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.

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.

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.

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial. species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique. (c) 2007 Elsevier GmbH. All rights reserved.

Use of the checkerboard DNA-DNA hybridisation technique for in vivo detection of cariogenic microorganisms on metallic brackets, with or without use of an antimicrobial agent

Objective: Using checkerboard DNA-DNA hybridisation (CDDH) assay, this randomised clinical study evaluated the contamination of metallic brackets by four cariogenic bacterial strains (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei and Lactobacillus acidophilus) and the efficacy of 0.12% chlorhexidine gluconate (CHX) mouthwashes in reducing bacterial contamination. Methods: Thirty-nine 11-33-year-old patients under treatment with fixed orthodontic appliances were enrolled in the study and had 2 new metallic brackets bonded to premolars. Nineteen patients used a 0.12% CHX mouthwash (Periogard (R)) and 20 patients used a placebo mouthwash (control) twice a week. After 30 days, the brackets were removed and samples were obtained for analysis by CDDH. Data were analysed statistically by the Kruskal-Wallis test (alpha = 0.05) using the SAS software. Results: S. mutans, S. sobrinus, L. casei and L. acidophilus were detected in 100% of the samples from both groups. However, brackets of the control group were more heavily contaminated by S. mutans and S. sobrinus (P < 0.01). In the experimental group, although all counts decreased after rinsing with the chlorhexidine solution, there was significant difference only for S. mutans (P = 0.03). Conclusions: The use of 0.12% chlorhexidine gluconate mouthwashes can be useful in clinical practice to reduce the levels of cariogenic microorganisms in patients under treatment with fixed orthodontic appliances. (C) 2011 Elsevier Ltd. All rights reserved.

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.

Oral colonization by Candida species in AIDS pediatric patients

The aim of this study was to assess the prevalence of factors associated with oral colonization by Candida spp. in pediatric patients with AIDS. The sample comprised of 117 children. Clinical status, medicines in use, and laboratory findings were obtained from hospital records; sociodemographic data were given by relatives. A dental examination assessed the prevalence of dental caries. The prevalence of oral colonization by Candida was 62%. Only seven children presented clinical manifestation of oral candidosis despite their high viral load index and low-for-age CD4 count. Candida colonization was directly associated with frequent use of antibiotics (prevalence ratio [PR] = 1.44), sulfa drugs (PR = 1.23), alteration in the oral mucosa (PR = 1.55), and untreated dental caries (PR = 1.93). It was inversely associated with the use of antiretroviral therapies (PR = 0.65). Candida albicans was the most frequently detected species (80%); phenotypic tests did not detect C. dubliniensis strains. This study observed a low prevalence of Candida-related oral lesions in these patients, which is compatible with the hypothesis that antiretroviral medicines may have contributed to reducing oral manifestations from Candida infection. The high prevalence of Candida colonization in HIV+/AIDS children with untreated dental caries reinforces the importance of oral health care in interdisciplinary health units that assist these patients.

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.

Influence of organic fertilization on the number of culturable diazotrophic endophytic bacteria isolated from sugarcane

The numbers of culturable diazotrophic endophytic bacteria (CDEB) from roots stems and leaves of sugarcane submitted to organic inorganic or no fertilization were compared In order to determine the size of the N(2) fixing populations the Most Probable Number technique (MPN) was used The quantification of diazotrophic bacteria by using the acetylene reduction assay (ARA) was more accurate than observing the bacterial growth in the vials to confirm N(2) fixing capability the detection of gene nifH was performed on a sample of 105 Isolated bacteria The production of extracellular enzymes involved in the penetration of the plants by the bacteria was also studied The results showed that organic fertilization enhances the number of CDEB when compared with conventional fertilization used throughout the growing season The maximum number of bacteria was detected in the roots Roots and stems presented the greatest number of CDEB in the middle of the cropping season and in leaves numbers varied according to the treatment Using two pairs of primers and two different methods the nifH gene was found in 104 of the 105 tested isolates Larger amounts of pectinase were released by isolates from sugarcane treated with conventional fertilizers (66%) whereas larger amounts of cellulase were released by strains isolated from sugarcane treated with organic fertilizers (80%) (C) 2010 Elsevier Masson SAS All rights reserved