Search

Counterfeiting of alcoholic beverages, particularly high-value spirits such as whiskey, presents significant challenges for regulators, manufacturers, and consumers. In this study, we introduce and validate a novel application of headspace extraction (HS) followed by gas chromatography with flame ionization detection (GC-FID) for the quantitative determination of ethanol content in 42 suspected counterfeit brazilian samples of whiskeys. This method, in conjunction with visual inspection of material inconsistencies, offers a combined approach to identify potential cases of fraud. The HS-GC-FID findings revealed that only 19% of the analyzed samples had ethanol content in the limits declared on the label, emphasizing the role of ethanol content as a chemical marker for suspected beverage fraud., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The lack of data regarding infant exposure to drugs of abuse consumed by lactating mothers has become a major health concern. Investigating psychoactive substances and their metabolites in breastmilk is an analytical approach to estimate the rate at which drugs of abuse are excreted and how much infants are exposed to them. In this study, we have developed and validated a GC-MS method using disposable pipette tips for simultaneously extracting ten analytes from breastmilk samples obtained from a milk bank in the city of Ribeirão Preto, Brazil. All the 67 analyzed samples tested negative for all the analytes. This is the first study that has applied disposable pipette extraction to analyze drugs of abuse in breastmilk samples.

Objective: To investigate how social and psychological characteristics differ between pregnant women who smoke and do not smoke. To explore associations between social and psychological features with changes of smoking habits by the end of pregnancy., Methods: A case-control study was set up. Smokers cases were never-smokers and ex-smokers controls. Pregnant women (n=328) from public prenatal services were interviewed. Socio-demographic data and psychological variables - personality traits, anxiety, depression, perceived stress, maternal fetal-attachment - were measured. Saliva samples were collected to measure cotinine and to check self-informed smoking status. In addition, 66 smokers were also assessed regarding smoking habits by late pregnancy. Smoking status was defined as a dependent variable. Exposure factors were analyzed through odds ratios. Logistic models and contingency tables were employed according to the nature of variables. "Qualitative change in smoking" was defined as a dependent variable for the last evaluation, and a logistic regression model was built., Results: Lower schooling, higher age, use of alcohol and drugs, living without a partner, and passive smoking showed associations with smoking. Anxiety, depression and perceived stress also exhibited positive association with smoking. Among personality traits, only Neuroticism was associated with smoking. None of the variables were associated with qualitative change in smoking by the end of pregnancy., Conclusion: Smoking during pregnancy is associated with more unfavorable social conditions. Pregnant women who smoke exhibit more negative psychological states than nonsmokers, including a profile of accentuated Neuroticism. None of the investigated variables could predict changes in smoking during pregnancy.

The interest in alternative biological matrices (e.g., hair and saliva) for forensic toxicology analysis has increased, and recent developments in sample preparation have targeted rapid, cheap, efficient and eco-friendly methods, including microextraction techniques. For this review, we have gathered information about these two hot topics. We discuss the composition, incorporation of analytes and advantages and disadvantages of different biological matrices, and also present the operation principles of the most reported microextraction procedures and their application in forensic toxicology. The outcome of this review may encourage future forensic researches into alternative samples and microextraction techniques.

Objectives: Candida parapsilosis is a healthcare-related fungal pathogen particularly common among immunocompromised patients. Our understanding of antifungal resistance mechanisms in C. parapsilosis remains very limited. We previously described an azole-resistant strain of C. parapsilosis (BC014R PSC ), obtained following exposure in vitro to posaconazole. Resistance was associated with overexpression of ergosterol biosynthetic genes (ERG genes), together with the transcription factors UPC2 (CPAR2-207280) and NDT80 (CPAR2-213640). The aim of this study was to identify the mechanisms underlying posaconazole resistance of the BC014R PSC strain., Methods: To identify the causative mutation, we sequenced the genomes of the susceptible (BC014S) and resistant (BC014R PSC ) isolates, using Illumina technology. Ergosterol content was assessed in both strains by mass spectrometry. UPC2 and NDT80 genes were deleted in BC014R PSC strain. Mutants were characterized regarding their azole susceptibility profile and ERG gene expression., Results: One homozygous missense mutation (R135I) was found in ERG3 (CPAR2-105550) in the azole-resistant isolate. We show that Erg3 activity is completely impaired, resulting in a build up of sterol intermediates and a failure to generate ergosterol. Deleting UPC2 and NDT80 in BC014R PSC reduces the expression of ERG genes and restores susceptibility to azole drugs., Conclusions: A missense mutation in the ERG3 gene results in azole resistance and up-regulation of ERG genes expression. We propose that this mutation prevents the formation of toxic intermediates when cells are treated with azoles. Resistance can be reversed by deleting Upc2 and Ndt80 transcription factors. UPC2 plays a stronger role in C. parapsilosis azole resistance than does NDT80., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction:: The implementation of the rapid test (RT) for syphilis increases access of vulnerable populations to early diagnosis and treatment, impacting the outcomes of infection. We aimed to assess the prevalence of and factors associated with syphilis in a Center for Testing and Counseling (CTC)., Methods:: We conducted a cross-sectional study at a Reference Center for sexually transmitted disease (STD) and acquired immune deficiency syndrome in Londrina, Northern Paraná State, Southern Brazil. Data regarding the 5,509 individuals who underwent RT from June 2012 to December 2014 were collected from patient records and the CTC Information System and served as the basis to check associations of syphilis cases (346) and cases without syphilis (5,163). Nine patients' records were not found. OpenEpi was used to perform a prevalence analysis and determine odds ratios to assess the associations between sociodemographic and behavioral variables (independent variables) and cases of syphilis (dependent variable). An alpha value <0.05 was considered statistically significant., Results:: The prevalence of syphilis was 6.3%; higher in males (7.5%) than in females (4.3%, p <0.001). Syphilis was associated with an age of 25-34 years, little education, and single marital status. The main associated behavioral factors were men who have sex with men, drug users, STD patients, and those presenting with an STD in the last year. The use of alcohol, marijuana, cocaine, and crack was significantly associated with syphilis., Conclusions:: Strategies for prevention and control of syphilis should be intensified, especially in populations identified as most vulnerable.

The present study assesses the diversity and composition of sediment bacterial and microeukaryotic communities from deep-sea mud volcanoes (MVs) associated with strike-slip faults in the South-West Iberian Margin (SWIM). We used a 16S/18S rRNA gene based pyrosequencing approach to characterize and correlate the sediment bacterial and microeukaryotic communities from MVs with differing gas seep regimes and from an additional site with no apparent seeping activity. In general, our results showed significant compositional changes of bacterial and microeukaryotic communities in sampling sites with different seepage regimes. Sediment bacterial communities were enriched with Methylococcales (putative methanotrophs) but had lower abundances of Rhodospirillales, Nitrospirales and SAR202 in the more active MVs. Within microeukaryotic communities, members of the Lobosa (lobose amoebae) were enriched in more active MVs. We also showed a strong correlation between Methylococcales populations and lobose amoeba in active MVs. This study provides baseline information on the diversity and composition of bacterial and microeukaryotic communities in deep-sea MVs associated with strike-slip faults.

In the present study, we compared communities of bacteria in two jellyfish species (the 'golden' jellyfish Mastigias cf.papua and the box jellyfish Tripedalia cf.cystophora) and water in three marine lakes located in the Berau region of northeastern Borneo, Indonesia. Jellyfish-associated bacterial communities were compositionally distinct and less diverse than bacterioplankton communities. Alphaproteobacteria, Gammaproteobacteria, Synechococcophycidae and Flavobacteriia were the most abundant classes in water. Jellyfish-associated bacterial communities were dominated by OTUs assigned to the Gammaproteobacteria (family Endozoicimonaceae), Mollicutes, Spirochaetes and Alphaproteobacteria (orders Kiloniellales and Rhodobacterales). Mollicutes were mainly restricted to Mastigias whereas Spirochaetes and the order Kiloniellales were most abundant in Tripedalia hosts. The most abundant OTU overall in jellyfish hosts was assigned to the family Endozoicimonaceae and was highly similar to organisms in Genbank obtained from various hosts including an octocoral, bivalve and fish species. Other abundant OTUs included an OTU assigned to the order Entomoplasmatales and mainly found in Mastigias hosts and OTUs assigned to the Spirochaetes and order Kiloniellales and mainly found in Tripedalia hosts. The low sequence similarity of the Entomoplasmatales OTU to sequences in Genbank suggests that it may be a novel lineage inhabiting Mastigias and possibly restricted to marine lakes., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study aimed to assess the effect of ultraviolet radiation (UVR) and chemical stress (triclosan-TCS; potassium dichromate-PD; prochloraz-PCZ) on bacterial communities of zebrafish (Danio rerio) embryos (ZEBC). Embryos were exposed to two UVR intensities and two chemical concentrations not causing mortality or any developmental effect (equivalent to the No-Observed-Effect Concentration-NOEC; NOEC diluted by 10-NOEC/10). Effects on ZEBC were evaluated using denaturing gradient gel electrophoresis (DGGE) and interpreted considering structure, richness and diversity. ZEBC were affected by both stressors even at concentrations/doses not affecting the host-organism (survival/development). Yet, some stress-tolerant bacterial groups were revealed. The structure of the ZEBC was always affected, mainly due to xenobiotic presence. Richness and diversity decreased after exposure to NOEC of PD. Interactive effects occurred for TCS and UVR. Aquatic microbiota imbalance might have repercussions for the host/aquatic system, particularly in a realistic scenario/climate change perspective therefore, future ecotoxicological models should consider xenobiotics interactions with UVR., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Dry-salted codfish and pollock are commercially important food products with a relatively long shelf life. To date, bacterial assemblages present in these products that are relevant for food safety have been monitored using only classical molecular and/or cultivation methods. The present study employed a rapid and accurate identification method involving PCR with denaturing gradient gel electrophoresis and pyrosequencing to characterize the bacterial assemblages in the skin of three closely related fishes: Gadus morhua, Gadus macrocephalus, and Theragra chalcogramma. This methodology can be crucial for timely identification of food spoilage, hazard analysis, and monitoring of critical control points during food production. Although all specimens were processed in the same factory, there were significant compositional differences in their skin bacterial communities. In general, the bacterial community was dominated by gram-negative species of the Gammaproteobacteria. Pyrosequencing yielded 90, 69, and 245 operational taxonomic units associated with G. morhua, G. macrocephalus, and T. chalcogramma, respectively. The most dominant operational taxonomic units were assigned in order to Pseudomonas sp., Serratia marcescens, Salinisphaera sp., and Psychrobacter pulmonis. Spoilage and pathogenic bacterial groups were detected in all the studied salted gadoid samples.

In the present study, we assessed the composition of Bacteria in four biotopes namely sediment, seawater and two sponge species (Stylissa massa and Xestospongia testudinaria) at four different reef sites in a coral reef ecosystem in West Java, Indonesia. In addition to this, we used a predictive metagenomic approach to estimate to what extent nitrogen metabolic pathways differed among bacterial communities from different biotopes. We observed marked differences in bacterial composition of the most abundant bacterial phyla, classes and orders among sponge species, water and sediment. Proteobacteria were by far the most abundant phylum in terms of both sequences and Operational Taxonomic Units (OTUs). Predicted counts for genes associated with the nitrogen metabolism suggested that several genes involved in the nitrogen cycle were enriched in sponge samples, including nosZ, nifD, nirK, norB and nrfA genes. Our data show that a combined barcoded pyrosequencing and predictive metagenomic approach can provide novel insights into the potential ecological functions of the microbial communities. Not only is this approach useful for our understanding of the vast microbial diversity found in sponges but also to understand the potential response of microbial communities to environmental change., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fish larvae in aquaculture have high mortality rates due to pathogenic bacteria, especially the Vibrio species, and ineffective prophylactic strategies. Vaccination is not feasible in larvae and antibiotics have reduced efficacy against multidrug resistant bacteria. A novel approach to controlling Vibrio infections in aquaculture is needed. The potential of phage therapy to combat vibriosis in fish larvae production has not yet been examined. We describe the isolation and characterization of two bacteriophages capable of infecting pathogenic Vibrio and their application to prevent bacterial infection in fish larvae. Two groups of zebrafish larvae were infected with V. anguillarum (∼106 CFU mL-1) and one was later treated with a phage lysate (∼108 PFU mL-1). A third group was only added with phages. A fourth group received neither bacteria nor phages (fish control). Larvae mortality, after 72 h, in the infected and treated group was similar to normal levels and significantly lower than that of the infected but not treated group, indicating that phage treatment was effective. Thus, directly supplying phages to the culture water could be an effective and inexpensive approach toward reducing the negative impact of vibriosis in larviculture.

In this study, two molecular techniques [denaturing gradient gel electrophoresis (DGGE) and barcoded pyrosequencing] were used to evaluate the composition of bacterial communities in salt marsh microhabitats [bulk sediment and sediment surrounding the roots (rhizosphere) of Halimione portulacoides and Sarcocornia perennis ssp. perennis] that have been differentially affected by oil hydrocarbon (OH) pollution. Both DGGE and pyrosequencing revealed that bacterial composition is structured by microhabitat. Rhizosphere sediment from both plant species revealed enrichment of operational taxonomic units closely related to Acidimicrobiales, Myxococcales and Sphingomonadales. The in silico metagenome analyses suggest that homologous genes related to OH degradation appeared to be more frequent in both plant rhizospheres than in bulk sediment. In summary, this study suggests that halophyte plant colonization is an important driver of hydrocarbonoclastic bacterial community composition in estuarine environments, which can be exploited for in situ phytoremediation of OH in salt marsh environments., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

For several years, knowledge on the microbiome associated with marine invertebrates was impaired by the challenges associated with the characterization of bacterial communities. With the advent of culture independent molecular tools it is possible to gain new insights on the diversity and richness of microorganisms associated with marine invertebrates. In the present study, we evaluated if different preservation and processing methodologies (prior to DNA extraction) can affect the bacterial diversity retrieved from snakelocks anemone Anemonia viridis. Denaturing gradient gel electrophoresis (DGGE) community fingerprints were used as proxy to determine the bacterial diversity retrieved (H'). Statistical analyses indicated that preservation significantly affects H'. The best approach to preserve and process A. viridis biomass for bacterial community fingerprint analysis was flash freezing in liquid nitrogen (preservation) followed by the use of a mechanical homogenizer (process), as it consistently yielded higher H'. Alternatively, biomass samples can be processed fresh followed by cell lyses using a mechanical homogenizer or mortar &pestle. The suitability of employing these two alternative procedures was further reinforced by the quantification of the 16S rRNA gene; no significant differences were recorded when comparing these two approaches and the use of liquid nitrogen followed by processing with a mechanical homogenizer.

Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 10(5) CFU ml(-1) and a phage concentration of ≈ 10(6-8) plaque forming unit ml(-1) were used. Phage survival was not significantly affected by the natural variability of pH (6.5-7.4), temperature (10-25 °C), salinity (0-30 g NaCl l(-1) ) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in non-marine systems through the addition of salt, whenever this option is feasible and does not affect the survival of aquatic species being cultured., (© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The surface microlayer (SML) is characterized by different physicochemical properties from underlying waters (UW). However, whether these differences in abiotic factors underlie the distinct sensitivity of bacterioneuston (i.e. SML bacteria) and bacterioplankton to environmental stressors remains to be addressed. We investigated the contribution of abiotic factors to the UV-B sensitivity of bacterioneuston and bacterioplankton. Nutrients (especially nitrogen and phosphate) emerged as important determinants of bacterial UV-B sensitivity. The role of particles, nutrients, and dissolved organic components on bacterial UV-B sensitivity was further evaluated using dilution cultures. Filtered samples were twofold more UV sensitive than unfiltered samples, suggesting a UV-protective effect of particles. High nutrient concentrations attenuated bacterial UV-B sensitivity (up to 40%), compared with unamended conditions, by influencing bacterial physiology and/or community composition. Suspending cells in natural water, particularly from the SML, also attenuated UV-B sensitivity (up to 23%), compared with suspension in an artificial mineral solution. Bioassays using Pseudomonas sp. strain NT5I1.2B revealed that chemical water properties influence UV-induced oxidative damage. UV-B sensitivity was associated with high cell-specific activities. The chemical environment of the SML and UW influences UV-B effects on the corresponding bacterial communities. Maintaining low cell activities might be advantageous in stressful environments, like the SML., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The present study combined a DGGE and barcoded 16S rRNA pyrosequencing approach to assess bacterial composition in the water of a recirculating aquaculture system (RAS) with a shallow raceway system (SRS) for turbot (Scophthalmus maximus) and sole (Solea senegalensis). Barcoded pyrosequencing results were also used to determine the potential pathogen load in the RAS studied. Samples were collected from the water supply pipeline (Sup), fish production tanks (Pro), sedimentation filter (Sed), biofilter tank (Bio), and protein skimmer (Ozo; also used as an ozone reaction chamber) of twin RAS operating in parallel (one for each fish species). Our results revealed pronounced differences in bacterial community composition between turbot and sole RAS, suggesting that in the systems studied there is a strong species-specific effect on water bacterial communities. Proteobacteria was the most abundant phylum in the water supply and all RAS compartments. Other important taxonomic groups included the phylum Bacteriodetes. The saltwater supplied displayed a markedly lower richness and appeared to have very little influence on bacterial composition. The following potentially pathogenic species were detected: Photobacterium damselae in turbot (all compartments), Tenacibaculum discolor in turbot and sole (all compartments), Tenacibaculum soleae in turbot (all compartments) and sole (Pro, Sed and Bio), and Serratia marcescens in turbot (Sup, Sed, Bio and Ozo) and sole (only Sed) RAS. Despite the presence of these pathogens, no symptomatic fish were observed. Although we were able to identify potential pathogens, this approach should be employed with caution when monitoring aquaculture systems, as the required phylogenetic resolution for reliable identification of pathogens may not always be possible to achieve when employing 16S rRNA gene fragments.

Marine lakes are unique ecosystems that contain isolated populations of marine organisms. Isolated from the surrounding marine habitat, many lakes house numerous endemic species. In this study, microbial communities of sponges inhabiting these lakes were investigated for the first time using barcoded pyrosequencing of 16S rRNA gene amplicons. Our main goals were to compare the bacterial richness and composition of two sponge species (Suberites diversicolor and Cinachyrella australiensis) inhabiting both marine lakes and adjacent open coastal systems. Host species and habitat explained almost 59% of the variation in bacterial composition. There was a significant difference in composition between both host species. Within S. diversicolor, there was little discernible difference between bacterial communities inside and outside lakes. The bacterial community of this species was, furthermore, dominated (63% of all sequences) by three very closely related alphaproteobacterial taxa identified as belonging to the recently described order Kiloniellales. Cinachyrella australiensis, in contrast, hosted markedly different bacterial communities inside and outside lakes with very few shared abundant taxa. Cinachyrella australiensis in open habitat only shared 9.4% of OTUs with C. australiensis in lake habitat. Bacteria were thus both highly species specific and, in the case of C. australiensis, habitat specific., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Studies have shown immunological and morphological alterations in the esophagus during the course of AIDS. Esophageal postmortem samples of 22 men with AIDS autopsied in a teaching hospital between 1982 and 2009 were collected. We carried out revision of the autopsy reports and medical records, morphometric analysis (Image J and KS-300 Kontron-Zeiss), and immunohistochemical (anti-S100, anti-IgA, anti-IgG, and anti-IgM) analysis of the esophagus. In accordance with most of the parameters evaluated, age and the smoking habit harmed the esophageal local immunity, whereas the use of antiretroviral therapy improved the immune characteristics of this organ. Patients with esophagitis also presented immunological fragility of the esophagus. This leads to the conclusion that alterations in the esophageal epithelium of patients with AIDS are not only caused by direct action of HIV but also the clinical and behavioral characteristics of the patient.

Global climate change has the potential to seriously and adversely affect marine ecosystem functioning. Numerous experimental and modeling studies have demonstrated how predicted ocean acidification and increased ultraviolet radiation (UVR) can affect marine microbes. However, researchers have largely ignored interactions between ocean acidification, increased UVR and anthropogenic pollutants in marine environments. Such interactions can alter chemical speciation and the bioavailability of several organic and inorganic pollutants with potentially deleterious effects, such as modifying microbial-mediated detoxification processes. Microbes mediate major biogeochemical cycles, providing fundamental ecosystems services such as environmental detoxification and recovery. It is, therefore, important that we understand how predicted changes to oceanic pH, UVR, and temperature will affect microbial pollutant detoxification processes in marine ecosystems. The intrinsic characteristics of microbes, such as their short generation time, small size, and functional role in biogeochemical cycles combined with recent advances in molecular techniques (e.g., metagenomics and metatranscriptomics) make microbes excellent models to evaluate the consequences of various climate change scenarios on detoxification processes in marine ecosystems. In this review, we highlight the importance of microbial microcosm experiments, coupled with high-resolution molecular biology techniques, to provide a critical experimental framework to start understanding how climate change, anthropogenic pollution, and microbiological interactions may affect marine ecosystems in the future.

A novel approach based on headspace solid-phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-ToFMS) was developed for the simultaneous screening of microbial and mite contamination level in cereals and coffee beans. The proposed approach emerges as a powerful tool for the rapid assessment of the microbial contamination level (ca. 70 min versus ca. 72 to 120 h for bacteria and fungi, respectively, using conventional plate counts), and mite contamination (ca. 70 min versus ca. 24 h). A full-factorial design was performed for optimization of the SPME experimental parameters. The methodology was applied to three types of rice (rough, brown, and white rice), oat, wheat, and green and roasted coffee beans. Simultaneously, microbiological analysis of the samples (total aerobic microorganisms, moulds, and yeasts) was performed by conventional plate counts. A set of 54 volatile markers was selected among all the compounds detected by GC×GC-ToFMS. Principal Component Analysis (PCA) was applied in order to establish a relationship between potential volatile markers and the level of microbial contamination. Methylbenzene, 3-octanone, 2-nonanone, 2-methyl-3-pentanol, 1-octen-3-ol, and 2-hexanone were associated to samples with higher microbial contamination level, especially in rough rice. Moreover, oat exhibited a high GC peak area of 2-hydroxy-6-methylbenzaldehyde, a sexual and alarm pheromone for adult mites, which in the other matrices appeared as a trace component. The number of mites detected in oat grains was correlated to the GC peak area of the pheromone. The HS-SPME/GC×GC-ToFMS methodology can be regarded as the basis for the development of a rapid and versatile method that can be applied in industry to the simultaneous assessment the level of microbiological contamination and for detection of mites in cereals grains and coffee beans.

To understand the functioning of sponges, knowledge of the structure of their associated microbial communities is necessary. However, our perception of sponge-associated microbiomes remains mainly restricted to marine ecosystems. Here, we report on the molecular diversity and composition of bacteria in the freshwater sponge Ephydatia fluviatilis inhabiting the artificial lake Vinkeveense Plassen, Utrecht, The Netherlands. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints revealed that the apparent diversities within the domain Bacteria and the phylum Actinobacteria were lower in E. fluviatilis than in bulk water. Enrichment of specific PCR-DGGE bands in E. fluviatilis was detected. Furthermore, sponge- and bulk water-derived bacterial clone libraries differed with respect to bacterial community composition at the phylum level. E. fluviatilis-derived sequences were affiliated with six recognized phyla, i.e., Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes, Chlamydiae and Verrucomicrobia, in order of relative abundance; next to the uncultured candidate phylum TM7 and one deeply rooted bacterial lineage of undefined taxonomy (BLUT). Actinobacteria, Proteobacteria, and Bacteroidetes were the dominant bacterial phyla in the freshwater clone library whereas sequences affiliated with Planctomycetes, Verrucomicrobia, Acidobacteria and Armatimonadetes were found at lower frequencies. Fine-tuned phylogenetic inference showed no or negligible overlaps between the E. fluviatilis and water-derived phylotypes within bacterial taxa such as Alphaproteobacteria, Bacteroidetes and Actinobacteria. We also ascertained the status of two alphaproteobacterial lineages as freshwater sponge-specific phylogenetic clusters, and report on high distinctiveness of other E. fluviatilis specific phylotypes, especially within the Bacteroidetes, Planctomycetes and Chlamydia taxa. This study supports the contention that the composition and diversity of bacteria in E. fluviatilis is partially driven by the host organism.

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.

Bacteria from the surface microlayer (bacterioneuston) and underlying waters (bacterioplankton) were isolated upon exposure to UV-B radiation, and their individual UV sensitivity in terms of CFU numbers, activity (leucine and thymidine incorporation), sole-carbon source use profiles, repair potential (light-dependent and independent), and photoadaptation potential, under different physiological conditions, was compared. Colony counts were 11.5-16.2% more reduced by UV-B exposure in bacterioplankton isolates (P < 0.05). Inhibition of leucine incorporation in bacterioneuston isolates was 10.9-11.5% higher than in bacterioplankton (P < 0.05). These effects were accompanied by a shift in sole-carbon source use profiles, assessed with Biolog(®) EcoPlates, with a reduction in consumption of amines and amino acids and increased use of polymers, particularly in bacterioneuston isolates. Recovery under starvation was generally enhanced compared with nourished conditions, especially in bacterioneuston isolates. Overall, only insignificant increases in the induction of antibiotic resistant mutant phenotypes (Rif(R) and Nal(R) ) were observed. In general, a potential for photoadaptation could not be detected among the tested isolates. These results indicate that UV effects on bacteria are influenced by their physiological condition and are accompanied by a shift in metabolic profiles, more significant in bacterioneuston isolates, suggesting the presence of bacterial strains adapted to high UV levels in the SML., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Bioremediation efforts often rely on the application of surfactants to enhance hydrocarbon bioavailability. However, synthetic surfactants can sometimes be toxic to degrading microorganisms, thus reducing the clearance rate of the pollutant. Therefore, surfactant-resistant bacteria can be an important tool for bioremediation efforts of hydrophobic pollutants, circumventing the toxicity of synthetic surfactants that often delay microbial bioremediation of these contaminants. In this study, we screened a natural surfactant-rich compartment, the estuarine surface microlayer (SML), for cultivable surfactant-resistant bacteria using selective cultures of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB). Resistance to surfactants was evaluated by colony counts in solid media amended with critical micelle concentrations (CMC) of either surfactants, in comparison with non-amended controls. Selective cultures for surfactant-resistant bacteria were prepared in mineral medium also containing CMC concentrations of either CTAB or SDS. The surfactantresistant isolates obtained were tested by PCR for the Pseudomonas genus marker gacA gene and for the naphthalene-dioxygenase-encoding gene ndo. Isolates were also screened for biosurfactant production by the atomized oil assay. A high proportion of culturable bacterioneuston was tolerant to CMC concentrations of SDS or CTAB. The gacA-targeted PCR revealed that 64% of the isolates were Pseudomonads. Biosurfactant production in solid medium was detected in 9.4% of tested isolates, all affiliated with genus Pseudomonas. This study shows that the SML is a potential source of surfactant-resistant and biosurfactant-producing bacteria in which Pseudomonads emerge as a relevant group.

The effects of UV radiation (UVR) on estuarine bacterioneuston and bacterioplankton were assessed in microcosm experiments. Bacterial abundance and DNA synthesis were more affected in bacterioplankton. Protein synthesis was more inhibited in bacterioneuston. Community analysis indicated that UVR has the potential to select resistant bacteria (e.g., Gammaproteobacteria), particularly abundant in bacterioneuston.

It is acknowledged that marine invertebrates produce bioactive natural products that may be useful for developing new drugs. By exploring untapped geographical sources and/or novel groups of organisms one can maximize the search for new marine drugs to treat human diseases. The goal of this paper is to analyse the trends associated with the discovery of new marine natural products from invertebrates (NMNPI) over the last two decades. The analysis considers different taxonomical levels and geographical approaches of bioprospected species. Additionally, this research is also directed to provide new insights into less bioprospected taxa and world regions. In order to gather the information available on NMNPI, the yearly-published reviews of Marine Natural Products covering 1990-2009 were surveyed. Information on source organisms, specifically taxonomical information and collection sites, was assembled together with additional geographical information collected from the articles originally describing the new natural product. Almost 10000 NMNPI were discovered since 1990, with a pronounced increase between decades. Porifera and Cnidaria were the two dominant sources of NMNPI worldwide. The exception was polar regions where Echinodermata dominated. The majority of species that yielded the new natural products belong to only one class of each Porifera and Cnidaria phyla (Demospongiae and Anthozoa, respectively). Increased bioprospecting efforts were observed in the Pacific Ocean, particularly in Asian countries that are associated with the Japan Biodiversity Hotspot and the Kuroshio Current. Although results show comparably less NMNPI from polar regions, the number of new natural products per species is similar to that recorded for other regions. The present study provides information to future bioprospecting efforts addressing previously unexplored taxonomic groups and/or regions. We also highlight how marine invertebrates, which in some cases have no commercial value, may become highly valuable in the ongoing search for new drugs from the sea.

Here, we use DGGE fingerprinting and barcoded pyrosequencing data, at six cut-off levels (85-100%), of all bacteria, Alphaproteobacteria and Betaproteobacteria to assess composition in the rhizosphere of nursery plants and nursery-raised transplants, native plants and bulk sediment in a mangrove habitat. When comparing compositional data based on DGGE fingerprinting and barcoded pyrosequencing at different cut-off levels, all revealed highly significant differences in composition among microhabitats. Procrustes superimposition revealed that ordination results using cut-off levels from 85-100% and DGGE fingerprint data were highly congruent with the standard 97% cut-off level. The various approaches revealed a primary gradient in composition from nursery to mangrove samples. The affinity between the nursery and transplants was greatest when using Betaproteobacteria followed by Alphaproteobacteria data. There was a distinct secondary gradient in composition from transplants to bulk sediment with native plants intermediate, which was most prevalent using all bacteria at intermediate cut-off levels (92-97%). Our results show that PCR-DGGE provides a robust and cost effective exploratory approach and is effective in distinguishing among a priori defined groups.

Bacterial communities of the surface microlayer (SML) of the estuary Ria de Aveiro (Portugal) were characterized in terms of abundance and activity during a 2-year survey at two sites with distinct hydrodynamic properties (marine and brackish water zones). The hydrodynamic conditions were simulated using a bidimensional numerical model and related to the microbiological observations. The pattern of variation of bacterial biomass productivity (BBP) was distinct between the two sampling sites. At the outer site, BBP was significantly lower at the SML, whereas at the inner site, it was significantly enhanced at the SML. Although the total bacterial abundance was similar in the SML and underlying water (UW), the fraction of cells attached to particles was significantly higher at the SML (two to three times). The integration of microbiological results with environmental and hydrological variables shows that strong currents in the marine zone promote the vertical mixing, inhibiting the establishment of an SML bacterial community distinct from that of UW. In contrast, in the brackish water zone, lower current velocities provide conditions for enhancing the bacterial activity in the enriched SML. Estuarine dynamics influence the distribution and activity of microorganisms at the SML and in the water column, with anticipated impacts for the carbon cycle in the estuarine environment., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Mangroves are complex and dynamic ecosystems varying in salinity, water level and nutrient availability; they also contain diverse and distinct microbial communities. Studies of microbes and their interactions with other ecosystem components (e.g., tree roots) are critical for our understanding of mangrove ecosystem functioning and remediation. Using a barcoding pyrosequencing approach, we previously noted the persistence of terrestrial bacterial populations on mangrove roots when nursery raised saplings were transplanted back to their natural environment. Here we go into further detail about the potential functional associations of bacterial guilds with distinct mangrove microhabitats including the rhizosphere. We also use a nonparametric richness estimator to show that estimated operational taxonomic unit (OTU) richness is more than twice that observed. In the transplant microhabitat, our estimate suggests that there are almost 7,000 OTU's for a sample size of 10,400 individual sequences with no sign of an asymptote, indicating that "true" richness for this microhabitat is substantially larger. Results on the number of bacterial OTU's should, however, be viewed with caution given that the barcoding pyrosequencing technique used can yield sequencing artifacts that may inflate richness estimates if not properly removed.

Background: Photodynamic antimicrobial chemotherapy (PACT) combines light, a light-absorbing molecule that initiates a photochemical or photophysical reaction, and oxygen. The combined action of these three components originates reactive oxygen species that lead to microorganisms' destruction. The aim was to evaluate the efficiency of PACT on Vibrio fischeri: 1) with buffer solution, varying temperature, pH, salinity and oxygen concentration values; 2) with aquaculture water, to reproduce photoinactivation (PI) conditions in situ., Methodology/principal Findings: To monitor the PI kinetics, the bioluminescence of V. fischeri was measured during the experiments. A tricationic meso-substituted porphyrin (Tri-Py(+)-Me-PF) was used as photosensitizer (5 µM in the studies with buffer solution and 10-50 µM in the studies with aquaculture water); artificial white light (4 mW cm(-2)) and solar irradiation (40 mW cm(-2)) were used as light sources; and the bacterial concentration used for all experiments was ≈10(7) CFU mL(-1) (corresponding to a bioluminescence level of 10(5) relative light units--RLU). The variations in pH (6.5-8.5), temperature (10-25°C), salinity (20-40 g L(-1)) and oxygen concentration did not significantly affect the PI of V. fischeri, once in all tested conditions the bioluminescent signal decreased to the detection limit of the method (≈7 log reduction). The assays using aquaculture water showed that the efficiency of the process is affected by the suspended matter. Total PI of V. fischeri in aquaculture water was achieved under solar light in the presence of 20 µM of Tri-Py(+)-Me-PF., Conclusions/significance: If PACT is to be used in environmental applications, the matrix containing target microbial communities should be previously characterized in order to establish an efficient protocol having into account the photosensitizer concentration, the light source and the total light dose delivered. The possibility of using solar light in PACT to treat aquaculture water makes this technology cost-effective and attractive.

Objectives: The length of stay after prolonged cardiac surgery has been associated with poor immediate outcomes and increased costs. This study aimed to evaluate the predictive power of the Ambler Score to anticipate the length of stay in the intensive care unit., Methods: This was a retrospective cohort study based on data collected from 110 patients undergoing valve replacement surgery alone or in combination with other procedures. Additive and logistic Ambler Scores were obtained and their predictive performances calculated using the Receiver Operating Characteristic curve. The normal length stay in the intensive care unit was assumed to be <3 days and prolonged >3 days. The areas under the receiver operating curves for both the additive and logistic models were compared using the Hanley-MacNeil test., Results: The mean intensive care unit length of stay was 4.2 days. Sixty-three patients were male. The logistic model showed areas under the receiver operating characteristic curve of 0.73 and 0.79 for hospitalization > 3 days and < 3 days, respectively, showing good discriminative power. For the additive model, the areas were 0.63 and 0.59 for hospitalization > 3 days and < 3 days, respectively, a poor discriminative power., Conclusions: In our database, prolonged length of stay in the intensive care unit was positively correlated with the logistic Ambler score. The performance of the logistic Ambler Score had good discriminative power for correlation with the intensive care unit length of stay.

Background: Mangrove forests are of global ecological and economic importance, but are also one of the world's most threatened ecosystems. Here we present a case study examining the influence of the rhizosphere on the structural composition and diversity of mangrove bacterial communities and the implications for mangrove reforestation approaches using nursery-raised plants., Methodology/principal Findings: A barcoded pyrosequencing approach was used to assess bacterial diversity in the rhizosphere of plants in a nursery setting, nursery-raised transplants and native (non-transplanted) plants in the same mangrove habitat. In addition to this, we also assessed bacterial composition in the bulk sediment in order to ascertain if the roots of mangrove plants affect sediment bacterial composition. We found that mangrove roots appear to influence bacterial abundance and composition in the rhizosphere. Due to the sheer abundance of roots in mangrove habitat, such an effect can have an important impact on the maintenance of bacterial guilds involved in nutrient cycling and other key ecosystem functions. Surprisingly, we also noted a marked impact of initial nursery conditions on the rhizosphere bacterial composition of replanted mangrove trees. This result is intriguing because mangroves are periodically inundated with seawater and represent a highly dynamic environment compared to the more controlled nursery environment., Conclusions/significance: In as far as microbial diversity and composition influences plant growth and health, this study indicates that nursery conditions and early microbial colonization patterns of the replants are key factors that should be considered during reforestation projects. In addition to this, our results provide information on the role of the mangrove rhizosphere as a habitat for bacteria from estuarine sediments.

In this study, the combination of culture enrichments and molecular tools was used to identify bacterial guilds, plasmids and functional genes potentially important in the process of petroleum hydrocarbon (PH) decontamination in mangrove microniches (rhizospheres and bulk sediment). In addition, we aimed to recover PH-degrading consortia (PHDC) for future use in remediation strategies. The PHDC were enriched with petroleum from rhizosphere and bulk sediment samples taken from a mangrove chronically polluted with oil hydrocarbons. Southern blot hybridization (SBH) assays of PCR amplicons from environmental DNA before enrichments resulted in weak positive signals for the functional gene types targeted, suggesting that PH-degrading genotypes and plasmids were in low abundance in the rhizosphere and bulk sediments. However, after enrichment, these genes were detected and strong microniche-dependent differences in the abundance and composition of hydrocarbonoclastic bacterial populations, plasmids (IncP-1α, IncP-1β, IncP-7 and IncP-9) and functional genes (naphthalene, extradiol and intradiol dioxygenases) were revealed by in-depth molecular analyses [PCR-denaturing gradient gel electrophoresis and hybridization (SBH and microarray)]. Our results suggest that, despite the low abundance of PH-degrading genes and plasmids in the environmental samples, the original bacterial composition of the mangrove microniches determined the structural and functional diversity of the PHDC enriched., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The effects of sewage sludge selectively enriched with Cd and Zn, both singly and in combination, on the bacterial, fungal, Alphaproteobacteria and Actinobacteria communities of a soil under arable or grassland management were studied with a PCR-DGGE approach. The effects of Cd and Zn were evaluated after a short time (7 d) when the Cd and Zn solubility were low and the C availability was high, and again after 180 d when the labile sludge C was mineralized and the effects of heavy metals predominated. In the arable soil all treatments induced significant short-term changes in the studied microbial groups, and long-term changes were observed in Actinobacteria and fungal communities. In the grassland soil, all treatments induced significant short-term changes in the studied microbial groups except for Alphaproteobacteria and fungi, and long-term effects on the actinobacteria and fungal communities. It was concluded that incorporation of Cd- and Zn-rich sludge into soils may have both short- and long-term effects on various bacterial phylogenetic groups whereas the metals may be better tolerated by the dominant soil fungi. In this study the impact was greater in arable than in grassland soil., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The aim of this study was to understand if two species of salt marsh plants, widely distributed in European estuaries (Spartina maritima and Halimione portulacoides) differently influence the distribution, activity, and metabolic physiology of sediment bacterial communities in monospecific banks, in comparison with uncolonized sediment (control). Microbiological descriptors of abundance and activity were assessed along vertical profiles of sediments. Rates of activity of the extracellular enzymes beta-glucosidase, alpha-glucosidase, aminopeptidase, arylsulfatase, and phosphatase were generally higher in the vegetation banks in relation to control sediments where they were also less variable with depth. This is interpreted as an indirect effect related to supply of plant-derived polymeric substrates for bacterial growth. Parameters related to sediment texture (grain size, percent of fines or water content) showed significant relations with cell abundance or maximum hydrolysis rates, pointing to an indirect effect of plant colonization exerted through the modification of sediment physical properties. The profiles of utilization of sole-carbon-source (Biolog Ecoplates) showed that only the communities from the upper sediment layer of the S. maritima and the H. portulacoides banks exhibit consistent differences in terms of physiological profiles. Bacterial communities in control sediments exhibited the lowest physiological variability between surface and sub-surface communities. The results indicate that microbial colonization and organic matter decomposition are enhanced under the influence of salt marsh plants and confirm that plant coverage is a major determinant of the processes of organic matter recycling in intertidal estuarine sediments.

Antimicrobial photodynamic therapy (aPDT) has emerged in the clinical field as a potential alternative to antibiotics to treat microbial infections. No cases of microbial viability recovery or any resistance mechanisms against it are yet known. 5,10,15-tris(1-Methylpyridinium-4-yl)-20-(pentafluorophenyl)-porphyrin triiodide (Tri-Py(+)-Me-PF) was used as photosensitizer. Vibrio fischeri and recombinant Escherichia coli were the studied bacteria. To determine the bacterial recovery after treatment, Tri-Py(+)-Me-PF (5.0 microM) was added to bacterial suspensions and the samples were irradiated with white light (40 W m(-2)) for 270 minutes. Then, the samples were protected from light, aliquots collected at different intervals and the bioluminescence measured. To assess the development of resistance after treatment, bacterial suspensions were exposed to white light (25 minutes), in presence of 5.0 microM of Tri-Py(+)-Me-PF (99.99% of inactivation) and plated. After the first irradiation period, surviving colonies were collected from the plate and resuspended in PBS. Then, an identical protocol was used and repeated ten times for each bacterium. The results suggest that aPDT using Tri-Py(+)-Me-PF represents a promising approach to efficiently destroy bacteria since after a single treatment these microorganisms do not recover their viability and after ten generations of partially photosensitized cells neither of the bacteria develop resistance to the photodynamic process.

Owing to the increasing importance of aquaculture to compensate for the progressive worldwide reduction of natural fish and to the fact that several fish farming plants often suffer from heavy financial losses due to the development of infections caused by microbial pathogens, including multidrug resistant bacteria, more environmentally-friendly strategies to control fish infections are urgently needed to make the aquaculture industry more sustainable. The aim of this review is to briefly present the typical fish farming diseases and their threats and discuss the present state of chemotherapy to inactivate microorganisms in fish farming plants as well as to examine the new environmentally friendly approaches to control fish infection namely phage therapy and photodynamic antimicrobial therapy.

The effects of genetically modified (GM), zeaxanthin-accumulating potato plants on microbial communities in the rhizosphere were compared to the effects of different potato cultivars. Two GM lines and their parental cultivar, as well as four other potato cultivars, were grown in randomized field plots at two sites and in different years. Rhizosphere samples were taken at three developmental stages during plant growth and analyzed using denaturing gradient gel electrophoresis (DGGE) fingerprints of Bacteria, Actinobacteria, Alpha- and Betaproteobacteria, Bacillus, Streptomycetaceae, Pseudomonas, gacA, Fungi, and Ascomycetes. In the bacterial DGGE gels analyzed, significant differences between the parental cultivar and the two GM lines were detected mainly for Actinobacteria but also for Betaproteobacteria and Streptomycetaceae, yet these differences occurred only at one site and in one year. Significant differences occurred more frequently for Fungi, especially Ascomycetes, than for bacteria. When all seven plant genotypes were compared, DGGE analysis revealed that different cultivars had a greater effect on both bacterial and fungal communities than genetic modification. The effects of genetic modification were detected mostly at the senescence developmental stage of the plants. The site was the overriding factor affecting microbial community structure compared to the plant genotype. In general, the fingerprints of the two GM lines were more similar to that of the parental cultivar, and the differences observed did not exceed natural cultivar-dependent variability.

Municipal sewage, urban runoff and accidental oil spills are common sources of pollutants in urban mangrove forests and may have drastic effects on the microbial communities inhabiting the sediment. However, studies on microbial communities in the sediment of urban mangroves are largely lacking. In this study, we explored the diversity of bacterial communities in the sediment of three urban mangroves located in Guanabara Bay (Rio de Janeiro, Brazil). Analysis of sediment samples by means of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments suggested that the overall bacterial diversity was not significantly affected by the different levels of hydrocarbon pollution at each sampling site. However, DGGE and sequence analyses provided evidences that each mangrove sediment displayed a specific structure bacterial community. Although primer sets for Pseudomonas, alphaproteobacterial and actinobacterial groups also amplified ribotypes belonging to taxa not intended to be enriched, sequence analyses of dominant DGGE bands revealed ribotypes related to Alteromonadales, Burkholderiales, Pseudomonadales, Rhodobacterales and Rhodocyclales. Members of these groups were often shown to be involved in aerobic or anaerobic degradation of hydrocarbon pollutants. Many of these sequences were only detected in the sampling sites with high levels of anthropogenic inputs of hydrocarbons. Many dominant DGGE ribotypes showed low levels of sequence identity to known sequences, indicating a large untapped bacterial diversity in mangrove ecosystems.

Antibiotic-resistant bacteria, nutrients and antibiotics that enter the soil by means of manure may enhance the proportion of bacteria displaying antibiotic resistance among soil bacteria and may affect bacterial community structure and function. To investigate the effect of manure and amoxicillin added to manure on soil bacterial communities, microcosm experiments were performed with two soil types and the following treatments: (1) nontreated, (2) manure-treated, (3) treated with manure supplemented with 10 mg amoxicillin kg(-1) soil and (4) treated with manure supplemented with 100 mg amoxicillin kg(-1) soil, with four replicates per treatment. Manure significantly increased the total CFU count and the amoxicillin-resistant CFU count of both soil types. However, only the soil with a history of manure treatment showed a significant increase in the relative number of amoxicillin-resistant bacteria as a result of amoxicillin amendment. The majority of plasmids exogenously isolated from soil originated from soil treated with amoxicillin-supplemented manure. All 16 characterized plasmids carried the bla-TEM gene, and 10 of them belonged to the IncN group. The bla-TEM gene was detected in DNA directly extracted from soil by dot-blot hybridization of PCR amplicons and showed an increased abundance in soil samples treated with manure. Molecular fingerprint analysis of 16S rRNA gene fragments amplified from soil DNA revealed significant effects of manure and amoxicillin on the bacterial community of both soils.

Polycyclic aromatic hydrocarbon (PAH) pollutants originating from oil spills and wood and fuel combustion are pollutants which are among the major threats to mangrove ecosystems. In this study, the composition and relative abundance in the sediment bacterial communities of naphthalene dioxygenase (ndo) genes which are important for bacterial adaptation to environmental PAH contamination were investigated. Three urban mangrove sites which had characteristic compositions and levels of PAH compounds in the sediments were selected. The diversity and relative abundance of ndo genes in total community DNA were assessed by a newly developed ndo denaturing gradient gel electrophoresis (DGGE) approach and by PCR amplification with primers targeting ndo genes with subsequent Southern blot hybridization analyses. Bacterial populations inhabiting sediments of urban mangroves under the impact of different sources of PAH contamination harbor distinct ndo genotypes. Sequencing of cloned ndo amplicons comigrating with dominant DGGE bands revealed new ndo genotypes. PCR-Southern blot analysis and ndo DGGE showed that the frequently studied nah and phn genotypes were not detected as dominant ndo types in the mangrove sediments. However, ndo genotypes related to nagAc-like genes were detected, but only in oil-contaminated mangrove sediments. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the abundance and diversity of ndo genes in sediments of urban mangroves.

The survival and colonization patterns of Pseudomonas putida PRD16 and Enterobacter cowanii PRF116 in the rhizosphere of greenhouse-grown tomato plants and the effects of their inoculation on the indigenous bacterial community were followed by selective plating, molecular fingerprinting, and confocal laser scanning microscopy (CLSM) over 3 weeks. Both strains, which showed in vitro antagonistic activity against Ralstonia solanacearum, were previously tagged with gfp. Seed and root inoculation were compared. Although plate counts decreased for both gfp-tagged antagonists, PRD16 showed a better survival in the rhizosphere of tomato roots independent of the inoculation method. Analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and CLSM confirmed the decrease in the relative abundance of the inoculant strains. Pronounced differences in the Pseudomonas community patterns for plants inoculated with PRD16 compared to the control were detected 3 weeks after root inoculation, indicating a longer-lasting effect. Analysis by CLSM showed rather heterogeneous colonization patterns for both inoculant strains. In comparison with seed inoculation, root inoculation led to a much better colonization as evidenced by all three methods. The colonization patterns observed by CLSM provide important information on the sampling strategy required for monitoring inoculant strains in the rhizosphere.

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.

The fungal population dynamics in soil and in the rhizospheres of two maize cultivars grown in tropical soils were studied by a cultivation-independent analysis of directly extracted DNA to provide baseline data. Soil and rhizosphere samples were taken from six plots 20, 40, and 90 days after planting in two consecutive years. A 1.65-kb fragment of the 18S ribosomal DNA (rDNA) amplified from the total community DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and by cloning and sequencing. A rhizosphere effect was observed for fungal populations at all stages of plant development. In addition, pronounced changes in the composition of fungal communities during plant growth development were found by DGGE. Similar types of fingerprints were observed in two consecutive growth periods. No major differences were detected in the fungal patterns of the two cultivars. Direct cloning of 18S rDNA fragments amplified from soil or rhizosphere DNA resulted in 75 clones matching 12 dominant DGGE bands. The clones were characterized by their HinfI restriction patterns, and 39 different clones representing each group of restriction patterns were sequenced. The cloning and sequencing approach provided information on the phylogeny of dominant amplifiable fungal populations and allowed us to determine a number of fungal phylotypes that contribute to each of the dominant DGGE bands. Based on the sequence similarity of the 18S rDNA fragment with existing fungal isolates in the database, it was shown that the rhizospheres of young maize plants seemed to select the Ascomycetes order Pleosporales, while different members of the Ascomycetes and basidiomycetic yeast were detected in the rhizospheres of senescent maize plants.

As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S.cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.