The role of lipid composition on the interaction between a tryptophan-rich protein and model bacterial membranes

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition and fluidity, on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

Evaluating and optimizing oral formulations of live bacterial vaccines using a gastro-small intestine model

Gastrointestinal (GI) models that mimic physiological conditions in vitro are important tools for developing and optimizing biopharmaceutical formulations. Oral administration of live attenuated bacterial vaccines (LBV) can safely and effectively promote mucosal immunity but new formulations are required that provide controlled release of optimal numbers of viable bacterial cells, which must survive gastrointestinal transit overcoming various antimicrobial barriers. Here, we use a gastro-small intestine gut model of human GI conditions to study the survival and release kinetics of two oral LBV formulations: the licensed typhoid fever vaccine Vivotif comprising enteric coated capsules; and an experimental formulation of the model vaccine Salmonella Typhimurium SL3261 dried directly onto cast enteric polymer films and laminated to form a polymer film laminate (PFL). Neither formulation released significant numbers of viable cells when tested in the complete gastro-small intestine model. The poor performance in delivering viable cells could be attributed to a combination of acid and bile toxicity plus incomplete release of cells for Vivotif capsules, and to bile toxicity alone for PFL. To achieve effective protection from intestinal bile in addition to effective acid resistance, bile adsorbent resins were incorporated into the PFL to produce a new formulation, termed BR-PFL. Efficient and complete release of 4.4x107 live cells per dose was achieved from BR-PFL at distal intestinal pH, with release kinetics controlled by the composition of the enteric polymer film, and no loss in viability observed in any stage of the GI model. Use of this in vitro GI model thereby allowed rational design of an oral LBV formulation to maximize viable cell release.

Linking alkaline phosphatase activity with bacterial phoD gene abundance in soil from a long-term management trial

Changes in land management practices may have significant implications for soil microbial communities important in organic P turnover. Soil bacteria can increase plant P availability by excreting phosphatase enzymes which catalyze the hydrolysis of ester-phosphate bonds. Examining the diversity and abundance of alkaline phosphatase gene harboring bacteria may provide valuable insight into alkaline phosphatase production in soils. This study examined the effect of 20 years of no input organic (ORG), organic with composted manure (ORG + M), conventional (CONV) and restored prairie (PRA) management on soil P bioavailability, alkaline phosphatase activity (ALP), and abundance and diversity of ALP gene (phoD) harboring bacteria in soils from the northern Great Plains of Canada. Management system influenced bioavailable P (P < 0.001), but not total P, with the lowest concentrations in the ORG systems and the highest in PRA. Higher rates of ALP were observed in the ORG and ORG + M treatments with a significant negative correlation between bioavailable P and ALP in 2011 (r2 = 0.71; P = 0.03) and 2012 (r2 = 0.51; P = 0.02), suggesting that ALP activity increased under P limiting conditions. The phoD gene abundance was also highest in ORG and ORG + M resulting in a significant positive relationship between bacterial phoD abundance and ALP activity (r2 = 0.71; P = 0.009). Analysis of phoD bacterial community fingerprints showed a higher number of species in CONV compared to ORG and ORG + M, contrary to what was expected considering greater ALP activity under ORG management. In 2012, banding profiles of ORG + M showed fewer phoD bacterial species following the second manure application, although ALP activity is higher than in 2011. This indicates that a few species may be producing more ALP and that quantitative gene analysis was a better indicator of activity than the number of species present.

Soil bacterial phoD gene abundance and expression in response to applied phosphorus and long-term management

Bacterial transformation of phosphorus (P) compounds in soil is largely dependent on soil microbial community function, and is therefore sensitive to anthropogenic disturbances such as fertilization or cropping systems. However, the effect of soil management on the transcription of bacterial genes that encode phosphatases, such as phoD, is largely unknown. This greenhouse study examined the effect of long-term management and P amendment on potential alkaline phosphatase (ALP) activity and phoD gene (DNA) and transcript (RNA) abundance. Soil samples (0–15 cm) were collected from the Glenlea Long-term Rotation near Winnipeg, Manitoba, to compare organic, conventional and prairie management systems. In the greenhouse, pots of soil from each management system were amended with P as either soluble mineral fertilizer or cattle manure and then planted with Italian ryegrass (Lolium multiforum). Soils from each pot were sampled for analysis immediately and after 30 and 106 days. Significant differences among the soil/P treatments were detected for inorganic P, but not the organic P in NaHCO3-extracts. At day 0, ALP activity was similar among the soil/P treatments, but was higher after 30 days for all P amendments in soil from organically managed plots. In contrast, ALP activity in soils under conventional and prairie management responded to increasing rates of manure only, with significant effects from medium and high manure application rates at 30 and 106 days. Differences in ALP activity at 30 days corresponded to the abundance of bacterial phoD genes, which were also significantly higher in soils under organic management. However, this correlation was not significant for transcript abundance. Next-generation sequencing allowed the identification of 199 unique phoD operational taxonomic units (OTUs) from the metagenome (soil DNA) and 35 unique OTUs from the metatranscriptome (soil RNA), indicating that a subset of phoD genes was being transcribed in all soils.

Impact of increasing fruit and vegetables and flavonoid intake on the human gut microbiota

Epidemiological studies have shown protective effects of fruits and vegetables (F&V) in lowering the risk of developing cardiovascular diseases (CVD) and cancers. Plant-derived dietary fibre (non-digestible polysaccharides) and/or flavonoids may mediate the observed protective effects particularly through their interaction with the gut microbiota. The aim of this study was to assess the impact of fruit and vegetable (F&V) intake on gut microbiota, with an emphasis on the role of flavonoids, and further to explore relationships between microbiota and factors associated with CVD risk. In the study, a parallel design with 3 study groups, participants in the two intervention groups representing high-flavonoid (HF) and low flavonoid (LF) intakes were asked to increase their daily F&V intake by 2, 4 and 6 portions for a duration of 6 weeks each, while a third (control) group continued with their habitual diet. Faecal samples were collected at baseline and after each dose from 122 subjects. Faecal bacteria enumeration was performed by fluorescence in situ hybridisation (FISH). Correlations of dietary components, flavonoid intake and markers of CVD with bacterial numbers were also performed. A significant dose X treatment interaction was only found for Clostidium leptum-Ruminococcus bromii/flavefaciens with a significant increase after intake of 6 additional portions in the LF group. Correlation analysis of the data from all 122 subjects independent from dietary intervention indicated an inhibitory role of F&V intake, flavonoid content and sugars against the growth of potentially pathogenic clostridia. Additionally, we observed associations between certain bacterial populations and CVD risk factors including plasma TNF-α, plasma lipids and BMI/waist circumference.

Wheat seed embryo excision enables the creation of axenic seedlings and Koch’s postulates testing of putative bacterial endophytes

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments

To evaluate the checkerboard DNA-DNA hybridization method for detection and quantitation of bacteria from the internal parts of dental implants and to compare bacterial leakage from implants connected either to cast or to pre-machined abutments. Nine plastic abutments cast in a Ni-Cr alloy and nine pre-machined Co-Cr alloy abutments with plastic sleeves cast in Ni-Cr were connected to Branemark-compatible implants. A group of nine implants was used as control. The implants were inoculated with 3 mu l of a solution containing 10(8) cells/ml of Streptococcus sobrinus. Bacterial samples were immediately collected from the control implants while assemblies were completely immersed in 5 ml of sterile Tripty Soy Broth (TSB) medium. After 14 days of anaerobic incubation, occurrence of leakage at the implant-abutment interface was evaluated by assessing contamination of the TSB medium. Internal contamination of the implants was evaluated with the checkerboard DNA-DNA hybridization method. DNA-DNA hybridization was sensitive enough to detect and quantify the microorganism from the internal parts of the implants. No differences in leakage and in internal contamination were found between cast and pre-machined abutments. Bacterial scores in the control group were significantly higher than in the other groups (P < 0.05). Bacterial leakage through the implant-abutment interface does not significantly differ when cast or pre-machined abutments are used. The checkerboard DNA-DNA hybridization technique is suitable for the evaluation of the internal contamination of dental implants although further studies are necessary to validate the use of computational methods for the improvement of the test accuracy. To cite this article:do Nascimento C, Barbosa RES, Issa JPM, Watanabe E, Ito IY, Albuquerque Junior RF. Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments.Clin. Oral Impl. Res. 20, 2009; 571-577.doi: 10.1111/j.1600-0501.2008.01663.x.

Bacterial leakage along the implant-abutment interface of premachined or cast components

In recent clinical studies, contamination of the inner parts of dental implants through bacterial penetration along the implant components has been observed. The aim of the present in-vitro study was to investigate leakage of Fusobacterium. nucleatum through the interface between implants and premachined or cast abutments. Both premachined (n = 10) and cast (n = 10) implant abutment assemblies were inoculated with 3.0 mu L of microbial inoculum. The assemblies were completely immersed in 5.0 mL of tryptic soy broth culture medium to observe leakage at the implant-abutment interface after 14 days of anaerobic incubation. Bacterial growth in the medium, indicative of microbial leakage, was found only in 1 out of 9 samples (11.1%) in each group. Both premachined and cast abutments connected to external hexagonal implants provide low percentages of bacterial leakage through the interface in in vitro unloaded conditions if the manufacturer`s instructions and casting procedures are properly followed.

Seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region

Community-acquired pneumonia (CAP) is a common cause of morbidity among children. Evidence on seasonality, especially on the frequency of viral and bacterial causative agents is scarce; such information may be useful in an era of changing climate conditions worldwide. To analyze the frequency of distinct infections, meteorological indicators and seasons in children hospitalized for CAP in Salvador, Brazil, nasopharyngeal aspirate and blood were collected from 184 patients aged < 5 y over a 21-month period. Fourteen microbes were investigated and 144 (78%) cases had the aetiology established. Significant differences were found in air temperature between spring and summer (p = 0.02) or winter (p < 0.001), summer and fall (p = 0.007) or winter (p < 0.001), fall and winter (p = 0.002), and on precipitation between spring and fall (p = 0.01). Correlations were found between: overall viral infections and relative humidity (p = 0.006; r = 0.6) or precipitation (p = 0.03; r = 0.5), parainfluenza and precipitation (p = 0.02; r = -0.5), respiratory syncytial virus (RSV) and air temperature (p = 0.048; r = -0.4) or precipitation (p = 0.045; r = 0.4), adenovirus and precipitation (p = 0.02; r = 0.5), pneumococcus and air temperature (p = 0.04; r = -0.4), and Chlamydia trachomatis and relative humidity (p = 0.02; r = -0.5). The frequency of parainfluenza infection was highest during spring (32.1%; p = 0.005) and that of RSV infection was highest in the fall (36.4%; p < 0.001). Correlations at regular strength were found between several microbes and meteorological indicators. Parainfluenza and RSV presented marked seasonal patterns.

Seed Cell Wall Storage Polysaccharides: Models to Understand Cell Wall Biosynthesis and Degradation

Cell wall storage polysaccharides (CWSPs) are found as the principal storage compounds in seeds of many taxonomically important groups of plants. These groups developed extremely efficient biochemical mechanisms to disassemble cell walls and use the products of hydrolysis for growth. To accumulate these storage polymers, developing seeds also contain relatively high activities of noncellulosic polysaccharide synthases and thus are interesting models to seek the discovery of genes and enzymes related to polysaccharide biosynthesis. CWSP systems offer opportunities to understand phenomena ranging from polysaccharide deposition during seed maturation to the control of source-sink relationship in developing seedlings. By studying polysaccharide biosynthesis and degradation and the consequences for cell and physiological behavior, we can use these models to develop future biotechnological applications.

Cell wall polysaccharides from cell suspension cultures of the Atlantic Forest tree Rudgea jasminoides (Rubiaceae)

Rudgea jasminoides (Rubiaceae) is a tropical tree species native of the Atlantic Forest in the south of Brazil. Previous studies with leaf cell walls of R. jasminoides showed a different proportion of cross-linked glycans compared to what is usually reported for eudicots. However, due to the difficulties of working with whole plant organs, cell suspensions of R. jasminoides, consisting of predominantly undifferentiated cells with mainly primary cell walls, were used to examine cell walls and extracellular soluble polysaccharides (EP) released into the culture medium. Sugar composition and linkage analysis showed homogalacturonans, xylogalacturonans and arabinogalactans to be the predominant EP. In the cell wall, homogalacturonans and arabinogalactans are the major pectins, and xyloglucans and xylans are the major cross-linking glycans. The presence of xylogalacturonans in the R. jasminoides cell cultures seems to be related to the occurrence of a homogeneous cell suspension with loosely attached cells. Although all alkali extractions from the cell walls yielded amounts of xyloglucan that exceed those of the xylans, the latter was found in a proportion that is higher than what has been usually reported for primary cell walls of most eudicots. The xyloglucan from cell walls of cell suspension cultures of R. jasminoides has low fucosylation levels and high proportion of galactosyl residues, a branching pattern commonly found in storage cell-wall xyloglucans.

Exploring Bacterial Diversity of Endodontic Microbiota by Cloning and Sequencing 16S rRNA

Introduction: The characterization of microbial communities infecting the endodontic system in each clinical condition may help on the establishment of a correct prognosis and distinct strategies of treatment. The purpose of this study was to determine the bacterial diversity in primary endodontic infections by 16S ribosomal-RNA (rRNA) sequence analysis. Methods: Samples from root canals of untreated asymptomatic teeth (n = 12) exhibiting periapical lesions were obtained, 165 rRNA bacterial genomic libraries were constructed and sequenced, and bacterial diversity was estimated. Results: A total of 489 clones were analyzed (mean, 40.7 +/- 8.0 clones per sample). Seventy phylotypes were identified of which six were novel phylotypes belonging to the family Ruminococcaceae. The mean number of taxa per canal was 10.0, ranging from 3 to 21 per sample; 65.7% of the cloned sequences represented phylotypes for which no cultivated isolates have been reported. The most prevalent taxa were Atopobium rimae (50.0%), Dialister invisus, Pre-votella oris, Pseudoramibacter alactolyticus, and Tannerella forsythia (33.3%). Conclusions: Although several key species predominate in endodontic samples of asymptomatic cases with periapical lesions, the primary endodontic infection is characterized by a wide bacterial diversity, which is mostly represented by members of the phylum Firmicutes belonging to the class Clostridia followed by the phylum Bacteroidetes. (J Ended 2011;37:922-926)

The pst operon of enteropathogenic Escherichia coli enhances bacterial adherence to epithelial cells

Enteropathogenic Escherichia coli (EPEC) adheres in vivo and in vitro to epithelial cells. Two main adhesins, the bundle-forming pilus and intimin, encoded by the Up operon and eae, respectively, are responsible for the localized and the intimate adherence phenotypes. Deletion of the pst operon of EPEC abolishes the transport of inorganic phosphate through the phosphate-specific transport system and causes the constitutive expression of the PHO regulon genes. In the absence of pst there is a decrease in the expression of the main EPEC adhesins and a reduction in bacterial adherence to epithelial cells in vitro. This effect is not related to PHO constitutivity, because a Delta pst phoB double mutant that is defective in the transcription of the PHO genes also displayed low levels of adherence and expression of adhesins. Likewise, a PHO-constitutive phoR mutation did not affect bacterial adherence. The expression of the per operon, which encodes the Up and ler regulators PerA and PerC, is also negatively affected by the pst deletion. Overall, the data presented here demonstrate that the pst operon of EPEC plays a positive role in the bacterial adherence mechanism by increasing the expression of perA and perC and consequently the transcription of bfp and eae.

Phylogenetic Validation of the Genera Angomonas and Strigomonas of Trypanosomatids Harboring Bacterial Endosymbionts with the Description of New Species of Trypanosomatids and of Proteobacterial Symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia cuiicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history. (C) 2011 Elsevier GmbH. All rights reserved.