Molecular detection of in-vivo microbial contamination of metallic orthodontic brackets by checkerboard DNA-DNA hybridization

Introduction: Knowing the microbiota that colonizes orthodontic appliances is important for planning strategies and implementing specific preventive measures during treatment. The purpose of this clinical trial was to evaluate in vivo the contamination of metallic orthodontic brackets with 40 DNA probes for different bacterial species by using the checkerboard DNA-DNA hybridization (CDDH) technique. Methods: Eighteen patients, 11 to 29 years of age having fixed orthodontic treatment, were enrolled in the study. Each subject had 2 new metallic brackets bonded to different premolars in a randomized manner. After 30 days, the brackets were removed and processed for analysis by CDDH. Data on bacterial contamination were analyzed descriptively and with the Kruskal-Wallis and Dunn post tests (alpha = 0.05). Forty microbial species (cariogenic microorganisms, bacteria of the purple, yellow, green, orange complexes, "red complex + Treponema socranskii," and the cluster of Actinomyces) were assessed. Results: Most bacterial species were present in all subjects, except for Streptococcus constellatus, Campylobacter rectus, Tannerella forsythia, T socranskii, and Lactobacillus acidophillus (94.4%), Propionibacterium acnes I and Eubacterium nodatum (88.9%), and Treponema denticola (77.8%). Among the cariogenic microorganisms, Streptococcus mutans and Streptococcus sobrinus were found in larger numbers than L acidophillus and Lactobacillus casei (P < 0.001). The periodontal pathogens of the orange complex were detected in larger numbers than those of the "red complex + T socranskii" (P < 0.0001). Among the bacteria not associated with specific pathologies, Veillonella parvula (purple complex) was the most frequently detected strain (P < 0.0001). The numbers of yellow and green complex bacteria and the cluster of Actinomyces were similar (P > 0.05). Conclusions: Metallic brackets in use for 1 month were multi-colonized by several bacterial species, including cariogenic microorganisms and periodontal pathogens, reinforcing the need for meticulous oral hygiene and additional preventive measures to maintain oral health in orthodontic patients. (Am J Orthod Dentofacial Orthop 2012;141:24-9)

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.

In vitro evaluation, in vivo quantification, and microbial diversity studies of nutritional strategies for reducing enteric methane production

The main objective of the present work was to study nutritive strategies for lessening the CH4 formation associated to ruminant tropical diets. In vitro gas production technique was used for evaluating the effect of tannin-rich plants, essential oils, and biodiesel co-products on CH4 formation in three individual studies and a small chamber system to measure CH4 released by sheep for in vivo studies was developed. Microbial rumen population diversity from in vitro assays was studied using qPCR. In vitro studies with tanniniferous plants, herbal plant essential oils derived from thyme, fennel, ginger, black seed, and Eucalyptus oil (EuO) added to the basal diet and cakes of oleaginous plants (cotton, palm, castor plant, turnip, and lupine), which were included in the basal diet to replace soybean meal, presented significant differences regarding fermentation gas production and CH4 formation. In vivo assays were performed according to the results of the in vitro assays. , when supplemented to a basal diet (Tifton-85 hay sp, corn grain, soybean meal, cotton seed meal, and mineral mixture) fed to adult Santa Ines sheep reduced enteric CH4 emission but the supplementation of the basal diet with EuO did not affect ( > 0.05) methane released. Regarding the microbial studies of rumen population diversity using qPCR with DNA samples collected from the in vitro trials, the results showed shifts in microbial communities of the tannin-rich plants in relation to control plant. This research demonstrated that tannin-rich , essential oil from eucalyptus, and biodiesel co-products either in vitro or in vivo assays showed potential to mitigate CH4 emission in ruminants. The microbial community study suggested that the reduction in CH4 production may be attributed to a decrease in fermentable substrate rather than to a direct effect on methanogenesis.

Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria

Biofilms represent a great concern for food industry, since they can be a source of persistent contamination leading to food spoilage and to the transmission of diseases. To avoid the adhesion of bacteria and the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants, microbial compounds that can modify the physicochemical properties of surfaces changing bacterial interactions and consequently adhesion. Different concentrations of the biosurfactants, surfactin from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated to reduce the adhesion and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Staphylococcus aureus, Listeria monocytogenes and Salmonella Enteritidis were studied using polystyrene as the model surface. The pre-conditioning with surfactin 0.25% reduced by 42.0% the adhesion of L monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1.0% reduced by 57.8% adhesion of L monocytogenes and by 67.8% adhesion of S. aureus to polystyrene.Biosurfactants were less effective to avoid adhesion of mixed cultures of the bacteria when compared with individual cultures. After 2 h contact with surfactin at 0.1% concentration, the pre-formed biofilms of S. aureus were reduced by 63.7%, L. monocytogenesby 95.9%, S. Enteritidis by 35.5% and the mixed culture biofilm by 58.5%. The rhamnolipids at 0.25% concentration removed 58.5% the biofilm of S. aureus, 26.5% of L monocytogenes, 23.0% of S. Enteritidis and 24.0% the mixed culture after 2 h contact. In general, the increase in concentration of biosurfactants and in the time of contact decreased biofilm removal percentage. These results suggest that surfactin and rhamnolipids can be explored to control the attachment and to disrupt biofilms of individual and mixed cultures of the food-borne pathogens. (C) 2011 Elsevier Ltd. All rights reserved.

Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes

A taxonomic and annotated functional description of microbial life was deduced from 53 Mb of metagenomic sequence retrieved from a planktonic fraction of the Neotropical high Andean (3,973 meters above sea level) acidic hot spring El Coquito (EC). A classification of unassembled metagenomic reads using different databases showed a high proportion of Gammaproteobacteria and Alphaproteobacteria (in total read affiliation), and through taxonomic affiliation of 16S rRNA gene fragments we observed the presence of Proteobacteria, micro-algae chloroplast and Firmicutes. Reads mapped against the genomes Acidiphilium cryptum JF-5, Legionella pneumophila str. Corby and Acidithiobacillus caldus revealed the presence of transposase-like sequences, potentially involved in horizontal gene transfer. Functional annotation and hierarchical comparison with different datasets obtained by pyrosequencing in different ecosystems showed that the microbial community also contained extensive DNA repair systems, possibly to cope with ultraviolet radiation at such high altitudes. Analysis of genes involved in the nitrogen cycle indicated the presence of dissimilatory nitrate reduction to N2 (narGHI, nirS, norBCDQ and nosZ), associated with Proteobacteria-like sequences. Genes involved in the sulfur cycle (cysDN, cysNC and aprA) indicated adenylsulfate and sulfite production that were affiliated to several bacterial species. In summary, metagenomic sequence data provided insight regarding the structure and possible functions of this hot spring microbial community, describing some groups potentially involved in the nitrogen and sulfur cycling in this environment. Citation: Jimenez DJ, Andreote FD, Chaves D, Montana JS, Osorio-Forero C, et al. (2012) Structural and Functional Insights from the Metagenome of an Acidic Hot Spring Microbial Planktonic Community in the Colombian Andes. PLoS ONE 7(12): e52069. doi:10.1371/journal.pone.0052069

COMPARISON OF DIFFERENT PROTOCOLS FOR THE EXTRACTION OF MICROBIAL DNA FROM REEF CORALS

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Modeling the growth rate and lag time of different strains of Salmonella enterica and Listeria monocytogenes in ready-to-eat lettuce

The growth parameters (growth rate, mu and lag time, lambda) of three different strains each of Salmonella enterica and Listeria monocytogenes in minimally processed lettuce (MPL) and their changes as a function of temperature were modeled. MPL were packed under modified atmosphere (5% O-2, 15% CO2 and 80% N-2), stored at 7-30 degrees C and samples collected at different time intervals were enumerated for S. enterica and L monocytogenes. Growth curves and equations describing the relationship between mu and lambda as a function of temperature were constructed using the DMFit Excel add-in and through linear regression, respectively. The predicted growth parameters for the pathogens observed in this study were compared to ComBase, Pathogen modeling program (PMP) and data from the literature. High R-2 values (0.97 and 0.93) were observed for average growth curves of different strains of pathogens grown on MPL Secondary models of mu and lambda for both pathogens followed a linear trend with high R2 values (>0.90). Root mean square error (RMSE) showed that the models obtained are accurate and suitable for modeling the growth of S. enterica and L monocytogenes in MP lettuce. The current study provides growth models for these foodborne pathogens that can be used in microbial risk assessment. (C) 2011 Elsevier Ltd. All rights reserved.

In vitro evaluation of the microbial contamination on new toothbrushes: A preliminary study

Objective: The presence and survival of microorganisms on toothbrush bristles might play a role on the etiology of oral infections. The aim of this in vitro study was to evaluate the presence of bacterial contamination on new toothbrushes before oral contact. Materials and methods: Forty toothbrushes from five different manufacturers were used in this experimental study. Each manufacturer was divided according to conventional local of obtaining: industry, drugstore, market, and perfumery. The toothbrush heads were completely immersed into tubes containing 5.0 mL of sterile peptonated water (dilution 1:10). A group of eight tubes containing the sterile solution was used as control. After 21 days of anaerobic incubation, occurrence of contamination was visually evaluated and confirmed by light microscopy. Results: Bacterial growth in the medium, indicative of bristles contamination, was found in a total of 19 out of 40 samples (47.5%) evaluated: 6 out of 14 samples (42.85%) from industry group, 4 out of 8 samples (50.0%) from drugstore, 5 out of 10 samples (50.0%) from market, and 4 out of 8 samples (50.0%) from perfumery. Only the toothbrushes with bristles coated with chlorhexidine did not show contamination. The Gram-negative sporulating bacilli were the most prevalent form recovered. Conclusions: Except for chlorhexidine group, bacterial growth was observed in all groups evaluated irrespective local of obtaining. Microsc. Res. Tech., 2012. (c) 2011 Wiley Periodicals, Inc.

Clinical and Microbiologic Evaluation, by Real-Time Polymerase Chain Reaction, of Non-Surgical Treatment of Aggressive Periodontitis Associated With Amoxicillin and Metronidazole

Background: The aim of the present study is to evaluate the clinical and microbiologic changes resulting from non-surgical periodontal treatment associated with amoxicillin and metronidazole in individuals with aggressive periodontitis. Methods: Fifteen individuals with aggressive periodontitis received non-surgical periodontal treatment and 45 days after completion of treatment were treated with antibiotics. Clinical data and samples of subgingival plaque were collected at baseline, 45 days after the non-surgical periodontal treatment, and 1 month after the use of antimicrobial agents. After 3 and 6 months, only clinical data were collected. The presence and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Dialister pneumosintes were determined by real-time polymerase chain reaction. Results: All clinical parameters, with the exception of clinical attachment level (CAL), had significantly (P<0.05) improved at the end of the third month after non-surgical therapy associated with antibiotics. There was significant (P<0.05) reduction in the quantities of Td and Tf. After 1 month, there were significant (P<0.05) reductions in the frequencies of Pg and Tf. Conclusion: Non-surgical mechanical treatment associated with the use of amoxicillin and metronidazole led to an improvement in all clinical parameters studied, except for CAL, and significantly reduced the amount of subgingival Tf and Td. J Periodontal 2012;83:744-752.

Phenol biodegradation by a microbial consortium: application of artificial neural network (ANN) modelling

In this study, an effective microbial consortium for the biodegradation of phenol was grown under different operational conditions, and the effects of phosphate concentration (1.4 g L-1, 2.8 g L-1, 4.2 g L-1), temperature (25 degrees C, 30 degrees C, 35 degrees C), agitation (150 rpm, 200 rpm, 250 rpm) and pH (6, 7, 8) on phenol degradation were investigated, whereupon an artificial neural network (ANN) model was developed in order to predict degradation. The learning, recall and generalization characteristics of neural networks were studied using data from the phenol degradation system. The efficiency of the model generated by the ANN was then tested and compared with the experimental results obtained. In both cases, the results corroborate the idea that aeration and temperature are crucial to increasing the efficiency of biodegradation.

Correlation of soil microbial community responses to contamination with crude oil with and without chromium and copper

Soil microcosms contaminated with crude oil with or without chromium and copper were monitored over a period of 90 days for microbial respiration, biomass, and for dehydrogenase, lipase, acid phosphatase, and arylsulfatase activities. In addition, the community structure was followed by enumerating the total heterotrophic and oil-degrading viable bacteria and by performing a denaturing gradient gel electrophoresis (DGGE) of the PCR amplified 16S rDNA. A significant difference was observed for biochemical activities and microbial community structures between the microcosms comprised of uncontaminated soil, soil contaminated with crude oil and soil contaminated with crude oil and heavy metals. The easily measured soil enzyme activities correlated well with microbial population levels, community structures and rates of respiration (CO2 production). The estimation of microbial responses to soil contamination provides a more thorough understanding of the microbial community function in contaminated soil, in situations where technical and financial resources are limited and may be useful in addressing bioremediation treatability and effectiveness. (C) 2012 Published by Elsevier Ltd.

Impact of Land Degradation on Soil Microbial Biomass and Activity in Northeast Brazil

Land degradation causes great changes in the soil biological properties. The process of degradation may decrease soil microbial biomass and consequently decrease soil microbial activity. The study was conducted out during 2009 and 2010 at the four sites of land under native vegetation (NV), moderately degraded land (LDL), highly degraded land (HDL) and land under restoration for four years (RL) to evaluate changes in soil microbial biomass and activity in lands with different degradation levels in comparison with both land under native vegetation and land under restoration in Northeast Brazil. Soil samples were collected at 0-10 cm depth. Soil organic carbon (SOC), soil microbial biomass C (MBC) and N (MBN), soil respiration (SR), and hydrolysis of fluorescein diacetate (FDA) and dehydrogenase (DHA) activities were analyzed. After two years of evaluation, soil MBC, MBN, FDA and DHA had higher values in the NV, followed by the RL. The decreases of soil microbial biomass and enzyme activities in the degraded lands were approximately 8-10 times as large as those found in the NV. However, after land restoration, the MBC and MBN increased approximately 5-fold and 2-fold, respectively, compared with the HDL. The results showed that land degradation produced a strong decrease in soil microbial biomass. However, land restoration may promote short- and long-term increases in soil microbial biomass.

Periodic sterility assessment of materials stored for up to 6 months at continuous microbial contamination risk: Laboratory study

An investigation was conducted to test the hypothesis that the storage time of packaging sterility has no effect on contamination susceptibility even under deliberate bacterial exposure (Serratia marcescens). No growth of the test microorganisms was identified in the experimental group in any of the storage intervals (7, 14, 28, 90, and 180 days). Current recommendations/guidelines suggest that contamination of packaging occurs only because of events. This study, done in vitro, supports these recommendations. Copyright (c) 2012 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Hydrogen production and consumption of organic acids by a phototrophic microbial consortium

Alternative fuel sources have been extensively studied. Hydrogen gas has gained attention because its combustion releases only water, and it can be produced by microorganisms using organic acids as substrates. The aim of this study was to enrich a microbial consortium of photosynthetic purple non-sulfur bacteria from an Upflow Anaerobic Sludge Blanket reactor (UASB) using malate as carbon source. After the enrichment phase, other carbon sources were tested, such as acetate (30 mmol l(-1)), butyrate (17 mmol l(-1)), citrate (11 mmol l(-1)), lactate (23 mmol l(-1)) and malate (14.5 mmol l(-1)). The reactors were incubated at 30 degrees C under constant illumination by 3 fluorescent lamps (81 mu mol m(-2) s(-1)). The cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H-2 l(-1) culture for acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H-2 mmol(-1) substrate for acetate, butyrate, citrate, lactate and malate, respectively. The consumption of substrates was 43% for acetate, 37% for butyrate, 100% for citrate, 49% for lactate and 100% for malate. Approximately 26% of the clones obtained from the Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) were similar to Rhodobacter, Rhodospirillum and Rhodopseudomonas, which have been widely cited in studies of photobiological hydrogen production. Clones similar to the genus Sulfurospirillum (29% of the total) were also found in the microbial consortium. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Epicoccum nigrum P16, a Sugarcane Endophyte, Produces Antifungal Compounds and Induces Root Growth

Background: Sugarcane is one of the most important crops in Brazil, mainly because of its use in biofuel production. Recent studies have sought to determine the role of sugarcane endophytic microbial diversity in microorganism-plant interactions, and their biotechnological potential. Epicoccum nigrum is an important sugarcane endophytic fungus that has been associated with the biological control of phytopathogens, and the production of secondary metabolites. In spite of several studies carried out to define the better conditions to use E. nigrum in different crops, little is known about the establishment of an endophytic interaction, and its potential effects on plant physiology. Methodology/Principal Findings: We report an approach based on inoculation followed by re-isolation, molecular monitoring, microscopic analysis, plant growth responses to fungal colonization, and antimicrobial activity tests to study the basic aspects of the E. nigrum endophytic interaction with sugarcane, and the effects of colonization on plant physiology. The results indicate that E. nigrum was capable of increasing the root system biomass and producing compounds that inhibit the in vitro growth of sugarcane pathogens Fusarium verticillioides, Colletotrichum falcatum, Ceratocystis paradoxa, and Xanthomomas albilineans. In addition, E. nigrum preferentially colonizes the sugarcane surface and, occasionally, the endophytic environment. Conclusions/Significance: Our work demonstrates that E. nigrum has great potential for sugarcane crop application because it is capable of increasing the root system biomass and controlling pathogens. The study of the basic aspects of the interaction of E. nigrum with sugarcane demonstrated the facultative endophytism of E. nigrum and its preference for the phylloplane environment, which should be considered in future studies of biocontrol using this species. In addition, this work contributes to the knowledge of the interaction of this ubiquitous endophyte with the host plant, and also to a better use of microbial endophytes in agriculture.