Scielo RSS <![CDATA[Brazilian Journal of Microbiology]]> vol. 45 num. 1 lang. pt <![CDATA[SciELO Logo]]> <![CDATA[<b>An overview of antimicrobial resistance and its public health significance</b>]]> Multiple papers have been published regarding the bacterial resistance theme over the last years. A variety of information has reached general and scientific public, daily bringing up data on new resistant microorganisms, new drugs, outbreaks, epidemiological news, resistance gene dissemination, and the lack of information in a particular field has caught our attention: the public health department. Most of researchers, physicians and government employees interpret the public health field as a separate department, not linked to this antibiotic resistance era that we are living nowadays. In this paper we carefully tried to fill in the blanks between public health and the bacteria resistance issue, also considering historical, social, economical and biological problematic that come with this possible pre-antibiotic era. <![CDATA[<b>PCR and ELISA (VIDAS ECO O157<sup>®</sup>) <i>Escherichia coli </i>O157:H7 identification in <i>Minas Frescal </i>cheese commercialized in Goiânia, GO</b>]]> Escherichia coli O157:H7 has been incriminated in food poisoning outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in many countries. Considering the high susceptibility of Minas Frescal cheese to contamination by E. coli O157:H7, the aim of this study was to determine the occurrence of this pathogen through PCR (Polymerase Chain Reaction) and ELISA (VIDAS ECO O157®, bioMérieux, Lyon, France) test. Thirty cheese samples manufactured by artisan farmhouse producers were collected from open-air markets in Goiânia and thirty from industries under Federal Inspection located in Goiás State which trade their products in supermarkets in Goiânia. E. coli O157:H7 was detected in 6.67% samples collected in open air markets using ELISA, and 23,33% with PCR. The pathogen was not detected in samples from industries under Federal Inspection. <![CDATA[<b>Control of <i>Listeria monocytogenes </i>growth in soft cheeses by bacteriophage P100</b>]]> The purpose of this study was to determine the effect of bacteriophage P100 on strains of Listeria monocytogenes in artificially inoculated soft cheeses. A mix of L. monocytogenes 1/2a and Scott A was inoculated in Minas Frescal and Coalho cheeses (approximately 10(5) cfu/g) with the bacteriophage added thereafter (8.3 x 10(7) PFU/g). Samples were analyzed immediately, and then stored at 10 ºC for seven days. At time zero, 30 min post-infection, the bacteriophage P100 reduced L. monocytogenes counts by 2.3 log units in Minas Frescal cheese and by 2.1 log units in Coalho cheese, compared to controls without bacteriophage. However, in samples stored under refrigeration for seven days, the bacteriophage P100 was only weakly antilisterial, with the lowest decimal reduction (DR) for the cheeses: 1.0 log unit for Minas Frescal and 0.8 log units for Coalho cheese. The treatment produced a statistically significant decrease in the counts of viable cells (p < 0.05) and in all assays performed, we observed an increase of approximately one log cycle in the number of viable cells of L. monocytogenes in the samples under refrigeration for seven days. Moreover, a smaller effect of phages was observed. These results, along with other published data, indicate that the effectiveness of the phage treatment depends on the initial concentration of L. monocytogenes, and that a high concentration of phages per unit area is required to ensure sustained inactivation of target pathogens on food surfaces. <![CDATA[<b>Occurrence of <i>Salmonella </i>spp. and generic <i>Escherichia coli </i>on beef carcasses sampled at a brazilian slaughterhouse</b>]]> A total of 120 beef carcasses were analyzed during processing at a slaughterhouse in southern Brazil. The carcasses were sampled by swab at three different steps of the slaughter line and then they were tested for Salmonella and E. coli. The Salmonella isolates were also examined for antimicrobial susceptibility. Salmonella prevalence distribution was modeled and the probability of contamination was simulated using @Risk program and 10,000 interactions. Results demonstrated that 4 beef carcasses (3.3%) were positive for Salmonella only in the first point. The six isolates of Salmonella were classified: S. Newport (n = 3), S. Saintpaul (n = 2) and S. Anatum (n = 1). No Salmonella strains exhibited resistance to any of the antimicrobials tested. As expected, the most contaminated point with E. coli was the first point (hide), presenting counts from 0.31 to 5.07 log cfu/100 cm². Much smaller E. coli counts were observed in the other points. Results indicated low levels of Salmonella and E. coli on the beef carcasses analyzed and also low probability of contamination of the carcasses by Salmonella, suggesting adequate microbiological quality. <![CDATA[<b>Antibiotic resistance in lactic acid bacteria isolated from some pharmaceutical and dairy products</b>]]> A total of 244 lactic acid bacteria (LAB) strains were isolated from 180 dairy and pharmaceutical products that were collected from different areas in Minia governorate, Egypt. LAB were identified phenotypically on basis of morphological, physiological and biochemical characteristics. Lactobacillus isolates were further confirmed using PCR-based assay. By combination of phenotypic with molecular identification Lactobacillus spp. were found to be the dominant genus (138, 76.7%) followed by Streptococcus spp. (65, 36.1%) and Lactococcus spp. (27, 15%). Some contaminant organisms such as (Staphylococcus spp., Escherichia coli, Salmonella spp., mould and yeast) were isolated from the collected dairy samples but pharmaceutical products were free of such contaminants. Susceptibility of LAB isolates to antibiotics representing all major classes was tested by agar dilution method. Generally, LAB were highly susceptible to Beta-lactams except penicillin. Lactobacilli were resistant to vancomycin, however lactococci and streptococci proved to be very susceptible. Most strains were susceptible to tetracycline and showed a wide range of streptomycin MICs. The MICs of erythromycin and clindamycin for most of the LAB were within the normal range of susceptibility. Sixteen Lactobacillus,8 Lactococcus and 8 Streptococcus isolates including all tetracycline and/or erythromycin resistant strains were tested for the presence of tetracycline and/or erythromycin resistant genes [tet(M) and/or erm(B)]. PCR assays shows that some resistant strains harbor tet(M) and/or erm(B) resistance genes. <![CDATA[<b>Comparison of the efficiency between two sampling plans for aflatoxins analysis in maize</b>]]> Variance and performance of two sampling plans for aflatoxins quantification in maize were evaluated. Eight lots of maize were sampled using two plans: manual, using sampling spear for kernels; and automatic, using a continuous flow to collect milled maize. Total variance and sampling, preparation, and analysis variance were determined and compared between plans through multifactor analysis of variance. Four theoretical distribution models were used to compare aflatoxins quantification distributions in eight maize lots. The acceptance and rejection probabilities for a lot under certain aflatoxin concentration were determined using variance and the information on the selected distribution model to build the operational characteristic curves (OC). Sampling and total variance were lower at the automatic plan. The OC curve from the automatic plan reduced both consumer and producer risks in comparison to the manual plan. The automatic plan is more efficient than the manual one because it expresses more accurately the real aflatoxin contamination in maize. <![CDATA[<b>Methods to preserve potentially toxigenic fungi</b>]]> Microorganisms are a source of many high-value compounds which are useful to every living being, such as humans, plants and animals. Since the process of isolating and improving a microorganism can be lengthy and expensive, preserving the obtained characteristic is of paramount importance, so the process does not need to be repeated. Fungi are eukaryotic, achlorophyllous, heterotrophic organisms, usually filamentous, absorb their food, can be either macro or microscopic, propagate themselves by means of spores and store glycogen as a source of storage. Fungi, while infesting food, may produce toxic substances such as mycotoxins. The great genetic diversity of the Kingdom Fungi renders the preservation of fungal cultures for many years relevant. Several international reference mycological culture collections are maintained in many countries. The methodologies that are most fit for preserving microorganisms for extended periods are based on lowering the metabolism until it reaches a stage of artificial dormancy . The goal of this study was to analyze three methods for potentially toxigenic fungal conservation (Castellani's, continuous subculture and lyophilization) and to identify the best among them. <![CDATA[<b>Survey of molds, yeast and <i>Alicyclobacillus </i>spp. from a concentrated apple juice productive process</b>]]> Bacteria and molds may spoil and/or contaminate apple juice either by direct microbial action or indirectly by the uptake of metabolites as off-flavours and toxins. Some of these microorganisms and/or metabolites may remain in the food even after extensive procedures. This study aim to identify the presence of molds (including heat resistant species) and Alicyclobacillus spp., during concentrated apple juice processing. Molds were isolated at different steps and then identified by their macroscopic and microscopic characteristics after cultivation on standard media at 5, 25 and 37ºC, during 7 days. Among the 19 isolated found, 63% were identified as Penicillium with 50% belonging to the P. expansum specie. With regards to heat resistant molds, the species Neosartorya fischeri, Byssochlamys fulva and also the genus Eupenicillium sp., Talaromyces sp. and Eurotium sp. were isolated. The thermoacidophilic spore-forming bacteria were identified as A. acidoterrestris by a further investigation based on 16S rRNA sequence similarity. The large contamination found indicates the need for methods to eliminate or prevent the presence of these microorganisms in the processing plants in order to avoid both spoilage of apple juice and toxin production. <![CDATA[<b>Simultaneous and successive inoculations of yeasts and lactic acid bacteria on the fermentation of an unsulfited Tannat grape must</b>]]> Interactions between yeasts and lactic acid bacteria are strain specific, and their outcome is expected to change in simultaneous alcoholic -malolactic fermentations from the pattern observed in successive fermentations. One Oenococcus oeni strain Lalvin VP41TM was inoculated with two Saccharomyces cerevisiae strains either simultaneously, three days after the yeast inoculation, or when alcoholic fermentation was close to finish. Early bacterial inoculations with each yeast strain allowed for the growth of the bacterial populations, and the length of malolactic fermentation was reduced to six days. Alcoholic fermentation by Lalvin ICV D80® yeast strain left the highest residual sugar, suggesting a negative effect of the bacterial growth and malolactic activity on its performance. In sequential inoculations the bacterial populations did not show actual growth with either yeast strain. In this strategy, both yeast strains finished the alcoholic fermentations, and malolactic fermentations took longer to finish. Lalvin ICV D80® allowed for higher viability and activity of the bacterial strain than Fermicru UY4® under the three inoculation strategies. This was beneficial for the sequential completion of both fermentations, but negatively affected the completion of alcoholic fermentation by Lalvin ICV D80® in the early bacteria additions. Conversely, Fermicru UY4®, which was rather inhibitory towards the bacteria, favored the timely completion of both fermentations simultaneously. As bacteria in early inoculations with low or no SO2 addition can be expected to multiply and interact with fermenting yeasts, not only are the yeast-bacterium strains combination and time point of the inoculation to be considered, but also the amount of bacteria inoculated. <![CDATA[<b>Biofilm formation by <i>Staphylococcus aureus </i>from food contact surfaces in a meat-based broth and sensitivity to sanitizers</b>]]> This study assessed the capacity of adhesion, the detachment kinetic and the biofilm formation by Staphylococcus aureus isolated from food services on stainless steel and polypropylene surfaces (2 x 2 cm) when cultivated in a meat-based broth at 28 and 7 ºC. It was also to study the efficacy of the sanitizers sodium hypochlorite (250 mg/L) and peracetic acid (30 mg/L) in inactivating the bacterial cells in the preformed biofilm. S. aureus strains adhered in high numbers regardless the assayed surface kind and incubation temperature over 72 h. Cells detachment of surfaces revealed high persistence over the incubation period. Number of cells needed for biofilm formation was noted at all experimental systems already after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered on polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacity to adhere and form biofilm on polypropylene and stainless steel surfaces under different growth conditions. Moreover, the cells in biofilm matrix were resistant for total removal when submitted to the exposure to sanitizers. <![CDATA[<b><i>Campylobacter jejuni </i></b><b>in commercial eggs</b>]]> This study evaluated the ability of Campylobacter jejuni to penetrate through the pores of the shells of commercial eggs and colonize the interior of these eggs, which may become a risk factor for human infection. Furthermore, this study assessed the survival and viability of the bacteria in commercial eggs. The eggs were placed in contact with wood shavings infected with C. jejuni to check the passage of the bacteria. In parallel, the bacteria were inoculated directly into the air chamber to assess the viability in the egg yolk. To determine whether the albumen and egg fertility interferes with the entry and survival of bacteria, we used varying concentrations of albumen and SPF and commercial eggs. C. jejuni was recovered in SPF eggs (fertile) after three hours in contact with contaminated wood shavings but not in infertile commercial eggs. The colonies isolated in the SPF eggs were identified by multiplex PCR and the similarity between strains verified by RAPD-PCR. The bacteria grew in different concentrations of albumen in commercial and SPF eggs. We did not find C. jejuni in commercial eggs inoculated directly into the air chamber, but the bacteria were viable during all periods tested in the wood shavings. This study shows that consumption of commercial eggs infected with C. jejuni does not represent a potential risk to human health. <![CDATA[<b>Optimization of nutritional and non-nutritional factors involved for production of antimicrobial compounds from <i>Lactobacillus pentosus </i>SJ65 using response surface methodology</b>]]> Bacteriocins from lactic acid bacteria are ribosomal synthesized antibacterial proteins/ peptides having wide range of applications. Lactobacillus pentosus SJ65, isolated from fermented Uttapam batter (used to prepare south Indian pan cake), produces bacteriocin having a broad spectrum of activity against pathogens. Optimization studies are of utmost important to understand the source of utilization and the conditions to enhance the production of metabolites. In the present study, an attempt was made to identify the parameters involved for maximal production of antimicrobial compounds especially bacteriocin from the isolate L. pentosus SJ65. Initially, optimal conditions, such as incubation period, pH, and temperature were evaluated. Initial screening was done using methodology onevariable-at-a-time (OVAT) for various carbon and nitrogen sources. Further evaluation was carried out statistically using Plackett-Burman design and the variables were analyzed using response surface methodology using central composite design. The optimum media using tryptone or soy peptone, yeast extract, glucose, triammonium citrate, MnSO4, dipotassium hydrogen phosphate and tween 80 produced maximum bacteriocin activity. <![CDATA[<b>Use <i>Carum copticum </i>essential oil for controlling the <i>Listeria monocytogenes </i>growth in fish model system</b>]]> This study was conducted to evaluate the antibacterial effect of Carum copticum essential oil (Ajowan EO) against Listeria monocytogenes in fish model system. Ajowan EO chemical composition was determined by gas chromatography/mass spectral analysis and the highest concentration of Carum copticum essential oil without any significant changes on sensory properties of kutum fish (Rutilus frisii kutum) was assigned. Then the inhibitory effect of Ajowan EO at different concentrations in presence of salt and smoke component was tested on L. monocytogenes growth in fish peptone broth (FPB), kutum broth and cold smoked kutum broth at 4 ºC for 12 days. Ajowan EO completely decreased the number of L. monocytogenes in FPB after 12 days of storage, however, antimicrobial effect of EO significantly reduced in kutum and cold smoked kutum broth. Addition of 4% NaCl and smoke component improved the anti-listerial activity of Ajowan EO in all fish model broths. <![CDATA[<b>Application of decolourized and partially purified polygalacturonase and</b> <b>α</b><b>-amylase in apple juice clarification</b>]]> Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T660nm = 97.0%) of juice was attained after 2 h of incubation at 50 ºC in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property. <![CDATA[<b>Occurrence of <i>Candida orthopsilosis </i>in Brazilian tomato fruits (<i>Lycopersicum esculentum </i>Mill.)</b>]]> We aimed to isolate and identify yeasts found in the tomato fruit in order to obtain isolates with biotechnological potential, such as in control of fungal diseases that damage postharvest fruits. We identified Candida orthopsilosis strains LT18 and LT24. This is the first report of this yeast on Lycopersicum esculentum fruits in Brazil. <![CDATA[<b>Prevalence and phenotypic characterization of <i>Enterococcus </i>spp. isolated from food in Brazil</b>]]> We evaluated the frequency of enterococci from food and found 95.2% of positivity, being E. faecium and E. faecalis the most frequent species. High-level streptomycin resistance was observed, as well as gelatinase and hemolysis activity, showing the potential role of environmental strains as reservoir of virulence and resistance traits. <![CDATA[<b>Best conditions for biodegradation of diesel oil by chemometric tools</b>]]> Diesel oil biodegradation by different bacteria-yeast-rhamnolipids consortia was tested. Chromatographic analysis of post-biodegradation residue was completed with chemometric tools (ANOVA, and a novel ranking procedure based on the sum of ranking differences). These tools were used in the selection of the most effective systems. The best results of aliphatic fractions of diesel oil biodegradation were observed for a yeast consortia with Aeromonas hydrophila KR4. For these systems the positive effect of rhamnolipids on hydrocarbon biodegradation was observed. However, rhamnolipids addition did not always have a positive influence on the biodegradation process (e.g. in case of yeast consortia with Stenotrophomonas maltophila KR7). Moreover, particular differences in the degradation pattern were observed for lower and higher alkanes than in the case with C22. Normally, the best conditions for "lower" alkanes are Aeromonas hydrophila KR4 + emulsifier independently from yeasts and e.g. Pseudomonas stutzeri KR7 for C24 alkane. <![CDATA[<b>Aflatoxin detoxification by manganese peroxidase purified from <i>Pleurotus ostreatus</i></b>]]> Manganese peroxidase (MnP) was produced from white rot edible mushroom Pleurotus ostreatus on the culture filtrate. The enzyme was purified to homogeneity using (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 column chromatography. The final enzyme activity achieved 81UmL-1, specific activity 78 U mg-1 with purification fold of 130 and recovery 1.2% of the crude enzyme. SDS-PAGE indicated that the pure enzyme have a molecular mass of approximately 42 kDa. The optimum pH was between 4-5 and the optimum temperature was 25 ºC. The pure MnP activity was enhanced by Mn2+,Cu2+,Ca2+ and K+ and inhibited by Hg+2 and Cd+2.H2O2 at 5 mM enhanced MnP activity while at 10 mM inhibited it significantly. The MnP-cDNA encoding gene was sequenced and determined (GenBank accession no. AB698450.1). The MnP-cDNA was found to consist of 497 bp in an Open Reading Frame (ORF) encoding 165 amino acids. MnP from P. ostreatus could detoxify aflatoxin B1 (AFB1) depending on enzyme concentration and incubation period. The highest detoxification power (90%) was observed after 48 h incubation at 1.5 U mL-1 enzyme activities. <![CDATA[<b>Yeast diversity associated to sediments and water from two Colombian artificial lakes</b>]]> In Colombia, knowledge of the yeast and yeast-like fungi community is limited because most studies have focused on species with clinical importance. Sediments and water represent important habitats for the study of yeast diversity, especially for yeast species with industrial, biotechnological, and bioremediation potential. The main purpose of this study was to identify and compare the diversity of yeast species associated with sediment and water samples from two artificial lakes in Universidad del Valle (Cali-Colombia). Yeast samplings were performed from fifteen sediment samples and ten water samples. Grouping of similar isolates was initially based on colony and cell morphology, which was then complemented by micro/mini satellite primed PCR banding pattern analysis by using GTG5 as single primer. A representative isolate for each group established was chosen for D1/D2 domain sequencing and identification. In general, the following yeast species were identified: Candida albicans, Candida diversa, Candida glabrata, Candida pseudolambica, Cryptococcus podzolicus, Cryptococcus rajasthanensis, Cryptococcus laurentii, Williopsis saturnus, Hanseniaspora thailandica, Hanseniaspora uvarum, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Torulaspora delbrueckii, Torulaspora pretoriensis, Tricosporon jirovecii, Trichosporon laibachii and Yarrowia lypolitica. Two possible new species were also found, belonging to the Issatchenkia sp. and Bullera sp. genera. In conclusion, the lakes at the Universidad del Valle campus have significant differences in yeast diversity and species composition between them. <![CDATA[<b>Protozoans bacterivory in a subtropical environment during a dry/cold and a rainy/warm season</b>]]> In aquatic ecosystems, bacteria are controlled by several organisms in the food chain, such as protozoa, that use them as food source. This study aimed to quantify the ingestion and clearance rates of bacteria by ciliates and heterotrophic nanoflagellates (HNF) in a subtropical freshwater reservoir (Monjolinho reservoir -São Carlos -Brazil) during one year period, in order to verify their importance as consumers and controllers of bacteria in two seasons, a dry/cold and a rainy/warm one. For this purpose, in situ bacterivory experiments were carried out bimonthly using fluorescently labeled bacteria with 5-(4,6 diclorotriazin-2yl) aminofluorescein (DTAF). Although ciliates have shown the highest individual ingestion and clearance rates, bacterivory was dominated by HNF, who showed higher population ingestion rates (mean of 9,140 bacteria h-1mL-1) when compared to ciliates (mean of 492 bacteria h-1mL-1). The greater predation impact on bacterial communities was caused mainly by the small HNF (< 5 µm) population, especially in the rainy season, probably due to the abundances of these organisms, the precipitation, trophic index state and water temperature that were higher in this period. Thus, the protozoan densities together with environmental variables were extremely relevant in determining the seasonal pattern of bacterivory in Monjolinho reservoir. <![CDATA[<b>Endophytic fungi from <i>Myrcia guianensis </i>at the Brazilian Amazon</b>: <b>distribution and bioactivity</b>]]> Beneficial interactions between plants and microorganisms have been investigated under different ecological, physiological, biochemical, and genetic aspects. However, the systematic exploration of biomolecules with potential for biotechnological products from this interaction still is relatively scarce. Therefore, this study aimed the evaluation of the diversity and antimicrobial activity of the endophytic fungi obtained from roots, stems and leafs of Myrcia guianensis (Myrtaceae) from the Brazilian Amazon. 156 endophytic fungi were isolated and above 80% were identified by morphological examination as belonging to the genera Pestalotiopsis, Phomopsis, Aspergillus, Xylaria, Nectria, Penicillium and Fusarium. Fermented broth of those fungi were assayed for antimicrobial activity and four inhibited the growth of Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Penicillium avellaneum. As the strain named MgRe2.2.3B (Nectria haematococca) had shown the most promising results against those pathogenic strains, its fermented broth was fractioned and only its two low polar fractions demonstrated to be active. Both fractions exhibited a minimum bactericidal concentration of 50 µg.mL-1 against S. aureus and a minimum fungicidal concentration of 100 µg.mL-1 against P. avellaneum. These results demonstrate the diversity of fungal genera in M. guianensis and the potential of these endophytic fungi for the production of new antibiotics. <![CDATA[<b>A primary assessment of the endophytic bacterial community in a xerophilous moss (<i>Grimmia montana</i>) using molecular method and cultivated isolates</b>]]> Investigating the endophytic bacterial community in special moss species is fundamental to understanding the microbial-plant interactions and discovering the bacteria with stresses tolerance. Thus, the community structure of endophytic bacteria in the xerophilous moss Grimmia montana were estimated using a 16S rDNA library and traditional cultivation methods. In total, 212 sequences derived from the 16S rDNA library were used to assess the bacterial diversity. Sequence alignment showed that the endophytes were assigned to 54 genera in 4 phyla (Proteobacteria, Firmicutes, Actinobacteria and Cytophaga/Flexibacter/Bacteroids). Of them, the dominant phyla were Proteobacteria (45.9%) and Firmicutes (27.6%), the most abundant genera included Acinetobacter, Aeromonas, Enterobacter, Leclercia, Microvirga, Pseudomonas, Rhizobium, Planococcus, Paenisporosarcina and Planomicrobium. In addition, a total of 14 species belonging to 8 genera in 3 phyla (Proteobacteria, Firmicutes, Actinobacteria) were isolated, Curtobacterium, Massilia, Pseudomonas and Sphingomonas were the dominant genera. Although some of the genera isolated were inconsistent with those detected by molecular method, both of two methods proved that many different endophytic bacteria coexist in G. montana. According to the potential functional analyses of these bacteria, some species are known to have possible beneficial effects on hosts, but whether this is the case in G. montana needs to be confirmed. <![CDATA[<b>Comparison of DNA extraction protocols for microbial communities from soil treated with biochar</b>]]> Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit MiniprepTM (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples. <![CDATA[<b>Tank bromeliad water</b>: <b>similar or distinct environments for research of bacterial bioactives?</b>]]> The Atlantic Rainforest does not have a uniform physiognomy, its relief determines different environmental conditions that define the composition of its flora and fauna. Within this ecosystem, bromeliads that form tanks with their leaves hold water reservoirs throughout the year, maintaining complex food chains, based mainly on autotrophic and heterotrophic bacteria. Some works concluded that the water held by tank bromeliads concentrate the microbial diversity of their ecosystem. To investigate the bacterial diversity and the potential biotechnology of these ecosystems, tank bromeliads of the Neoregelia cruenta species from the Atlantic Rainforest in Brazil were used as models for this research. Bacteria isolated from these models were tested for production of bioactive compounds. DGGE of the water held by tank bromeliads was performed in different seasons, locations and sun exposure to verify whether these environmental factors affect bacterial communities. The DGGE bands profile showed no grouping of bacterial community by the environmental factors tested. Most of the isolates demonstrated promising activities in the tests performed. Collectively, these results suggest that tank bromeliads of the N. cruenta species provide important habitats for a diverse microbial community, suggesting that each tank forms a distinct micro-habitat. These tanks can be considered excellent sources for the search for new enzymes and/or new bioactive composites of microbial origin. <![CDATA[<b>Screening of endoglucanase-producing bacteria in the saline rhizosphere of <i>Rhizophora mangle</i></b>]]> In screening the culturable endoglucanase-producing bacteria in the rhizosphere of Rhizophora mangle, we found a prevalence of genera Bacillus and Paenibacillus. These bacteria revealed different activities in endoglucolysis and biofilm formation when exposed to specific NaCl concentrations, indicating modulated growth under natural variations in mangrove salinity. <![CDATA[<b>Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against <i>Anaplasma marginale </i>in cattle</b>]]> The recombinant protein MSP5 has been established as an important antigen for serological diagnosis of Anaplasma marginale by enzyme-linked immunosorbent assay (ELISA). However, due to the high cost of specialized equipment, this technique is not accessible to all laboratories, especially in developing countries in areas where the disease is endemic. The present study describes the standardization of a latex agglutination test (LAT) to detect antibodies against A. marginale based on recombinant MSP5. Compared with indirect enzyme-linked immunosorbent assay (iELISA), the relative sensitivity and specificity of the LAT were 95.21% and 91.86% respectively, with an almost perfect agreement between tests (kappa index = 0.863). These results can be considered important for the serological diagnosis of A. marginale, as they indicate that the test represents a rapid and low cost alternative to ELISA. <![CDATA[<b>Isolation of <i>Actinobacillus seminis </i>from a goat with clinical epididymo-orchitis in Brazil</b>]]> The present study reports the first isolation of Actinobacillus seminis from a goat in Brazil. A four-year-old Moxotó breeding goat in a flock of 70 goats and 65 sheep reared together in the county of Patos, semiarid region of Northeastern Brazil, showed clinical signs of unilateral orchitis and epididymitis. Diagnosis of A. seminis infection was confirmed by association of clinical findings, bacterial isolation and 16S rRNA gene sequencing. This result suggests that A. seminis may be an additional cause of infertility in goats, and that sheep may be the source of infection because the mixed farming system allows the contact between sheep and goats in the semiarid region of Northeastern Brazil. <![CDATA[<b>In vitro inhibition of the bovine viral diarrhoea virus by the essential oil of <i>Ocimum basilicum </i>(basil) and monoterpenes</b>]]> The bovine viral diarrhoea virus (BVDV) is suggested as a model for antiviral studies of the hepatitis C virus (HCV). The antiviral activity of the essential oil of Ocimum basilicum and the monoterpenes camphor, thymol and 1,8-cineole against BVDV was investigated. The cytotoxicities of the compounds were measured by the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) test, and the antiviral activities were tested by the plaque reduction assay. The oil or compounds were added to the assay in three different time points: a) pre-treatment of the virus (virucidal assay); b) pre-treatment of the cells; or c) post-treatment of the cells (after virus inoculation). The percentage of plaques inhibition for each compound was determined based on the number of plaques in the viral control. The results were expressed by CC50 (50% cytotoxic concentration), IC50 (inhibitory concentration for 50% of plaques) and SI (selectivity index = CC50/IC50). Camphor (CC50 = 4420.12 µgmL-1) and 1,8-cineole (CC50 = 2996.10 µgmL-1) showed the lowest cytotoxicities and the best antiviral activities (camphor SI = 13.88 and 1,8-cineol SI = 9.05) in the virucidal assay. The higher activities achieved by the monoterpenes in the virucidal assay suggest that these compounds act directly on the viral particle. <![CDATA[<b>Methicillin-resistant coagulase-negative staphylococci from healthy dogs in Nsukka, Nigeria</b>]]> The occurrence, resistance phenotype and molecular mechanisms of resistance of methicillin-resistant staphylococci from groin swabs of 109 clinically healthy dogs in Nsukka, Nigeria were investigated. The groin swab samples were cultured on mannitol salt agar supplemented with 10 µgof cloxacillin. Sixteen methicillin-resistant coagulase negative staphylococci (MRCoNS), all harbouring the mecA gene were isolated from 14 (12.8%) of the 109 dogs studied. The MRCoNS isolated were: S. sciuri subspecies rodentium, S. lentus, S. haemolyticus, and S. simulans with S. sciuri subspecies rodentium (62.5%) being the predominant species. Thirteen (81.3%) of the MRCoNS were resistant to tetracycline while 12 (75%) and 10 (62.5%) were resistant to kanamycin and trimthoprim-sulphamethoxazole respectively. None of the isolates was resistant to fusidic acid, linezolid and vancomycin. Thirteen (81.3%) of the MRCoNS were multi-drug resistance (MDR). Other antimicrobial genes detected were: blaZ, tet(K), tet(M), tet(L), erm(B), lnu(A), aacA-aphD, aphA3, str, dfr(G), cat pC221,and cat pC223. Methicillin-resistant staphylococci are common colonizers of healthy dogs in Nigeria with a major species detected being S. sciuri subsp. rodentium. <![CDATA[<b>Acute myonecrosis in horse caused by <i>Clostridium novyi </i>type A</b>]]> The objective of this study was to describe the first report involving a case of equine acute myonecrosis caused by C. novyi type A with an emphasis on clinical signs, the pathological and bacteriological analysis, and molecular identification of the microorganisms as the key of the definitive diagnosis. <![CDATA[<b>Thin layer microcolony culture associated with PCR for early identification of <i>Mycobacterium bovis</i></b>]]> The initial growth of mycobacteria from 49 samples of cattle and buffalo organs collected in commercial slaughterhouses was compared between modified Middlebrook 7H11 thin layer microcolony culture and Stonebrink medium used in the isolation of Mycobacterium bovis. Aliquots were decontaminated by Petroff's method, processed and cultured in both media. The identity of the acid-fast bacilli stained by Ziehl-Neelsen was confirmed by PCR. Optical microscopy showed that results of the early observation of Mycobacterium bovis colonies in thin layer culture were similar to those obtained in macroscopic observation of the colonies in Stonebrink medium. However, early observation of the colonies enabled early confirmation by PCR, given the shorter time to the visualization of colonies when thin layer culture was used (between the 12nd and 25th day of culture). <![CDATA[<b>Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens</b>]]> Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens. <![CDATA[<b>Comparison of cefoxitin disk diffusion test and <i>mecA </i>gene PCR results for methicillin resistance detection in <i>Staphylococcus intermedius </i>group isolates from canine origin in Brazil</b>]]> The study evaluated cefoxitin disk diffusion tests breakpoints and their correlation to mecA gene PCR results for detecting Methicillin-resistant Staphylococcus intermedius Group (MRSP) isolates from dogs in Brazil. Agreement using proposed breakpoint (resistant < 30 mm) was encouraging. The current study reinforces that an epidemiological breakpoint can be established to predict presence of MRSP. <![CDATA[<b>Molecular mechanism of fluoroquinolones resistance in <i>Mycoplasma hominis </i>clinical isolates</b>]]> To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH) clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX), intermediate resistant to Levofloxacin (LVX) and Sparfloxacin (SFX), and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV. <![CDATA[<b>Paracoccidioidomycosis in southern Rio Grande do Sul</b>: <b>a retrospective study of histopathologically diagnosed cases</b>]]> Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis and is endemic to Brazil. The aim of this study was to perform a retrospective analysis of the PCM cases in the countryside south of Rio Grande do Sul, Brazil. The files from four histopathology laboratories located in the city of Pelotas were obtained, and all of the epidemiological and clinical data from the PCM diagnosed cases were collected for analysis. A total of 123 PCM cases diagnosed between 1966 and 2009 were selected. Of these patients, 104 (84.5%) were male, and 17 were female. The patients ranged from 02 to 92 years of age. Fifty-two cases (41.9%) were obtained from the oral pathology laboratory, and the remaining 71 cases (58.1%) were obtained from the three general pathology laboratories. Of all of the patients studied, 65.2% lived in rural zones and worked in agriculture or other related fields. Data on the evolution of this disease was available for 43 cases, and the time frame ranged from 20 to 2920 days (mean = 572.3 days). An accurate diagnosis performed in less than 30 days only occurred in 21% of the cases. PCM is endemic to the countryside of Rio Grande do Sul. Therefore, it is recommended that PCM be included as a differential diagnosis, mainly for individuals between 30 and 60 years of age, living in rural zones and who have respiratory signs and associated-oropharyngeal lesions. <![CDATA[<b>Antifungal activity of metabolites of the endophytic fungus <i>Trichoderma brevicompactum </i>from garlic</b>]]> The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8SITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4β-acetoxy-12,13-epoxy-Δ9-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 µgmL-1. Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 µgmL-1. However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 µgmL-1). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin. <![CDATA[<b>Evaluation of chromogenic media and seminested PCR in the identification of <i>Candida </i>species</b>]]> Identification of Candida cultured from various clinical specimens to the species level is increasingly necessary for clinical laboratories. Although sn PCR identifies the species within hours but its cost-effectiveness is to be considered. So there is always a need for media which help in the isolation and identification at the species level. The study aimed to evaluate the performance of different chromogenic media and to compare the effectiveness of the traditional phenotypic methods vs. seminested polymerase chain reaction (sn PCR) for identification of Candida species. One hundred and twenty seven Candida strains isolated from various clinical specimens were identified by conventional methods, four different chromogenic media and sn PCR. HiCrome Candida Differential and CHROMagar Candida media showed comparably high sensitivities and specificities in the identification of C. albicans, C. tropicalis, C. glabrata and C. krusei. CHROMagar Candida had an extra advantage of identifying all C. parapsilosis isolates. CHROMagar-Pal's medium identified C. albicans, C. tropicalis and C. krusei with high sensitivities and specificities, but couldn't identify C. glabrata or C. parapsilosis. It was the only medium that identified C. dubliniensis with a sensitivity and specificity of 100%. Biggy agar showed the least sensitivities and specificities. The overall concordance of the snPCR compared to the conventional tests including CHROMAgar Candida in the identification of Candida species was 97.5%. The use of CHROMAgar Candida medium is an easy and accurate method for presumptive identification of the most commonly encountered Candida spp. <![CDATA[<b>Newly-synthesized chalcones-inhibition of adherence and biofilm formation of methicillin-resistant <i>Staphylococcus aureus</i></b>]]> Biofilm formation and adherence of bacteria to host tissue are one of the most important virulence factors of methicillin-resistant strains of Staphylococcus aureus (MRSA). The number of resistant strains is seriously increasing during the past years and bacteria have become resistant, not only to methicillin, but also to other commonly used antistaphylococcal antibiotics. There is a great need for discovering a novel antimicrobial agent for the treatment of staphylococcal infections. One of the most promising groups of compounds appears to be chalcones. In present study we evaluated the in vitro effect of three newly synthesized chalcones: 1,3-Bis-(2-hydroxy-phenyl)-propenone, 3-(3Hydroxy-phenyl)-1-(2-hydroxy-phenyl)-propenone and 3-(4-Hydroxy-phenyl)-1-(2-hydroxyphenyl)-propenone on glycocalyx production, biofilm formation and adherence to human fibronectin of clinical isolates and laboratory control strain of MRSA (ATCC 43300). Subinhibitory concentrations of the tested compounds reduced the production of glycocalyx, biofilm formation and adherence to human fibronectin of all MRSA strains. Inhibition of biofilm formation was dose dependent and the most effective was 1,3-Bis-(2-hydroxy-phenyl)-propenone. In our study we demonstrated that three newly-synthesized chalcones exhibited significant effect on adherence and biofilm formation of MRSA strains. Chalcones may be considered as promising new antimicrobial agents that can be used for prevention of staphylococcal infections or as adjunct to antibiotics in conventional therapy. <![CDATA[<b>Characterization of the virulence, growth temperature and antibiotic resistance of the <i>Campylobacter jejuni </i>IAL 2383 strain isolated from humans</b>]]> The objective of this study was to characterize the C. jejuni IAL2383 strain isolated from humans in Brazil. Transcripts for the racR, dnaJ and ciaB genes were found and flaA, plda and cadF genes were present in the genome and bacteria was sensitive to most of the important antimicrobials used to treat humans. C. jejuni IAL2383 is a good experimental model to analyze the interactions with cells. <![CDATA[<b>A case of extensive chromoblastomycosis from North India</b>]]> A case of extensive chromoblastomycosis of the right leg and thigh with verruciform to nodular lesions evolving rapidly over five years duration is reported. The diagnosis was confirmed by visualizing pathognomonic pigmented muriform bodies with unique septate hyphae and mycological culture yielding Fonsecaea pedrosoi. <![CDATA[<b>Filamentous fungi and media for cellulase production in solid state cultures</b>]]> Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77. <![CDATA[<b>Optimization of zofimarin production by an endophytic fungus, <i>Xylaria </i>sp. Acra L38</b>]]> To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µ/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium. <![CDATA[<b>Concentration, characterization and application of lipases from <i>Sporidiobolus pararoseus </i>strain</b>]]> Lipases produced by a newly isolated Sporidiobolus pararoseus strain have potential catalytic ability for esterification reactions. After production, the enzymatic extracts (conventional crude and precipitated, 'CC' and 'CP', and industrial crude and precipitated, 'IC' e 'IP') were partially characterized. The enzymes presented, in general, higher specificity for short chain alcohols and fatty acids. The precipitated extract showed a good thermal stability, higher than that for crude enzymatic extracts. The 'CC' and 'CP' enzymes presented high activities after exposure to pH 6.5 and 40 ºC. On the other hand, the 'IC' and 'IP' extracts kept their activities in a wide range of pH memory but presented preference for higher reaction temperatures. Preliminary studies of application of the crude lipase extract in the enzymatic production of geranyl propionate using geraniol and propionic acid as substrates in solvent-free system led to a reaction conversion of 42 ± 1.5%. <![CDATA[<b>Production of polypeptide antibiotic from <i>Streptomyces parvulus </i>and its antibacterial activity</b>]]> A highly potent secondary metabolite producing actinomycetes strain is isolated from marine soil sediments of Visakhapatnam sea coast, Bay of Bengal. Over all ten strains are isolated from the collected soil sediments. Among the ten actinomycetes strains the broad spectrum strain RSPSN2 was selected for molecular characterization, antibiotic production and its purification. The nucleotide sequence of the 1 rRNA gene (1261 base pairs) of the most potent strain evidenced a 96% similarity with Streptomyces parvulus 1044 strain, Streptomyces parvulus NBRC 13193 and Streptomyces parvulus BY-F. From the taxonomic features, the actinomycetes isolate RSPSN2 matches with Streptomyces parvulus in the morphological, physiological and biochemical characters. Thus, it was given the suggested name Streptomyces parvulus RSPSN2. The active metabolite was extracted using ethyl acetate (1:3, v/v) at pH 7.0. The separation of active ingredient and its purification was performed by using both thin layer chromatography (TLC) and column chromatography (CC) techniques. Spectrometric studies such as UV-visible, FTIR, and NMR and mass were performed. The antibacterial activity of pure compound was performed by cup plate method against some pathogenic bacteria including of streptomycin resistant bacteria like (Pseudomonas mirabilis. Pseudomonas putida and Bacillus cereus). In conclusion, the collected data emphasized the fact that a polypeptide antibiotic (Actinomycin D) was produced by Streptomyces parvulus RSPSN2. <![CDATA[<b>Modulation of antimicrobial metabolites production by the fungus <i>Aspergillus parasiticus</i></b>]]> Biosynthesis of active secondary metabolites by fungi occurs as a specific response to the different growing environments. Changes in this environment alter the chemical and biological profiles leading to metabolites diversification and consequently to novel pharmacological applications. In this work, it was studied the influence of three parameters (fermentation length, medium composition and aeration) in the biosyntheses of antimicrobial metabolites by the fungus Aspergillus parasiticus in 10 distinct fermentation periods. Metabolism modulation in two culturing media, CYA and YES was evaluated by a 2² full factorial planning (ANOVA) and on a 2³ factorial planning, role of aeration, medium composition and carbohydrate concentration were also evaluated. In overall, 120 different extracts were prepared, their HPLC profiles were obtained and the antimicrobial activity against A. flavus, C. albicans, E. coli and S. aureus of all extracts was evaluated by microdilution bioassay. Yield of kojic acid, a fine chemical produced by the fungus A. parasiticus was determined in all extracts. Statistical analyses pointed thirteen conditions able to modulate the production of bioactive metabolites by A. parasiticus. Effect of carbon source in metabolites diversification was significant as shown by the changes in the HPLC profiles of the extracts. Most of the extracts presented inhibition rates higher than that of kojic acid as for the extract obtained after 6 days of fermentation in YES medium under stirring. Kojic acid was not the only metabolite responsible for the activity since some highly active extracts showed to possess low amounts of this compound, as determined by HPLC. <![CDATA[<b>DesinFix TM 135 in fermentation process for bioethanol production</b>]]> Brazil has the world's largest ethanol production from sugarcane, but bacterial contamination decreases the ethanol yields. It was shown that the biocide DesinFixTM 135 can reduce the contamination without decreasing the yeasts' viability or negatively affecting the ethanol production. <![CDATA[<b>Molecular detection of virulence factors among food and clinical <i>Enterococcus faecalis </i>strains in South Brazil</b>]]> The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains. <![CDATA[<b>Genome-wide transcription analyses in <i>Mycobacterium tuberculosis </i>treated with lupulone</b>]]> Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, still causes higher mortality than any other bacterial pathogen until now. With the emergence and spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR-TB) strains, it becomes more important to search for alternative targets to develop new antimycobacterial drugs. Lupulone is a compound extracted from Hops (Hurnulus lupulus), which exhibits a good antimicrobial activity against M. tuberculosis with minimal inhibitory concentration (MIC) value of 10 µg/mL, but the response mechanisms of lupulone against M. tuberculosis are still poorly understood. In this study, we used a commercial oligonucleotide microarray to determine the overall transcriptional response of M. tuberculosis H37Rv triggered by exposure to MIC of lupulone. A total of 540 genes were found to be differentially regulated by lupulone. Of these, 254 genes were upregulated, and 286 genes were downregulated. A number of important genes were significantly regulated which are involved in various pathways, such as surface-exposed lipids, cytochrome P450 enzymes, PE/PPE multigene families, ABC transporters, and protein synthesis. Real-time quantitative RT-PCR was performed for choosed genes to verified the microarray results. To our knowledge, this genome-wide transcriptomics approach has produced the first insights into the response of M. tuberculosis to a lupulone challenge. <![CDATA[<b>Detection of enteric viruses in activated sludge by feasible concentration methods</b>]]> Human enteric viruses are responsible to cause several diseases, including gastroenteritis and hepatitis, and can be present in high amounts in sewage sludge. This study compared virus recovery efficiency of two feasible concentration methods used for detecting human adenovirus (HAdV), rotavirus species A (RV-A), norovirus genogroup II (NoV GII) and hepatitis A virus (HAV) in sewage sludge from an activated sludge process. Twelve sewage sludge samples were collected bi-monthly from January to July, 2011. Ultracentrifugation was compared with a simplified protocol based on beef extract elution for recovering enteric viruses. Viruses were quantified by quantitative real-time PCR assays and virus recovery efficiency and limits of detection were determined. Methods showed mean recovery rates lower than 7.5%, presenting critical limits of detection (higher than 10² 10³ genome copies -GC L-1 for all viruses analyzed). Nevertheless, HAdV were detected in 90% of the analyzed sewage sludge samples (range: 1.8 x 10(4) to 1.1 x 10(5) GC L-1), followed by RV-A and NoV (both in 50%) and HAV (8%). Results suggesting that activated sludge is contaminated with high viral loads and HAdV are widely disseminated in these samples. The low virus recovery rates achieved, especially for HAV, indicate that other feasible concentration methods could be developed to improve virus recovery efficiency in these environmental matrices. <![CDATA[<b>Cloning, characterization and expression of a novel laccase gene <i>Pclac</i>2 from <i>Phytophthora capsici</i></b>]]> Laccases are blue copper oxidases (E.C. that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2'-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 ºC, respectively . The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes. <![CDATA[<b>Chemical management in fungicide sensivity of <i>Mycosphaerella fijiensis </i>collected from banana fields in México</b>]]> The chemical management of the black leaf streak disease in banana caused by Mycosphaerella fijiensis (Morelet) requires numerous applications of fungicides per year. However this has led to fungicide resistance in the field. The present study evaluated the activities of six fungicides against the mycelial growth by determination of EC50 values of strains collected from fields with different fungicide management programs: Rustic management (RM) without applications and Intensive management (IM) more than 25 fungicide application/year. Results showed a decreased sensitivity to all fungicides in isolates collected from IM. Means of EC50 values in mg L-1 for RM and IM were: 13.25 ± 18.24 and 51.58 ± 46.14 for azoxystrobin, 81.40 ± 56.50 and 1.8575 ± 2.11 for carbendazim, 1.225 ± 0.945 and 10.01 ± 8.55 for propiconazole, 220 ± 67.66 vs. 368 ± 62.76 for vinclozolin, 9.862 ± 3.24 and 54.5 ± 21.08 for fludioxonil, 49.2125 ± 34.11 and 112.25 ± 51.20 for mancozeb. A molecular analysis for β-tubulin revealed a mutation at codon 198 in these strains having an EC50 greater than 10 mg L-1 for carbendazim. Our data indicate a consistency between fungicide resistance and intensive chemical management in banana fields, however indicative values for resistance were also found in strains collected from rustic fields, suggesting that proximity among fields may be causing a fungus interchange, where rustic fields are breeding grounds for development of resistant strains. Urgent actions are required in order to avoid fungicide resistance in Mexican populations of M. fijiensis due to fungicide management practices.