counterpart. These features make bacterial fatty acid synthesis a desirable target for antibiotic discovery. The structural divergence of the conserved enzymes and the presence of different isozymes catalyzing the same reactions in the pathway make bacterial fatty acid synthesis a narrow spectrum target rather than the traditional broad spectrum target. Furthermore, bacterial fatty acid synthesis inhibitors are single-targeting, rather than multi-targeting like traditional monotherapeutic, broad-spectrum antibiotics. The single-targeting nature of bacterial fatty acid synthesis inhibitors makes overcoming fast-developing, target-based resistance a necessary consideration for antibiotic development. Target-based resistance can be overcome through multi-targeting inhibitors, a cocktail of single-targeting inhibitors, or by making the single targeting inhibitor sufficiently high affinity through a pathogen selective approach such that target-based mutants are still susceptible to therapeutic concentrations of drug. Many of the pathogens requiring new antibiotic treatment options encode for essential bacterial fatty acid synthesis enzymes. This review will evaluate the most promising targets in bacterial fatty acid metabolism for antibiotic therapeutics development and review the potential and challenges in advancing each of these targets to the clinic and circumventing target-based resistance. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Seasonal features of consumption of lysine by uncultivated bacterial plankton of Eutrophic water reservoir

Dynamics of bacterial plankton community of eutrophic water reservoir in laboratory microecosystems with amino acid lysine was studied using PCR-DGGE technique. The addition of lysine to the microecosystems resulted in changes in the composition of the bacterial plankton in summer; in particular, a number of Lys1 and Lys2 species (genotypes) that consume this amino acid grew fast in the bacterial community. The plank tonic bacterial communities did not respond to the addition of lysine in spring and late summer. The obtained data confirm the hypothesis for the narrow specialization of bacterial plankton species to the consumption of individual organic substances.     

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.     

Screening of bacterial associates of marine sponges for single cell oil and PUFA

AIM: To screen bacterial associates from marine sponges for single cell oil (SCO)/polyunsaturated fatty acid (PUFA) production. METHODS AND RESULTS: Using Sudan black 'B' staining technique the bacterial associates were screened for cellular lipid accumulation, effect of culture media, incubation period and C : N ratio. Extraction of the bacterial lipids was carried out by Floch's method and fatty acid methyl esters were analysed by GC and GC/MS. Four bacterial associates of 50 isolated from eight marine sponges tested positive for lipid accumulation. Two bacterial associates, viz. Bacillus subtilis (RRL-8) from Aurora globostellata and Pseudomonas spp. (RRL-28) from Heteronema erecta were found to produce total lipids 16.9 and 31.7%, respectively, of their dry biomass. CONCLUSIONS: Increase in C:N ratio significantly improved lipid production to 33.4 and 42.7%. Both the isolates produced gamma-linolenic acid (18:3 omega6; 4.5 and 1.12% respectively), whereas B. subtilis showed 3.8% of eicosapentaenoic acid (20:5 omega3) along with branched chain fatty acids. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of oleaginous bacterial associates from marine sponges.     

Analysis of differences in amino acid substitution patterns, using multilevel G-tests

In this paper, a new algorithm is presented, which makes possible multilevel comparison of BLOSUM protein substitution matrices based on data from different groups of organisms. As an example, a comparison between substitution matrices based on data from two groups of bacterial genomes with different GC content is presented. Our approach includes evaluating the number of amino acid pairs in BLOCKS databases created separately for the two groups of bacteria using protein sequences deposited in the COG database. Differences of distributions of amino acid pair counts are tested using the chi-squared based G-test. Different analysis levels make it possible to distinguish different patterns of amino acid substitution. Application of the algorithm reveals statistically significant differences in amino acid substitution patterns between AT-rich and GC-rich groups of bacterial organisms. The differences are particularly visible in the overall substitution pattern, amino acid conservation pattern and in comparison of substitution patterns for single amino acids. The algorithm presented in this paper can be considered a novel method for multi-level comparison of amino acid substitution patterns. The presented approach is not limited to bacterial organisms and BLOSUM substitution matrices. Statistically significant differences between substitution patterns in the two groups of bacterial organisms with respect to amino acid conservation pattern can be the evidence of different rate of evolutionary change between AT-rich and GC-rich bacterial organisms.     

Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.     

The role of indoleacetaldehyde in IAA production from tryptophan by plants and by their epiphytic bacteria

Tryptophan, tryptamine, or indolepyruvic acid were applied to 2 systems: a bacterial (pea stem sections containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). In the plant system, the production of indoleacetic acid and indoleethanol (tryptophol) from each applied indole derivative is clearly reduced by the aldehyde reagents bisulfite and dimedon, respectively. Indoleacetaldehyde is chromatographically detected after alkaline liberation from its bisulfite addition product. In the bacterial system, the production of indoleacetic acid and indoleethanol is likewise reduced by bisulfite and dimedon. However, after tryptophan or tryptamine application, we could not detect indoleacetaldehyde in the described way. In one case only, namely tryptamine application to the bacterial system, indoleethanol production (contrary to indoleacetic acid production) is scarcely reduced by the aldehyde reagents. This indicates a bacterial pathway tryptamine → indoleethanol which bypasses indoleacetaldehyde.     

Microbiology and chemistry of acid lakes in Florida: I. Effects of drought and post-drought conditions

Short term changes in acid loading and dissolved organic carbon (DOC) content were studied in relation to water column bacteria of ten acid lakes on the Katharine Ordway Preserve, Florida. Five clear oligotrophic lakes and five dark dystrophic lakes were sampled during and after a drought period in July and September, 1985. Water column bacterial densities, light extinction, chlorophyll a, DOC, pH, dissolved oxygen, nutrients, and other chemical variables were measured. Significant positive correlations existed among DOC, chlorophyll a, pH, and water column bacterial densities during the drought period.There were no significant changes in water column bacterial densities or pH of clear lakes in the post-drought period, despite a 4.6 fold increase in acid loading from rainfall. A 3 fold increase of DOC, a decline in pH, and decreased bacterial densities in dark lakes suggested inhibition of bacteria by DOC and pH. A decrease in the relationship of DOC to bacterial numbers in all lakes was also noted. The correlations among DOC, chlorophyll a, and pH were no longer significant.Using data from both time periods significant polynomial regressions were observed between DOC and bacterial density and DOC and chlorophyll a. Maximum bacterial numbers occurred at 20 mg C 1^–1 of DOC. Above this bacterial numbers decreased also suggesting an inhibitory effect of DOC. Because pH was lower after DOC had increased in the dark lakes, the increase in acid conditions may have enhanced this inhibitory effect. The short term effects of DOC on the dark-lake bacteria greatly exceeded the influence of acid loading on clear-lake bacteria.     

Role of Capsular Colanic Acid in Adhesion of Uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Role of capsular colanic acid in adhesion of uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Effects of a dietary organic acid mixture and of dietary fibre levels on ileal and faecal nutrient apparent digestibility, bacterial nitrogen flow, microbial metabolite concentrations and rate of passage in the digestive tract of pigs

Six 34-kg barrows were fitted with a post-valve T-caecum cannula and assigned to six dietary treatments according to a 6 × 5 change-over design to study how a mixture of formic acid, sorbate, and benzoate (0 or 8.4 g/kg feed) influences apparent ileal and faecal digestibility coefficients, bacterial nitrogen (N) flow, microbial metabolite concentrations, and passage rate in pigs fed isoenergetic diets with medium, high, or very high fibre content (neutral-detergent fibre (NDF): 199, 224, and 248 g/kg dry matter, respectively). These barley and soya-bean meal based diets contained 0, 75, and 150 g/kg barley fibre (NDF: 577 g/kg) and 0, 8, and 16 g/kg rapeseed oil, respectively. The dietary organic acid mixture improved the apparent ileal digestibility of 14 of the 17 amino acids analysed (P < 0.05). Increasing levels of dietary fibre linearly decreased the apparent ileal digestibility of six of the 17 amino acids analysed (P < 0.05). Ileal flows of bacterial N and amino acids as assessed on the basis of purine flow were decreased by the dietary organic acid mixture (P < 0.05) but were not affected by dietary fibre level (P>0.05). As assessed on the basis of diaminopimelic acid flow, bacterial N flow was increased by both the dietary organic acid mixture and increased dietary fibre levels (P < 0.05). The dietary organic acid mixture reduced the concentration of lactic acid and increased that of acetic acid in ileal digesta (P < 0.05), while dietary fibre levels had a quadratic effect on concentrations of acetic, propionic, and butyric acid (P < 0.05). The mean retention time of Co (solute marker) and Yb (particle marker) in the large intestine decreased in a linear manner by increasing dietary fibre levels (P < 0.05) but was not affected by the dietary organic acid mixture (P>0.05). The results show that a dietary organic acid mixture has a positive effect on the apparent ileal digestibility of most amino acids irrespective of dietary fibre levels. This could be at least partly related to changes in bacterial N flow in the ileum. However, different bacterial markers showed opposite effects on bacterial N flow, which makes it questionable to use a constant bacterial marker / bacterial N ratio to estimate bacterial N flow. Increasing levels of dietary fibre had negative effects on the apparent ileal amino acid digestibilities and shortened the mean retention time of digesta in the large intestine.     

乙醇胁迫处理对酒酒球菌SD-2a生理特性的影响

为制备高效的葡萄酒乳酸菌发酵剂,以酒酒球菌SD-2a为试验材料,研究了5%、10%不同浓度的乙醇胁迫处理对菌体生长、细胞内MLE活性、冻干存活率及细胞膜脂肪酸组分的影响。试验结果表明乙醇胁迫处理明显降低了菌体的生长速率和生物产量。与对照相比,5%乙醇胁迫处理降低了菌体的冻干存活率,而10%乙醇处理却显著增加了菌体的冻干存活率。细胞膜脂肪酸组分的测定结果表明,前者细胞膜U/S比值为1.12,比对照降低了26.3%,而后者细胞膜U/S比值为2.29,比对照增加了50.6%。故认为酒酒球菌SD-2a为适应不同浓度的乙醇胁迫环境,在细胞膜脂肪酸水平上所采用的机制不同,且菌体的冻干存活率与其细胞膜脂肪酸组分密切相关。     

18-氨基酸注射液的动态浊度法检测研究

建立 18 -氨基酸注射液细菌内毒素定量测定方法 ,控制药品质量。采用动态浊度法 ,对 18-氨基酸注射稀释液进行干扰预实验、干扰实验定量检测。结果 18-氨基酸注射液在 8倍稀释时无干扰作用。因此用动态浊度法定量检查 18-氨基酸注射液细菌内毒素的含量 ,结果准确 ,在实际应用中完全可行。     

Bacterial decolorization and detoxification of black liquor from rayon grade pulp manufacturing paper industry and detection of their metabolic products

This study deals with the decolorization of black liquor (BL) by isolated potential bacterial consortium comprising Serratia marcescens (GU193982), Citrobacter sp. (HQ873619) and Klebsiella pneumoniae (GU193983). The decolorization of BL was studied by using the different nutritional as well as environmental parameters. In this study, result revealed that the ligninolytic activities were found to be growth associated and the developed bacterial consortium was efficient for the reduction of COD, BOD and color up to 83%, 74% and 85%, respectively. The HPLC analysis of degraded samples of BL has shown the reduction in peak area compared to control. Further, the GC-MS analysis showed that, most of the compounds detected in control were diminished after bacterial treatment while, formic acid hydrazide, 4-cyclohexane-1,2-dicarboxylic acid, carbamic acid, 1,2-benzenedicarboxylic acid and erythropentanoic acid were found as new metabolites. Further, the seed germination test using Phaseolus aureus has supported the detoxification of bacterial decolorized BL.     

Improved method using muramic acid to estimate biomass of bacteria in sediments

Summary A method, which depends on the measurement of muramic acid content to estimate bacterial biomass, has been improved in sensitivity by two orders of magnitude. It is now applicable to any aquatic sediment, whereas previously it was mainly useful in the analysis of gut contents of deposit-feeding animals. Reduced NAD, a product of the oxidation of d-lactate derived from muramic acid, is assayed using bacterial luciferase. The amount of muramic acid in a number of terrestrial and marine bacteria was measured, and found to be lower than that obtained with the previous, less specific, assay procedure. The muramic acid content of a blue-green alga has been measured, thus allowing blue-green algae to be taken into account when estimating bacterial biomass. Experimental evidence is presented which shows that muramic acid in cell wall fragments of bacteria is rapidly degraded by microorganisms in a marine sediment.     

Impact of oleic acid (cis-9-octadecenoic acid) on bacterial viability and biofilm production in Staphylococcus aureus

Staphylococcus aureus is responsible for a broad variety of chronic infections. Most S. aureus clinical isolates show the capacity to adhere to abiotic surfaces and to develop biofilms. Because S. aureus growing in a biofilm is highly refractory to treatment, inhibition of biofilm formation represents a major therapeutic objective. We evaluated the effects of oleic acid on primary adhesion and biofilm production in eight genotypically different S. aureus strains as well as in the biofilm-negative Staphylococcus carnosus strain TM300. Oleic acid inhibited primary adhesion but increased biofilm production in every S. aureus strain tested. Staphylococcus aureus strain UAMS-1 was then selected as a model organism for studying the mechanisms triggered by oleic acid on the formation of a biofilm in vitro. Oleic acid inhibited the primary adhesion of UAMS-1 dose dependently with an IC(50) around 0.016%. The adherent bacterial population decreased proportionally with increasing concentrations of oleic acid whereas an opposite effect was observed on the planktonic population. Overall, the total bacterial counts remained stable. Macroscopic detachments and clumps were visible from the adherent bacterial population. In the presence of oleic acid, the expression of sigB, a gene potentially involved in bacterial survival through an effect on fatty acid composition, was not induced. Our results suggest a natural protective effect of oleic acid against primary adhesion.