Scielo RSS <![CDATA[Brazilian Journal of Microbiology]]> http://www.scielo.br/rss.php?pid=1517-838220160002&lang=es vol. 47 num. 2 lang. es <![CDATA[SciELO Logo]]> http://www.scielo.br/img/en/fbpelogp.gif http://www.scielo.br <![CDATA[<em>Sphingomonas</em> from petroleum-contaminated soils in Shenfu, China and their PAHs degradation abilities]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200271&lng=es&nrm=iso&tlng=es Abstract Members of the Sphingomonas genus are often isolated from petroleum-contaminated soils due to their unique abilities to degrade polycyclic aromatic hydrocarbons (PAHs), which are important for in situ bioremediation. In this study, a combined phenotypic and genotypic approach using streptomycin-containing medium and Sphingomonas -specific PCR was developed to isolate and identify culturable Sphingomonas strains present in petroleum-contaminated soils in the Shenfu wastewater irrigation zone. Of the 15 soil samples examined, 12 soils yielded yellow streptomycin-resistant colonies. The largest number of yellow colony-forming units (CFUs) could reach 105 CFUs g-1 soil. The number of yellow CFUs had a significant positive correlation (p &lt; 0.05) with the ratio of PAHs to total petroleum hydrocarbons (TPH), indicating that Sphingomonas may play a key role in degrading the PAH fraction of the petroleum contaminants at this site. Sixty yellow colonies were selected randomly and analyzed by colony PCR using Sphingomonas -specific primers, out of which 48 isolates had PCR-positive signals. The 48 positive amplicons generated 8 distinct restriction fragment length polymorphism (RFLP) patterns, and 7 out of 8 phylotypes were identified as Sphingomonas by 16S rRNA gene sequencing of the representative strains. Within these 7 Sphingomonas strains, 6 strains were capable of using fluorene as the sole carbon source, while 2 strains were phenanthrene-degrading Sphingomonas. To the best of our knowledge, this is the first report to evaluate the relationship between PAHs contamination levels and culturable Sphingomonas in environmental samples. <![CDATA[<em>Eichhornia azurea</em> decomposition and the bacterial dynamic: an experimental research]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200279&lng=es&nrm=iso&tlng=es Abstract Organic decomposition is a complex interaction between chemical, physical and biological processes, where the variety of aquatic vascular plants is essential for the trophic dynamics of freshwater ecosystems. The goal of this study was to determine the aquatic macrophyte Eichhornia azurea (Sw.) Kunth decomposition rate, the time relation with the limnological parameters, and whether this relationship is a result of decomposition processes. To that end, we collected water and leaves of E. azurea in Surf Leopoldo, PR. The experiment consisted of two treatments: 25 containers with 450 mL of water and 0.8 g of biomass dry weight were used with or without the addition of macrophytes. Samples were collected in triplicate at times 0, 3 h, 6 h, 12 h, 24 h, 72 h, 120 h, 168 h and 240 h. When the container was removed, the plant material was dried in an oven. After 48 h, the material was measured to obtain the final dry weight. Analyses of pH, conductivity, dissolved oxygen, total phosphorus N-ammonia (NH4), soluble reactive phosphorus (PO4) and dissolved organic carbon were performed, and the decomposition rate was calculated. The results showed significant temporal variation of limnological parameters in the study. Additionally, dissolved oxygen, conductivity, dissolved organic carbon and total phosphorus were correlated with the dry weight of the biomass, suggesting that E. azurea decomposition significantly interferes with the dynamics of these variables. <![CDATA[Preliminary studies of new strains of <em>Trametes</em> sp. from Argentina for laccase production ability]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200287&lng=es&nrm=iso&tlng=es Abstract Oxidative enzymes secreted by white rot fungi can be applied in several technological processes within the paper industry, biofuel production and bioremediation. The discovery of native strains from the biodiverse Misiones (Argentina) forest can provide useful enzymes for biotechnological purposes. In this work, we evaluated the laccase and manganese peroxidase secretion abilities of four newly discovered strains of Trametes sp. that are native to Misiones. In addition, the copper response and optimal pH and temperature for laccase activity in culture supernatants were determined.The selected strains produced variable amounts of laccase and MnP; when Cu2+ was added, both enzymes were significantly increased. Zymograms showed that two isoenzymes were increased in all strains in the presence of Cu2+. Strain B showed the greatest response to Cu2+ addition, whereas strain A was more stable at the optimal temperature and pH. Strain A showed interesting potential for future biotechnological approaches due to the superior thermo-stability of its secreted enzymes. <![CDATA[Role of pH on antioxidants production by <em>Spirulina</em> (<em>Arthrospira</em>) <em>platensis</em>]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200298&lng=es&nrm=iso&tlng=es Abstract Algae can tolerate a broad range of growing conditions but extreme conditions may lead to the generation of highly dangerous reactive oxygen species (ROS), which may cause the deterioration of cell metabolism and damage cellular components. The antioxidants produced by algae alleviate the harmful effects of ROS. While the enhancement of antioxidant production in blue green algae under stress has been reported, the antioxidant response to changes in pH levels requires further investigation. This study presents the effect of pH changes on the antioxidant activity and productivity of the blue green alga Spirulina (Arthrospira) platensis. The algal dry weight (DW) was greatly enhanced at pH 9.0. The highest content of chlorophyll a and carotenoids (10.6 and 2.4 mg/g DW, respectively) was recorded at pH 8.5. The highest phenolic content (12.1 mg gallic acid equivalent (GAE)/g DW) was recorded at pH 9.5. The maximum production of total phycobiliprotein (159 mg/g DW) was obtained at pH 9.0. The antioxidant activities of radical scavenging activity, reducing power and chelating activity were highest at pH 9.0 with an increase of 567, 250 and 206% compared to the positive control, respectively. Variation in the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) was also reported. While the high alkaline pH may favor the overproduction of antioxidants, normal cell metabolism and membrane function is unaffected, as shown by growth and chlorophyll content, which suggests that these conditions are suitable for further studies on the harvest of antioxidants from S. platensis. <![CDATA[Screening of <em>Trichoderma</em> isolates for their potential of biosorption of nickel and cadmium]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200305&lng=es&nrm=iso&tlng=es Abstract Fourteen Trichoderma isolates were evaluated for their tolerance to two heavy metals, nickel and cadmium. Three isolates, MT-4, UBT-18, and IBT-I, showed high levels of nickel tolerance, whereas MT-4, UBT-18, and IBT-II showed better tolerance of cadmium than the other isolates. Under nickel stress, biomass production increased up to a Ni concentration of 60 ppm in all strains but then decreased as the concentrations of nickel were further increased. Among the nickel-tolerant isolates, UBT-18 produced significantly higher biomass upon exposure to nickel (up to 150 ppm); however, the minimum concentration of nickel required to inhibit 50% of growth (MIC50) was highest in IBT-I. Among the cadmium-tolerant isolates, IBT-II showed both maximum biomass production and a maximum MIC50 value in cadmium stress. As the biomass of the Trichoderma isolates increased, a higher percentage of nickel removal was observed up to a concentration of 40 ppm, followed by an increase in residual nickel and a decrease in biomass production at higher nickel concentrations in the medium. The increase in cadmium concentrations resulted in a decrease in biomass production and positively correlated with an increase in residual cadmium in the culture broth. Nickel and cadmium stress also influenced the sensitivity of the Trichoderma isolates to soil fungistasis. Isolates IBT-I and UBT-18 were most tolerant to fungistasis under nickel and cadmium stress, respectively. <![CDATA[Rhizobial characterization in revegetated areas after bauxite mining]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200314&lng=es&nrm=iso&tlng=es Abstract Little is known regarding how the increased diversity of nitrogen-fixing bacteria contributes to the productivity and diversity of plants in complex communities. However, some authors have shown that the presence of a diverse group of nodulating bacteria is required for different plant species to coexist. A better understanding of the plant symbiotic organism diversity role in natural ecosystems can be extremely useful to define recovery strategies of environments that were degraded by human activities. This study used ARDRA, BOX-PCR fingerprinting and sequencing of the 16S rDNA gene to assess the diversity of root nodule nitrogen-fixing bacteria in former bauxite mining areas that were replanted in 1981, 1985, 1993, 1998, 2004 and 2006 and in a native forest. Among the 12 isolates for which the 16S rDNA gene was partially sequenced, eight, three and one isolate(s) presented similarity with sequences of the genera Bradyrhizobium, Rhizobium and Mesorhizobium, respectively. The richness, Shannon and evenness indices were the highest in the area that was replanted the earliest (1981) and the lowest in the area that was replanted most recently (2006). <![CDATA[Sugarcane trash levels in soil affects the fungi but not bacteria in a short-term field experiment]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200322&lng=es&nrm=iso&tlng=es Abstract The sugarcane in Brazil is passing through a management transition that is leading to the abolition of pre-harvest burning. Without burning, large amounts of sugarcane trash is generated, and there is a discussion regarding the utilization of this biomass in the industry versus keeping it in the field to improve soil quality. To study the effects of the trash removal on soil quality, we established an experimental sugarcane plantation with different levels of trash over the soil (0%, 50% and 100% of the original trash deposition) and analyzed the structure of the bacterial and fungal community as the bioindicators of impacts. The soil DNA was extracted, and the microbial community was screened by denaturing gradient gel electrophoresis in two different seasons. Our results suggest that there are no effects from the different levels of trash on the soil chemistry and soil bacterial community. However, the fungal community was significantly impacted, and after twelve months, the community presented different structures among the treatments. <![CDATA[Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, <em>Plutella xylostella</em> (Linnaeus), from India and its possible role in indoxacarb degradation]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200327&lng=es&nrm=iso&tlng=es Abstract Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM. <![CDATA[Characterization of the variable region in the class 1 integron of antimicrobial-resistant <em>Escherichia coli</em> isolated from surface water]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200337&lng=es&nrm=iso&tlng=es Abstract Fecal bacteria are considered to be a potential reservoir of antimicrobial resistance genes in the aquatic environment and could horizontally transfer these genes to autochthonous bacteria when carried on transferable and/or mobile genetic elements. Such circulation of resistance genes constitutes a latent public health hazard. The aim of this study was to characterize the variable region of the class 1 integron and relate its genetic content to resistance patterns observed in antimicrobial-resistant Escherichia coli isolated from the surface waters of Patos Lagoon, Southern Brazil. Genetic diversity of the isolates and presence of the qacEΔ1 gene, which confers resistance to quaternary ammonium compounds, were also investigated. A total of 27 isolates were analyzed. The variable region harbored dfrA17, dfrA1 and dfrA12 genes, which confer resistance to trimethoprim, and aadA1, aadA5 and aadA22 genes that encode resistance to streptomycin/spectinomycin. Most of the isolates were considered resistant to quaternary ammonium compounds and all of them carried the qacE Δ1 gene at the 3′ conserved segment of the integron. ERIC-PCR analyses of E. coli isolates that presented the integrons showed great genetic diversity, indicating diverse sources of contamination in this environment. These results suggest that fecal bacteria with class 1 integrons in aquatic environments are potentially important reservoirs of antibiotic-resistance genes and may transfer these elements to other bacteria that are capable of infecting humans. <![CDATA[Proteobacteria, extremophiles and unassigned species dominate in a tape-like showerhead biofilm]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200345&lng=es&nrm=iso&tlng=es Abstract The development of showerhead biofilms exposes the user to repeated contact with potentially pathogenic microbes, yet we know relatively little about the content of these aggregates. The aim of the present study was to examine the microbial content of tape-like films found protruding from a domestic showerhead. Culturing showed that the films were dominated by aerobic α- and β-proteobacteria. Three isolates made up almost the entire plate count. These were a Brevundimonas species, a metalophilic Cupriavidus species and a thermophile, Geobacillus species. Furthermore, it was shown that the Cupriavidus isolate alone had a high capacity for biofilm formation and thus might be the initiator of biofilm production. A clone library revealed the same general composition. However, half of the 70 clones analyzed could not be assigned to a particular bacterial phylum and of these 29 differed from one another by only 1–2 base pairs, indicating a single species. Thus both the culture dependent and culture independent characterizations suggest a simple yet novel composition. The work is important as the biofilm is fundamentally different in form (tape-like) and content from that of all previously reported ones, where variously Mycobacterium, Methylobacterium and Xanthomonas species have dominated, and extremophiles were not reported. <![CDATA[Cellulase activity and dissolved organic carbon release from lignocellulose macrophyte-derived in four trophic conditions]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200352&lng=es&nrm=iso&tlng=es Abstract Considering the importance of lignocellulose macrophyte-derived for the energy flux in aquatic ecosystems and the nutrient concentrations as a function of force which influences the decomposition process, this study aims to relate the enzymatic activity and lignocellulose hydrolysis in different trophic statuses. Water samples and two macrophyte species were collected from the littoral zone of a subtropical Brazilian Reservoir. A lignocellulosic matrix was obtained using aqueous extraction of dried plant material (≈40 °C). Incubations for decomposition of the lignocellulosic matrix were prepared using lignocelluloses, inoculums and filtered water simulating different trophic statuses with the same N:P ratio. The particulate organic carbon and dissolved organic carbon (POC and DOC, respectively) were quantified, the cellulase enzymatic activity was measured by releasing reducing sugars and immobilized carbon was analyzed by filtration. During the cellulose degradation indicated by the cellulase activity, the dissolved organic carbon daily rate and enzyme activity increased. It was related to a fast hydrolysable fraction of cellulose that contributed to short-term carbon immobilization (ca. 10 days). After approximately 20 days, the dissolved organic carbon and enzyme activity were inversely correlated suggesting that the respiration of microorganisms was responsible for carbon mineralization. Cellulose was an important resource in low nutrient conditions (oligotrophic). However, the detritus quality played a major role in the lignocelluloses degradation (i.e., enzyme activity) and carbon release. <![CDATA[Arbuscular mycorrhizal fungi in <em>Mimosa tenuiflora</em> (Willd.) Poir from Brazilian semi-arid]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200359&lng=es&nrm=iso&tlng=es Abstract Many plant species from Brazilian semi-arid present arbuscular mycorrhizal fungi (AMF) in their rhizosphere. These microorganisms play a key role in the establishment, growth, survival of plants and protection against drought, pathogenic fungi and nematodes. This study presents a quantitative analysis of the AMF species associated with Mimosa tenuiflora, an important native plant of the Caatinga flora. AMF diversity, spore abundance and root colonization were estimated in seven sampling locations in the Ceará and Paraíba States, during September of 2012. There were significant differences in soil properties, spore abundance, percentage of root colonization, and AMF diversity among sites. Altogether, 18 AMF species were identified, and spores of the genera Acaulospora, Claroideoglomus, Dentiscutata, Entrophospora, Funneliformis, Gigaspora, Glomus, Racocetra, Rhizoglomus and Scutellospora were observed. AMF species diversity and their spore abundance found in M. tenuiflora rhizosphere shown that this native plant species is an important host plant to AMF communities from Brazilian semi-arid region. We concluded that: (a) during the dry period and in semi-arid conditions, there is a high spore production in M. tenuiflora root zone; and (b) soil properties, as soil pH and available phosphorous, affect AMF species diversity, thus constituting key factors for the similarity/dissimilarity of AMF communities in the M. tenuiflora root zone among sites. <![CDATA[<em>Candida glabrata</em> among <em>Candida</em> spp. from environmental health practitioners of a Brazilian Hospital]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200367&lng=es&nrm=iso&tlng=es Abstract The incidence of the species Candida albicans and non-albicans Candida was evaluated in a Brazilian Tertiary Hospital from the environment and health practitioners. In a 12-month period we had a total positivity of 19.65% of Candida spp. The most recurring non-albicans Candida species was C. glabrata (37.62%), generally considered a species of low virulence, but with a higher mortality rate than C. albicans. Subsequently, C. parapsilosis (25.74%) and C. tropicalis (16.86%) were the second and third most commonly isolated species. Considering the total samples collected from the emergency room and from the inpatient and the pediatric sector, 19.10% were positive for Candida spp., with the predominance of non-albicans Candida species (89.42%). The high percentage of positivity occurred in the hands (24.32%) and the lab coats (21.88%) of the health care assistants. No sample of C. albicans presented a profile of resistance to the drugs. All the non-albicans Candida species presented a decreased susceptibility to miconazole and itraconazole, but they were susceptible to nystatin. Most of the isolates were susceptible to fluconazole and amphotericin B. As expected, a high resistance rate was observed in C. glabrata and C. krusei, which are intrinsically less susceptible to this antifungal agent. The contamination of environmental surfaces by Candida spp. through hand touching may facilitate the occurrence of Candida infections predominantly in immunocompromised patients. In addition to that, the antifungal agents used should be carefully evaluated considering local epidemiologic trends in Candida spp. infections, so that therapeutic choices may be better guided. <![CDATA[Susceptibility and molecular characterization of <em>Candida</em> species from patients with vulvovaginitis]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200373&lng=es&nrm=iso&tlng=es Abstract Vulvovaginal candidiasis affects women of reproductive age, which represents approximately 15–25% of vaginitis cases. The present study aimed to isolate and characterize yeast from the patients irrespective of the presentation of clinical symptoms. The isolates were subjected to in vitro susceptibility profile and characterization by molecular markers, which intended to assess the distribution of species. A total of 40 isolates were obtained and identified through the CHROMagar, API20aux and by ITS and D1/D2 regions sequencing of DNAr gene. Candida albicans strains were genotyped by the ABC system and the isolates were divided into two genotypic groups. The identity of the C. albicans, C. glabrata, C. guilliermondii, C. kefyr and Saccharomyces cerevisiae isolates was confirmed by the multilocus analysis. The strains of Candida, isolated from patients with complications, were found to be resistant to nystatin but sensitive to fluconazole, amphotericin B and ketoconazole, as observed by in vitro sensitivity profile. The isolates from asymptomatic patients, i.e., the colonized group, showed a dose-dependent sensitivity to the anti-fungal agents, fluconazole and amphotericin B. However, the isolates of C. albicans that belong to distinct genotypic groups showed the same in vitro susceptibility profile. <![CDATA[Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200381&lng=es&nrm=iso&tlng=es Abstract Pan-drug resistant Gram-negative bacteria, being resistant to most available antibiotics, represent a huge threat to the medical community. Colistin is considered the last therapeutic option for patients in hospital settings. Thus, we were concerned in this study to demonstrate the membrane permeabilizing activity of colistin focusing on investigating its efficiency toward those pan-drug resistant isolates which represent a critical situation. We determined the killing dynamics of colistin against pan-drug resistant isolates. The permeability alteration was confirmed by different techniques as: leakage, electron microscopy and construction of an artificial membrane model; liposomes. Moreover, selectivity of colistin against microbial cells was also elucidated. Colistin was proved to be rapid bactericidal against pan-drug resistant isolates. It interacts with the outer bacterial membrane leading to deformation of its outline, pore formation, leakage of internal contents, cell lysis and finally death. Furthermore, variations in membrane composition of eukaryotic and microbial cells provide a key for colistin selectivity toward bacterial cells. Colistin selectively alters membrane permeability of pan-drug resistant isolates which leads to cell lysis. Colistin was proved to be an efficient last line treatment for pan-drug resistant infections which are hard to treat. <![CDATA[<em>DEFB1</em> gene polymorphisms and tuberculosis in a Northeastern Brazilian population]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200389&lng=es&nrm=iso&tlng=es Abstract β-Defensin-1, an antimicrobial peptide encoded by the DEFB1 gene, is known to play an important role in lung mucosal immunity. In our association study we analyzed three DEFB1 functional polymorphisms -52G&gt;A (rs1799946), -44C&gt;G (rs1800972) and -20G&gt;A (rs11362) in 92 tuberculosis patients and 286 healthy controls, both from Northeast Brazil: no association was found between the studied DEFB1 polymorphisms and the disease. However we cannot exclude that this lack of association could be due to the low number of subjects analyzed, as suggested by the low statistical power achieved for the three analyzed SNPs (values between 0.16 and 0.50). <![CDATA[Isolation of recombinant antibodies directed against surface proteins of <em>Clostridium difficile</em>]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200394&lng=es&nrm=iso&tlng=es Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility. <![CDATA[Evaluation of a fungal collection as certified reference material producer and as a biological resource center]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200403&lng=es&nrm=iso&tlng=es Abstract Considering the absence of standards for culture collections and more specifically for biological resource centers in the world, in addition to the absence of certified biological material in Brazil, this study aimed to evaluate a Fungal Collection from Fiocruz, as a producer of certified reference material and as Biological Resource Center (BRC). For this evaluation, a checklist based on the requirements of ABNT ISO GUIA34:2012 correlated with the ABNT NBR ISO/IEC17025:2005, was designed and applied. Complementing the implementation of the checklist, an internal audit was performed. An evaluation of this Collection as a BRC was also conducted following the requirements of the NIT-DICLA-061, the Brazilian internal standard from Inmetro, based on ABNT NBR ISO/IEC 17025:2005, ABNT ISO GUIA 34:2012 and OECD Best Practice Guidelines for BRCs. This was the first time that the NIT DICLA-061 was applied in a culture collection during an internal audit. The assessments enabled the proposal for the adequacy of this Collection to assure the implementation of the management system for their future accreditation by Inmetro as a certified reference material producer as well as its future accreditation as a Biological Resource Center according to the NIT-DICLA-061. <![CDATA[Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry for identification of <em>Clostridium</em> species isolated from Saudi Arabia]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200410&lng=es&nrm=iso&tlng=es Abstract The aim of this study was to identify different Clostridium spp. isolated from currency notes from the Ha’il region of Saudi Arabia in September 2014 using MALDI–TOF-MS. Clostridium spp. were identified by Bruker MALDI–TOF-MS and compared with VITEK 2. The confirmation of the presence of different Clostridium spp. was performed by determining the sequence of the 16S ribosomal RNA gene. In this study, 144 Clostridium spp. were isolated. Among these specimens, MALDI–TOF-MS could identify 88.8% (128/144) of the isolates to the species level and 92.3% (133/144) to the genus level, whereas, VITEK 2 identified 77.7% of the (112/144) isolates. The correct identification of the 144 isolates was performed by sequence analysis of the 500 bp 16S rRNA gene. The most common Clostridium spp. identified were Clostridium perfringens (67.36%), Clostridium subterminale (14.58%), Clostridium sordellii (9%) and Clostridium sporogenes (9%). The results of this study demonstrate that MALDI–TOF-MS is a rapid, accurate and user friendly technique for the identification of Clostridium spp. Additionally, MALDI–TOF-MS has advantages over VITEK 2 in the identification of fastidious micro-organisms, such as Clostridium spp. Incorporating this technique into routine microbiology would lead to more successful and rapid identification of pathogenic and difficult to identify micro-organisms. <![CDATA[Curli fimbria: an <em>Escherichia coli</em> adhesin associated with human cystitis]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200414&lng=es&nrm=iso&tlng=es Abstract Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (ΔcsgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of α-D-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis. <![CDATA[Enhanced production of erythritol and mannitol by <em>Yarrowia lipolytica</em> in media containing surfactants]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200417&lng=es&nrm=iso&tlng=es Abstract Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L-1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L-1 after 5 days, which corresponded to 0.47 g g-1 yield and productivity of 1.1 g L-1 h-1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol. <![CDATA[Antimicrobial activities of six essential oils commonly used as condiments in Brazil against <em>Clostridium perfringens</em>]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200424&lng=es&nrm=iso&tlng=es Abstract Despite recent advances in food production technology, food-borne diseases (FBD) remain a challenging public health concern. In several countries, including Brazil, Clostridium perfringens is among the five main causative agents of food-borne diseases. The present study determines antimicrobial activities of essential oils of six condiments commonly used in Brazil, viz., Ocimum basilicum L. (basil), Rosmarinus officinalis L. (rosemary), Origanum majorana L. (marjoram), Mentha × piperita L. var. Piperita (peppermint), Thymus vulgaris L. (thyme) and Pimpinella anisum L. (anise) against C. perfringens strain A. Chemical compositions of the oils were determined by GC–MS (gas chromatography–mass spectrometry). The identities of the isolated compounds were established from the respective Kováts indices, and a comparison of mass spectral data was made with those reported earlier. The antibacterial activity was assessed from minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the microdilution method. Minimum inhibitory concentration values were 1.25 mg mL-1 for thyme, 5.0 mg mL-1 for basil and marjoram, and 10 mg mL-1 for rosemary, peppermint and anise. All oils showed bactericidal activity at their minimum inhibitory concentration, except anise oil, which was only bacteriostatic. The use of essential oils from these common spices might serve as an alternative to the use of chemical preservatives in the control and inactivation of pathogens in commercially produced food systems. <![CDATA[Effect of ultrasound on survival and growth of <em>Escherichia coli</em> in cactus pear juice during storage]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200431&lng=es&nrm=iso&tlng=es Abstract The aim of this study was to investigate the effectiveness of ultrasound as a conservation method for the inactivation of Escherichia coli inoculated into cactus pear juices (green and purple). Total soluble solids, pH, titratable acidity, and the kinetics of E. coli in cactus pear juices treated by ultrasound (60%, 70%, 80% and 90% amplitude levels for 1, 3 and 5 min) were evaluated over 5 days. Total inactivation was observed in both fruit juices after 5 min of ultrasound treatment at most amplitude levels (with the exception of 60% and 80%). After one and two days of storage, the recovery of bacteria counts was observed in all cactus pear juices. Ultrasound treatment at 90% amplitude for 5 min resulted in non-detectable levels of E. coli in cactus pear juice for 2 days. The parameters of pH, titratable acidity and soluble solids were unaffected. <![CDATA[Occurrence and antimicrobial resistance patterns of <em>Listeria monocytogenes</em> isolated from vegetables]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200438&lng=es&nrm=iso&tlng=es Abstract Although the consumption of fresh and minimally processed vegetables is considered healthy, outbreaks related to the contamination of these products are frequently reported. Among the food-borne pathogens that contaminate vegetables is Listeria monocytogenes, a ubiquitous organism that exhibits the ability to survive and multiply at refrigerated temperatures. This study aimed to evaluate the occurrence of L. monocytogenes in vegetables as well as the antimicrobial resistance of isolates. The results showed that 3.03% of samples were contaminated with L. monocytogenes, comprising 2.22% of raw vegetables and 5.56% of ready-to-eat vegetables. Multiplex PCR confirmed the virulence potential of the isolates. Antimicrobial resistance profiling showed that 50% of the isolates were susceptible to the antibiotics used. The resistance of one isolate to penicillin G, a commonly employed therapeutic agent, and the presence of serotype 4b, a serotype commonly associated with food-borne outbreaks, could be potential health hazards for consumers. <![CDATA[Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200444&lng=es&nrm=iso&tlng=es Abstract Presence of extended spectrum beta-lactamases (ESBL) in bacteria is a growing health concern of global significance. The local, regional, national, and international epidemiological studies for extended spectrum beta-lactamases-producing Enterobacteriaceae and their encoding genes in foods are still incomplete. The objective of this study was to determine the occurrence of extended spectrum beta-lactamases-producing Enterobacteriaceae and the characteristics of their encoding genes from a total of 250 samples of various foods of animal-origin (100 raw chicken meat, 100 raw cow milk, and 50 raw cow milk cheese) sold in Turkey. Overall, 55 isolates were positive as extended spectrum beta-lactamases-producing Enterobacteriaceae. The most prevalent extended spectrum beta-lactamases-producing strain were identified as Escherichia coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), Klebsiella pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) by Vitek® MS. The simultaneous production of extended spectrum beta-lactamases and AmpC was detected in five isolates (9.1%) in E. coli (80%) and E. cloacae (20%). The frequency rates of blaTEM, blaCTX-M, and blaSHV were 96.4%, 53.7%, and 34.5%, respectively. The co-existence of bla -genes was observed in 82% of extended spectrum beta-lactamases producers with a distribution of blaTEM &amp; blaCTX-M (52.7%), blaTEM &amp; blaSHV (20%), blaTEM &amp; blaCTX-M &amp; blaSHV (12.7%), and blaSHV &amp; blaCTX-M (1.8%). The most prevalent variant of blaCTX-M clusters was defined as blaCTX-M-1 (97.2%), followed by blaCTX-M-8 (2.8%). In summary, the analysed foods were found to be posing a health risk for Turkish consumers due to contamination by Enterobacteriaceae with a diversity of extended spectrum beta-lactamases encoding genes. <![CDATA[Bacteria isolated from Korean black raspberry vinegar with low biogenic amine production in wine]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200452&lng=es&nrm=iso&tlng=es Abstract A high concentration of histamine, one of the biogenic amines (BAs) usually found in fermented foods, can cause undesirable physiological side effects in sensitive humans. The objective of this study is to isolate indigenous Acetobacter strains from naturally fermented Bokbunja vinegar in Korea with reduced histamine production during starter fermentation. Further, we examined its physiological and biochemical properties, including BA synthesis. The obtained strain MBA-77, identified as Acetobacter aceti by 16S rDNA homology and biochemical analysis and named A. aceti MBA-77. A. aceti MBA-77 showed optimal acidity % production at pH 5; the optimal temperature was 25 °C. When we prepared and examined the BAs synthesis spectrum during the fermentation process, Bokbunja wine fermented with Saccharomyces cerevisiae showed that the histamine concentration increased from 2.72 of Bokbunja extract to 5.29 mg/L and cadaverine and dopamine was decreased to 2.6 and 10.12 mg/L, respectively. Bokbunja vinegar prepared by A. aceti MBA-77 as the starter, the histamine concentration of the vinegar preparation step was decreased up to 3.66 mg/L from 5.29 mg/L in the wine preparation step. To our knowledge, this is the first report to demonstrate acetic acid bacteria isolated from Bokbunja seed vinegar with low spectrum BA and would be useful for wellbeing vinegar preparation. <![CDATA[Surface response methodology for the optimization of lipase production under submerged fermentation by filamentous fungi]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200461&lng=es&nrm=iso&tlng=es Abstract A Plackett–Burman Factorial Design of 16 experiments was conducted to assess the influence of nine factors on the production of lipases by filamentous fungi. The factors investigated were bran type (used as the main carbon source), nitrogen source, nitrogen source concentration, inducer, inducer concentration, fungal strain (Aspergillus niger or Aspergillus flavus were selected as good lipase producers via submerged fermentation), pH and agitation. The concentration of the yeast extract and soybean oil and the pH had a significant effect (p &lt; 0.05) on lipase production and were consecutively studied through a Full Factorial Design 23, with the concentration of yeast extract and pH being significant (p &lt; 0.05). These variables were optimized using a central composite design, obtaining maximum lipolytic activities with the use of 45 g/L of yeast extract and pH 7.15. The statistical model showed a 94.12% correlation with the experimental data. <![CDATA[Isolation and expression of two polyketide synthase genes from <em>Trichoderma harzianum 88</em> during mycoparasitism]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200468&lng=es&nrm=iso&tlng=es Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88. <![CDATA[Endophytic fungus strain 28 isolated from <em>Houttuynia cordata</em> possesses wide-spectrum antifungal activity]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200480&lng=es&nrm=iso&tlng=es Abstract The aim of this paper is to identify and investigate an endophytic fungus (strain 28) that was isolated from Houttuynia cordata Thunb, a famous and widely-used Traditional Chinese Medicine. Based on morphological methods and a phylogenetic analysis of ITS sequences, this strain was identified as Chaetomium globosum. An antifungal activity bioassay demonstrated that the crude ethyl acetate (EtOAc) extracts of strain 28 had a wide antifungal spectrum and strong antimicrobial activity, particularly against Exserohilum turcicum (Pass.) Leonard et Suggs, Botrytis cinerea persoon and Botrytis cinerea Pers. ex Fr. Furthermore, the fermentation conditions, extraction method and the heat stability of antifungal substances from strain 28 were also studied. The results showed that optimal antifungal activity can be obtained with the following parameters: using potato dextrose broth (PDB) as the base culture medium, fermentation for 4–8 d (initial pH: 7.5), followed by extraction with EtOAc. The extract was stable at temperatures up to 80 °C. This is the first report on the isolation of endophytic C. globosum from H. cordata to identify potential alternative biocontrol agents that could provide new opportunities for practical applications involving H. cordata. <![CDATA[Sugarcane straw as a feedstock for xylitol production by <em>Candida guilliermondii</em> FTI 20037]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200489&lng=es&nrm=iso&tlng=es Abstract Sugarcane straw has become an available lignocellulosic biomass since the progressive introduction of the non-burning harvest in Brazil. Besides keeping this biomass in the field, it can be used as a feedstock in thermochemical or biochemical conversion processes. This makes feasible its incorporation in a biorefinery, whose economic profitability could be supported by integrated production of low-value biofuels and high-value chemicals, e.g., xylitol, which has important industrial and clinical applications. Herein, biotechnological production of xylitol is presented as a possible route for the valorization of sugarcane straw and its incorporation in a biorefinery. Nutritional supplementation of the sugarcane straw hemicellulosic hydrolyzate as a function of initial oxygen availability was studied in batch fermentation of Candida guilliermondii FTI 20037. The nutritional supplementation conditions evaluated were: no supplementation; supplementation with (NH4)2SO4, and full supplementation with (NH4)2SO4, rice bran extract and CaCl2·2H2O. Experiments were performed at pH 5.5, 30 °C, 200 rpm, for 48 h in 125 mL Erlenmeyer flasks containing either 25 or 50 mL of medium in order to vary initial oxygen availability. Without supplementation, complete consumption of glucose and partial consumption of xylose were observed. In this condition the maximum xylitol yield (0.67 g g-1) was obtained under reduced initial oxygen availability. Nutritional supplementation increased xylose consumption and xylitol production by up to 200% and 240%, respectively. The maximum xylitol volumetric productivity (0.34 g L-1 h-1) was reached at full supplementation and increased initial oxygen availability. The results demonstrated a combined effect of nutritional supplementation and initial oxygen availability on xylitol production from sugarcane straw hemicellulosic hydrolyzate. <![CDATA[Enhancement of pigment extraction from <em>B. braunii</em> pretreated using CO<sub>2</sub> rapid depressurization]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200497&lng=es&nrm=iso&tlng=es Abstract Extraction of compounds from microalgae requires cell disruption as a pretreatment to increase extraction yield. Botryococcus braunii is a microalga with a significant content of carotenoids and other antioxidant compounds, such as chlorophylls. Cell disruption of B. braunii using CO2 rapid depressurization was studied as a pretreatment for the extraction of carotenoid and chlorophyll pigments. We studied the effect of temperature (21–49 °C) and pressure (6–13 MPa) during static compression on pigment recovery with supercritical CO2 at 40 °C, 30 MPa and solvent flow of 4.7 L NPT/min. Within the experimental region, the extraction yield of carotenoids and chlorophylls increased by 2.4- and 2.2-fold respectively. Static compression conditions of high pressure and low temperature increased the extraction of carotenoids and especially chlorophylls. We selected 21 °C and 13 MPa as the cell disruption condition, which produced 1.91 g/kg d.s. of carotenoids and 14.03 mg/kg d.s. of chlorophylls. Pretreated microalga gave a 10-fold higher chlorophyll extraction yield compared to the untreated sample. While for carotenoids and tocopherols were 1.25 and 1.14-fold higher, respectively. Additionally, antioxidant activity of pretreated microalga (33.22 mmol TE/kg oil) was significantly higher than the value for the untreated samples (29.11 mmol TE/kg oil) (p ≤ 0.05). Confocal microscopy images showed morphological differences between micro-colonies with and without disruption treatment, suggesting that partial cell disruption by rapid depressurization improved the extraction of microalga compounds. <![CDATA[<em>Mycobacterium paratuberculosis</em> detection in cow's milk in Argentina by immunomagnetic separation-PCR]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200506&lng=es&nrm=iso&tlng=es Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (&lt;0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina. <![CDATA[Phylogenetic characterization of the first Ungulate tetraparvovirus 2 detected in pigs in Brazil]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200513&lng=es&nrm=iso&tlng=es Abstract Ungulate tetraparvovirus 2 (UTV2) , formerly known as porcine hokovirus due to its discovery in Hong Kong, is closely related to a Primate tetraparvovirus (human PARV-4) and Ungulate tetraparvovirus 1 (bovine hokovirus). Until now, UTV2 was detected in European, Asian and North American countries, but its occurrence in Latin America is still unknown. This study describes the first report of UTV2 in Brazil, as well as its phylogenetic characterization. Tissue samples (lymph node, lung, liver, spleen and kidney) of 240 piglets from eight different herds (30 animals each herd) were processed for DNA extraction. UTV2 DNA was detected by PCR and the entire VP1/VP2 gene was sequenced for phylogenetic analysis. All pigs from this study displayed postweaning multisystemic wasting syndrome (PMWS). UTV2 was detected in 55.3% of the samples distributed in the variety of porcine tissues investigated, as well as detected in almost all herds, with one exception. The phylogenetic analysis demonstrated that Brazilian UTV2 sequences were more closely related to sequences from Europe and United States. <![CDATA[A protein expression system for tandem affinity purification in <em>Xanthomonas citri</em> subsp. citri]]> http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822016000200518&lng=es&nrm=iso&tlng=es Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.