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Aim: To investigate the antifungal potential of Macrocybe titans extracts against Candida albicans. Material & methods: Extracts were obtained as aqueous (EfraMat-22 and EfraMat-45) and methanolic/ethyl acetate fractions. Results: Broth microdilution and disk diffusion assays showed that EfraMat-45 provided the best results in terms of minimum inhibitory concentration. Scanning electron microscopy analysis revealed morphological changes and slight damage on the surfaces of cells exposed to EfraMat-45 at the MIC. Fluorescence microscopy analysis of the yeasts showed cell elongation. EfraMat-45 presented high levels of phenolic compounds and flavonoids, high antioxidant activity and absence of in vitro cytotoxicity. Conclusion: The results indicated that the aqueous extract of M. titans is highly promising as an antifungal agent.

The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified. O objetivo deste trabalho foi comparar os métodos de esfregaço fecal direto (DFS) e centrífugo sedimentação (CS) para a pesquisa de oocistos de Cryptosporidium spp. em amostras fecais de bezerros leiteiros. Foram coletadas 114 amostras fecais de bezerros com até seis meses de idade, provenientes de dez propriedades leiteiras localizadas nos municípios de Palotina e Francisco Alves, Paraná. Por meio da análise microscópica, foi possível observar oocistos de Cryptosporidium spp. em 51,75% (59/114) das amostras. Pelo método da CS foi identificada positividade em 48,25% (55/114) das amostras, enquanto nas lâminas pelo método do esfregaço fecal direto (DFS), apenas 6,14% (7/114) foram positivas. Somente 4 amostras foram positivas exclusivamente no método do DFS. Para assegurar que na análise microscópica não houvesse resultados falso-negativos, as 55 amostras negativas para os dois métodos de confecção de lâminas (DFS e CS) foram selecionadas para análise molecular por meio da técnica de Nested PCR. Das 55 amostras submetidas a nPCR, 24% (13/55) apresentaram-se positivas e foram encaminhadas para o sequenciamento genético de uma porção do genoma, o qual possibilitou identificar as espécies de C. parvum, C. bovis e C. ryanae. Além da caracterização das espécies de Cryptosporidium, foi possível identificar a presença de bactérias do gênero Acinetobacter interferindo diretamente nas amostras analisadas. A análise microscópica revelou maior sensibilidade quando o método de CS foi usado para a confecção dos esfregaços fecais, entretanto, amostras negativas por meio dessa metodologia apresentaram resultados positivos na nPCR. Desta forma, para a confirmação dos casos de Cryptosporidium spp., é indicado a realização da análise molecular. Novos estudos são necessários para se comprovar a especificidade dos primers utilizados, uma vez que na nPCR o resultado obtido foi positivo, porém ao realizar o sequenciamento genético houve a identificação de Acinetobacter spp.

This work aimed to perform the molecular identification of fungi mango fruits, in addition to verifying the effect of the essential oils for microbial control. Pericarp segments containing injured and healthy areas of mango fruits were initially disinfested, and then incubated in PDA culture medium at 25 °C. From all isolated material, UDC03, UDC04, UDC09 and UDC12 were preselected, which presented homogeneous cultures. The UDC04 and UDC12 isolates were submitted to the sequencing of the ITS region for presenting mycelial growth both greater and morphologically distinct of fungi from Colletotrichum genus. When BLAST is used in species identification, it was observed that UDC04 and UDC12 belong to Botryosphaeria dothidea and Neofusicoccum parvum, respectively. Regarding the control of UDC04, it was observed that BDA culture medium containing 1.5 to 2% of lemongrass and eucalyptus oils resulted in complete inhibition. For UDC12, mycelial growth inhibition was detected with the use of 1% and 1.5% essential oils of eucalyptus and lemongrass, respectively. The chia essential oil was not effective for the isolates control. Finally, the results reveal the importance of molecular identification of these phytopathogens associated with the establishment of an alternative control of fruits due to problems of contamination with chemical products.

This work explores the production of biohydrogen from brewery wastewater using as inoculum a culture produced by natural fermentation of synthetic wastewater and Klebsiella pneumoniae isolated from the environment. Klebsiella pneumoniae showed good performance as inoculum, as evaluated using assays of between 9 and 16 cycles, with durations of 12 and 24 h, carbohydrate concentrations from 2.79 to 7.22 g L−1, and applied volumetric organic loads from 2.6 to 12.6 g carbohydrate L−1 day−1. The best results were achieved with applied volumetric organic loads of 12.6 g carbohydrate L−1 day−1 and cycle length of 12 h, resulting in mean volumetric productivity of 0.88 L H2 L−1 day−1, maximum molar flow of 10.80 mmol H2 h−1, and mean yield of 0.70 mol H2 mol−1 glucose consumed. The biogas H2 content was between 18 and 42%, while the mean organic compounds removal and carbohydrate conversion efficiencies were 23 and 81%, respectively. The inoculum produced by natural fermentation was not viable.

This paper presents a strategy for isolation, identification and evaluation of microalgae strains synthesizing biologically active compounds (BAC) with antimicrobial activity against food-borne and other pathogens. Water samples containing microalgae were initially submitted to the enrichment process, followed by standard procedure for obtaining pure culture. Purified DNA from the microalgae isolates was used for sequencing the amplified 18S rDNA genomic region, which resulted in the identification of Poterioochromonas malhamensis, Chlorella sp., Micractinium sp., Tetradesmus sp. and Desmodesmus sp. All isolated strains were cultivated in 1-L flat-plate PBRs, with the Poterioochromonas malhamensis being the only one to show antibacterial activity in agar diffusion assay. Further, PPEQ-01strain was cultured under different types and scales of PBR, intensity and spectrum of the incident light, in order to study and prove the antibacterial potential of the strain. Ethanolic and methanolic supernatants, or hydrophilic and methanolic extracts produced from biomass cultivated in different conditions led to formation of bacterial growth inhibition halos around the impregnated disks used in the agar diffusion test. The hydrophilic extract, obtained from biomass cultured under green illumination, exhibited stronger activity against all tested pathogenic bacteria as compared to the other extracts. The results obtained with the new isolated Poterioochromonas malhamensis strain proved efficiency of the innovative complex approach applied to BAC aiming the identification of microalgae that present antimicrobial activity.

Plant growth-promoting bacteria are ecological alternatives for fertilization, mainly for gramineous. Since plant x bacteria interaction is genotype and strain dependent, searching for new strains may contribute to the development of new biofertilizers. We aim to characterize plant growth-promoting capacity of Leclercia adecarboxylata strain Palotina, formerly isolated by our group in corn. A single isolated colony was taken and its genome was sequenced using Illumina technology. The whole genome was compared to other Leclercia adecarboxylata strains, and their biological and growth-promoting traits, such as P solubilization and auxin production, were tested. Following that, a 4.8 Mb genome of L. adecarboxylata strain Palotina was assembled and the functional annotation was carried out. This paper is the first to report the genes associated with plant growth promotion demonstrating in vitro indole acid production by this strain. These results project the endophyte as a potential biofertilizer for further commercial exploitation.

O genero Scopulariopsis spp. engloba fungos cosmopolitas que normalmente sao encontrados na poeira domestica ou no solo. Raramente causam infeccao em humanos, todavia podem ocasionar onicomicose, otomicose e panoftalmite, se introduzido nos olhos devido a feridas penetrantes. Em determinadas situacoes podem resultar em micoses cutâneas, profundas e potencialmente infeccoes sistemicas, estas normalmente associadas a pacientes imunodeprimidos (COX; IRVING, 1993). Relatar a ocorrencia de Scopulariopsis spp. proveniente de swab lesional de um felino ( Felis catus domesticus ) em tratamento para esporotricose na cidade de Guaira, estado do Parana, Brasil. Um felino macho, nao-castrado, escore corporal de 2,5/5 e dois anos de idade, foi submetido a colheita de material lesional para isolamento de colonia de Sporothrix spp. O mesmo residia em uma casa com outros 50 gatos, quatro galinaceos, tres caes e possuia acesso a rua, contudo ha uma semana se encontrava isolado juntamente com outros quatro felinos, tambem com esporotricose, em um comodo dentro do ambiente interno da casa da tutora que tambem estava em tratamento para a doenca. O animal apresentava lesoes ulcerativas em ponta de orelha, na regiao interescapular, regiao do metacarpo esquerdo, face e testiculo e se encontrava em tratamento utilizando itraconazol 100mg, iodeto de potassio 30mg e silimarina 100mg. Procedeu-se a colheita do material com swab nasal, swab de unhas e swab lesional, todas as amostras foram armazenadas em solucao salina 0,9% esteril e refrigerados, ate o momento do processamento. O material foi estriado em placa contendo agar Sabouraud com gentamicina, e submetido a crescimento em temperatura ambiente. Apos sete dias, verificou-se o crescimento de uma colonia fungica de coloracao brancacenta, irregular e algodonosa no estriado proveniente de swab lesional. Procedeu-se entao o teste da fita adesiva corada com lactophenol cotton blue e analisada por meio de microscopia optica. Na microscopia foi possivel verificar hifas septadas, conidioforos com aneis hialinos, conidios piriformes com base truncada, de parede lisa em corrente, sendo caracteristico do genero Scopulariopsis spp. Esses dados demonstram que mais estudos referentes aos mecanismos de patogenicidade deste fungo devem ser realizados, visto que a presenca deste no local da lesao causada pela multiplicacao do Sporothrix spp. pode estar relacionada ao desenvolvimento de micoses cutâneas, o que pode favorecer o aumento das lesoes ocasionadas pelo Sporothrix , levando ao questionamento sobre a eficacia do tratamento no paciente felino.

Background Yeasts of the Candida parapsilosis complex have frequently been reported as agents of fungal infection in Brazil and worldwide, most of the cases are related to hospital-acquired infection. C. parapsilosis is the third most common cause of candidemia, and the hands of hospital workers as well as hospital surfaces have been suggested as possible sources. Objectives In this study we verified the frequency of C. parapsilosis on the hands of workers and on surfaces in the adult intensive care unit (AICU) of a tertiary hospital in Parana-Brazil. Methods Surface samples were collected with swabs moistened with saline, and a plastic bag with distilled water was used to collect samples from hands. The yeasts were identified by morphology, MALDI-TOF mass spectrometry and PCR-RFLP of the secondary alcohol dehydrogenase-encoding gene (SADH) after digestion with the restriction enzyme BanI. Results and conclusions A total of 223 yeast were found, of which 101 (45.29%) were identified as C. parapsilosis sensu stricto. Of these, 46.66% (n = 35) were found on surfaces and 44.59% (n = 66) on the hands of the employees. The analysis of C. parapsilosis strains by microsatellite loci (CP1, CP4, CP6 and B5) showed 80 different genotypes. Their antifungal susceptibility profile, evaluated by the microdilution broth method, revealed that C. parapsilosis was sensitive to amphotericin B, fluconazole and voriconazole, but not to micafungin. The results revealed the heterogeneity of the yeast population, suggesting that there is no common source of contamination in the AICU of this hospital.

The COVID‐19 pandemic has led to the adoption of social quarantine measures in numerous countries. In this context, both COVID‐19 and quarantine can have a substantial impact in the mental health of affected populations. Health care services have had to adapt themselves to this new reality. Fil: Dorman, Guido. Instituto de Neurología Cognitiva; Argentina Fil: Alvarez Dengra, Agustín. Instituto de Neurología Cognitiva; Argentina Fil: Fiorini, Adriana. Instituto de Neurología Cognitiva; Argentina Fil: Failla, Belén. Instituto de Neurología Cognitiva; Argentina Fil: Vallejos, Florencia. Instituto de Neurología Cognitiva; Argentina Fil: Pontello, Noelia. Instituto de Neurología Cognitiva; Argentina Fil: Roca, María. Instituto de Neurología Cognitiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bustin, Julian. Instituto de Neurología Cognitiva; Argentina

This work aimed to use ultraviolet (UV) radiation to prevent microbial growth in diesel fuel with different compositions. Commercial samples of ultra-low-sulfur diesel (ULSD) and high-sulfur diesel (HSD) fuels with 11% of biodiesel were used as purchased. These samples were incorporated of 20% biodiesel and different amounts of free water (no addition, 2500, and 10,000 mL.m−3). All samples were stored for ten days and subjected to UV-C radiation treatment (55 W, 253 nm) for 360 min to reduce the number of microorganisms. Physicochemical parameters, such as water content, kinematic viscosity, density, and oxidative stability, were measured before and after UV-C treatment. The presence of microorganisms was detected by UV–vis spectrophotometry (600 nm) during the simulated storage, before and after UV-C treatment. An increase in the number of microorganisms was observed during the storage period due to the water content. The UV-C radiation reduced the microorganisms, especially in samples with high water content. Low sulfur levels did not affect the efficiency of the treatment. Significant variations in kinematic viscosity and oxidative stability were observed after the treatment. The presence of microorganisms was reduced by more than 50% after the incidence of UV-C radiation. Therefore, UV-C radiation is a promising technique for microbiological control in diesel fuel. These effects can last up to ten days after the first incidence of radiation.

Aim: To evaluate if radiation used in radiotherapy can cause changes in the virulence potential of Candida tropicalis ATCC 750. Materials & methods: C. tropicalis was exposed in vitro to identical dose and scheme of irradiation would be used in patients with head and neck cancer. Some virulence parameters were analyzed before and after irradiation. Results: Colony morphologies were irreversibly affected by irradiation. Increase in growth rate, filamentation, adhesion on cell lines and phagocytosis process were also observed. Overall the irradiated C. tropicalis cells became more efficient at causing systemic infection in mice. Conclusion: γ-radiation induced important changes in C. tropicalis increasing its virulence profile, which could directly affect the relationship between yeasts and hosts.

Vulvovaginal candidiasis (VVC) is one of the most frequent female genital disorders and Candida glabrata is the second most common agent. Current study was aimed to study the susceptibility to antifungal agents of C. glabrata isolated from vaginal samples and some virulence attributes in order to better understand why this species is emerging as the main VVC agents. A total of 60 C. glabrata vaginal isolates were included in this study. Firstly they were screened by susceptibility tests to antifungal agents. The isolates that showed sensitivity or resistance to fluconazole were evaluated for their virulence potential, including ability to adhere to polystyrene and vaginal ring, cell surface hydrophobicity (CSH) and capacity to form biofilm. Candida glabrata isolates varied significantly in adherence capacity, biofilm formation and CSH. However, it was possible to observe that isolates resistant to fluconazole adhered more efficiently to the vaginal ring and were statistically more able to form biofilm. These results allow hypothesizing that C. glabrata is an emergent agent in VVC probably because the treatment with fluconazole selects this species. But once adhered, yeasts remain on biotic or abiotic surfaces causing colonization or VVC symptomatology.

As bactérias são os mais abundantes organismos em nosso planeta, são encontradas em diversos habitats e podem ser usadas para o benefício da humanidade. Dentre elas, as bactérias celulolíticas que apresentam um metabolismo capaz de realizar a degradação da celulose, por meio da produção de enzimas celulases tem grande potencial no setor industrial. Para a identificação de novas estirpes de bactérias celulolíticas deve-se estabelecer um protocolo qualitativo. Neste trabalho, foi empregada uma metodologia modificada baseada no método de Theater e Wood (1989), obtendo-se estirpes bacterianas isoladas de solo na região oeste do Paraná. Como resultados, foi obtida a caracterização de estirpes positivas para a degradação da celulose, demostrando a interação da bactéria com a celulose (CMC) com a formação de halos de degradação.

Little is known about the use of microbial lipases for the treatment of beef industry effluent. It is essential to develop techniques for the sustainable management of this waste, given its high polluting potential, especially due to the high concentrations of oils and greases (O&G) present. Therefore, this study investigates the pretreatment of slaughterhouse effluent using lipase-producing microorganisms isolated from the waste itself. Firstly, a physicochemical characterization of the effluent was performed, followed by isolation and identification of a lipase-producing bacterium, which was used in a pretreatment step. A central composite rotational design (CCRD) was applied, considering the independent variables pH, temperature, and inoculum concentration, resulting in 17 assays, with lipolytic activity, O&G, and free fatty acids (measured during 72 h) as the response variables. The slaughterhouse effluent presented high polluting potential, with an O&G concentration of 2553.3 mg L−1 and COD of 8914.4 mg L−1. The isolated bacterium was identified as Aeromonas hydrophila. After 72 h of the experiment, lipolytic activity of 1.40 U mL−1, 59% O&G removal (pH: 7.0, temperature: 30.0 °C and inoculum concentration: 10%), and production of 36.0 μmol mL−1 of free fatty acids were obtained. It could be concluded that cattle slaughterhouse effluent is an excellent source of lipase-producing microorganisms and that the use of enzymes for the treatment of this waste is a promising technology.

Several waste sources have been studied as substrate sources for the production of biogas rich in hydrogen and for the isolation of bacteria capable of fermenting several substrates for the same purpose. Nonetheless, to simplify the process and minimize production costs, it is important to seek alternatives both for the use of microbial consortia using crude waste and for the use of substrates also in their crude form, without the need for purification. The aim of this study was to use only waste as inoculum and substrate for the biological production of hydrogen. Thus, samples from anaerobic ponds of a poultry slaughterhouse were used as inoculum. Sucrose, pure glycerol (in initial tests) and crude glycerol (inserted in blends with pure glycerol) were used as substrates. H2 production experiments were conducted in batches, using a reactor kept in an anaerobic environment for 11 days, at 35°C, under orbital agitation at 150 rpm. To analyse the composition of the biogas and the presence of soluble metabolic products (SMPs), samples of the headspace gases generated and of the reaction medium were collected. The results using sucrose as substrate indicated that the inoculum under study has potential for bio-H2 production, as it produced CH4-free biogas containing 50–60% H2. The inoculum was also shown to be adaptable to the use of glycerine as a substrate, producing biogas with similar characteristics to those obtained from sucrose degradation; however, it required a longer acclimatization period, and thus more in-depth study is required.

Staphylococcus aureus is one of the most common microorganisms responsible for high morbidity and mortality in humans and animals. Methicillin-resistant S. aureus are responsible for several outbreaks worldwide and therapeutic arsenal has become scarce. The present investigation verified the epidemiological profile of S. aureus strains isolated from the veterinary hospital staff, from dairy cattle workers and also from milk samples of dairy cattle presenting mastitis. Samples were characterized phenotypically by antibiogram, catalase, and coagulase tests, and also by Voges-Proskauer test. The isolated strains were characterized genotypically by specific Polymerase Chain Reaction and Amplified Ribosomal DNA Restriction Analysis (ARDRA). From the 218 isolated strains, 27 were identified as S. aureus (12%), four of them were resistant to oxacillin and two of them were classified as MRSA (Methicillin-resistant S. aureus). The prevalence of isolated strains among animal personnel care was low (2%) but all MRSA isolates were found among the clinical staff. Results of ARDRA pointed out that S. aureus strains isolated from different animal care personnel were grouped in the same cluster when HindIII and HinfII restriction enzymes were used. When ARDRA was performed with HaeIII enzyme, the formation of two clusters was observed, but the isolated strains were not correlated. The prevalence of S. aureus strains isolated was higher in clinical staff and the biochemical and molecular assays of them presented 100% of correlation. Staphylococcus aureus está entre os microrganismos que apresentam as maiores taxas de morbidade e mortalidade em seres humanos e animais. Linhagens de S. aureus resistentes a meticilina podem causar surtos de infecção em todo o mundo, o que contribui para a escassez de arsenal terapêutico. Este trabalho analisou o perfil epidemiológico de estirpes de S. aureus isoladas de pessoas que trabalham em contato com animais em um hospital veterinário com gado leiteiro e também em amostras de leite de vacas acometidas por mastite. As estirpes de S. aureus isoladas foram caracterizadas fenotipicamente por meio de antibiograma, testes de catalase e coagulase, e pelo teste de Voges-Proskauer. As amostras também foram caracterizadas genotipicamente pela técnica de Analise de Restricao de DNA Ribossomico Amplificado (ARDRA-PCR). Das 218 estirpes isoladas, 27 foram identificados como S. aureus (12%). Entre essas, quatro estirpes foram resistentes a oxacilina e duas classificadas como SARM (S. aureus resistente a meticilina). A ocorrência de estirpes de S.aureus isoladas entre o pessoal que trabalha em contato com os animais foi baixa (2%), mas estirpes identificadas como SARM foram encontradas na equipe clínica. As análises de ARDRA realizadas com as enzimas de restrição HindIII e HinfII demonstraram que S. aureus isolados de diferentes indivíduos que trabalhavam com animais foram agrupados no mesmo cluster. Quando a ARDRA foi realizada com HaeIII foi observada formação de dois grupos, mas os isolados não se correlacionaram. Conclusão: a ocorrência de estirpes de S. aureus isoladas foi maior na equipe clínica, apresentando também correlação de 100% nos ensaios bioquímicos e moleculares.

Background: In recent years, very few effective drugs against Mycobacterium tuberculosis have emerged, which motivates the research with drugs already used in the treatment of tuberculosis. Ethambutol is a bacteriostatic drug that affects cell wall integrity, but the effects of this drug on bacilli are not fully exploited. Objective: Based on the need to better investigate the complex mechanism of action of ethambutol, our study presented the proteome profile of M. tuberculosis after different times of ethambutol exposure, aiming to comprehend the dynamics of bacilli response to its effects. M. tuberculosis was exposed to ½ MIC of ethambutol at 24 and 48 hours. The proteins were identified by MALDI-TOF/TOF. Results: The main protein changes occurred in metabolic proteins as dihydrolipoyl dehydrogenase (Rv0462), glutamine synthetase1 (Rv2220), electron transfer flavoprotein subunit beta (Rv3029c) and adenosylhomocysteinase (Rv3248c). Conclusion: Considering the functions of these proteins, our results support that the intermediary metabolism and respiration were affected by ethambutol and this disturbance provided proteins that could be explored as additional targets for this drug.

The effect of a meropenem-ciprofloxacin combination (MCC) on the susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa (MRPA) clinical isolates was determined using checkerboard and time-kill curve techniques. Structural changes and differential gene expression that resulted from the synergistic action of the MCC against one of the P. aeruginosa isolates (1071-MRPA]) were evaluated using electron microscopy and representational difference analysis (RDA), respectively. The differentially expressed, SOS response-associated, and resistance-associated genes in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC were monitored by quantitative PCR. The MCC was synergistic against 25% and 40.6% of MDR P. aeruginosa isolates as shown by the checkerboard and time-kill curves, respectively. The morphological and structural changes that resulted from the synergistic action of the MCC against 1071-MRPA were a summation of the effects observed with each antimicrobial alone. One exception included outer membrane vesicles, which were seen in a greater amount upon ciprofloxacin exposure but were significantly inhibited upon MCC exposure. Cell wall- and DNA repair-associated genes were differentially expressed in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC. However, some of the RDA-detected, resistance-associated, and SOS response-associated genes were expressed at significantly lower levels in 1071-MRPA exposed to the MCC. The MCC may be an alternative for the treatment of MDR P. aeruginosa . The effect of this antimicrobial combination may be not only the result of a summation of the effects of meropenem and ciprofloxacin but also a result of differential action that likely inhibits protective mechanisms in the bacteria.

UNLABELLED This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty-seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Parana state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequence PCR (REP-PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC-PCR and REP-PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin-producing E. coli in milk produced in the region studied. PRACTICAL APPLICATION Knowledge about the presence of diarrheagenic Escherichia coli in pasteurized milk is important developing and implementing control measures in milk and dairy production.

Since abiotic and biotic factors can compromise the survival of bacteria and their viability, encapsulation of cells in biodegradable gel matrices, a biological macromolecule, is one alternative to have their shelf life extended. Here, it was developed a gel-based formulation of the bioinoculant Azospirillum brasilense strain AbV5 and determined the effect of trehalose and humic acid supplementation in viability and survival of bacteria. For each 2 ml of sodium alginate solution (3%), 1 ml of the inoculum was extruded in a solution containing sodium alginate complexed with calcium chloride, forming calcium alginate beads. Supplements were used in a ratio of 2:2:1. Treatments were peat; alginate; alginate + humic acid; alginate + trehalose 0.1 M; alginate + trehalose 1 M. Morphometric aspects, survival rate and viability were determined in 9 storage periods (3, 5, 7, 14, 21, 30, 45, 60, 90 days). As results, beads were able to sustain the growth of A. brasilense for 90 days. Shelf life quality decreased in all treatments and peat remained the best carrier. Encapsulation, despite promoting the greatest losses in the survival of bacteria in the first days, ensured better cell viability. Trehalose in low concentrations (0.1M) improved cell viability during storage, optimizing plant inoculation.

Identification of the nucleotide consensus sequence in mammalian replication origins is a difficult and controversial problem. The hypothesis that local DNA topology could be involved in recognition by replication proteins is an exciting possibility. Secondary DNA structures, including intrinsically bent DNA, can be easily detected, and they may indicate a specific pattern in or near mammalian replication origins. This work presents the entire mapping of the intrinsically bent DNA sites (IBDSs), using in silico analysis and a circular permutation assay, of the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the mammalian amplified AMPD2 gene domain. The results show that each origin presents an IBDS that flanks the straight core of these DNA replication sites. In addition, the in silico prediction of the nucleosome positioning reveals a strong indication that the center of an IBDS is localized in a nucleosome-free region (NFR). The structure of each of these curved sites is presented together with their helical parameters and topology. Together, the data that we present here indicate that the oriGNAI3 origin where preferential firing to the replication initiation events in the amplified AMPD2 domain occurs is the only origin that presents a straight, narrow region that is flanked on both sides by two intrinsically bent DNA sites within a short distance (~300 bp); however, all of the origins present at least one IBDS, which is localized in the NFR region. These results indicate that structural features could be implicated in the mammalian DNA replication origin and support the possibility of detecting and characterizing these segments.

DNA replication is a crucial process for species survival, nevertheless it is not clear which factors define origin selection in multicellular eukaryotes. Developmental gene amplification systems, such as the one described during ovarian follicles development in Drosophila melanogaster, are useful tools for studying of DNA replication process in these organisms. We previously described that the well characterized third chromosome amplified domain of D. melanogater displays three intrinsically bent DNA sites: b1, localized at an amplification control element (ACE3), b2 and b3, both localized at the preferential origin ori-β. This proposal aimed to construct a Drosophila transformation vector, which contains a short deletion at the ACE3, in order to reduce the intrinsically bent DNA site b1, and analyze the functional role of this site in the gene amplification process. Through a series of cloning steps, we obtained a Big Parent vector derivative, containing a deletion at the positions 176-180 bp, inside the ACE3. The generation of a Drosophila transformation vector displays a reduced intrinsically bent DNA site in the third chromosome amplified domain, it will allow the analysis of the functional role of this curvature in developmental gene amplification, providing new insights on replication initiation in D. melanogaster and the function of intrinsically bent DNA sites.

Aim: To study the proteomic and morphological changes in Mycobacterium tuberculosis H37Rv exposed to subinhibitory concentration of isoniazid (INH). Materials & methods: The bacillus was exposed to ½ MIC of INH at 12, 24 and 48 h. The samples’ cells were submitted to scanning electron microscopy. The proteins were separated by 2D gel electrophoresis and identified by MS. Results: INH exposure was able to alter the format, the multiplication and causing a cell swelling in the bacillus. The major altered proteins were related to the virulence, detoxification, adaptation, intermediary metabolism and lipid metabolism. Conclusion: The protein and morphological changes in M. tuberculosis induced by ½ MIC INH were related to defense mechanism of the bacillus or the action of INH therein.

HMGB-like proteins are architectural chromatin factors, and their function is heavily dependent on their ability to interact with DNA (especially non-canonical DNA structures). HMGB1 is involved in many DNA processes, and dysregulation of HMGB protein expression has profound effects on cellular transcription, resulting in severe developmental defects as well as cancer. During DNA replication, elements that form the origin are still not well defined in metazoans. Sites with A (adenine) or T (thymine) repeats cause intrinsic curvatures in the DNA and are described to be involved in the replication machinery by providing binding sites to replication proteins. As a result, the DNA molecule shows intrinsically bent DNA sites, caused by periodic repeats of 2 or more As/Ts (dA/dT) as well as intrinsically non-bent DNA sites (INBDs), due to a succession of curvatures that cancel each other. In the present study, we mapped 11 INBDSs present in the AMPD2 gene that are related to each replication origin (oriGNAI3, oriC, oriB, and oriA). Following characterization of INBDSs, we tested the ability of HMGB1 to bind to the bent (b1, b2, b4a, b4b, b5, b6, b7, and b8) and non-bent DNA fragments (nb7, nb11, nb1, nb2, nb4, and nb5) via electrophoretic mobility shift assays. All fragments showed efficient binding to HMGB1. However, the non-bent DNA fragments nb2, nb4, and nb5 showed slightly reduced binding efficiency.

Objectives To determine the frequency of yeast in the oral cavity of patients with chronic renal failure, undergoing hemodialysis (PCRFH); identification and antifungal susceptibility profile of yeast and demographic profile of patients. Methods We performed mouthwash in 146 PCRFH; the rinse fluid was collected and cultured, yeasts grown were identified by phenotypic and molecular methods. The antifungal susceptibility profile was determined against nystatin, amphotericin B, fluconazole, voriconazole, and caspofungin based in Clinical and Laboratory Standards Institute (document M27-A3). Results Positive culture was observed in 39% of patients, of whom 53% were women; the median of dialysis time was 2.9 years. The age of the colonized patients varied between 26 and 84 years, with a median of 52.5 years. PCRFH over 45 years were significantly more colonized (P = 0.0108) as well as denture wearers (84.0%). We isolated 81 yeasts, predominantly Candida albicans (63%) followed by Candida glabrata. In general, yeasts were sensitive to the evaluated antifungal agents, but there was significant variation in the minimum inhibitory concentration, especially among non-C. albicans Candida (NCAC) compared to fluconazole, caspofungin, and amphotericin B. NCAC required significantly higher concentrations of fluconazole (P

Previous reports demonstrated that actin is necessary for nucleocapsid transport and viral gene expression during nucleopolyhedrovirus infection of Bombyx mori. The first intron of B. mori A3 actin contains a cryptic promoter that drives expression of a rare isoform. We detected differences in the size and nucleotide composition of the first intron of the A3 actin gene from B. mori strain C24A, which is more resistant to nucleopolyhedrovirus than the M11A strain (22 and 95% lethality, respectively). We sought to determine if resistance to BmMNPV infection and the A3 actin promoter structure are correlated. Intrinsically bent DNA sites in these sequences, which determine curved structures, were analyzed by electrophoretic mobility assays and the helical parameters ENDS ratio, roll and twist. We found both fragments to have non-centralized bent DNA sites with distinct ENDS ratio values, nucleotide positions and two-dimensional structures. Additionally, a conformational-sensitive gel electrophoresis assay identified an allelic variation found in strain M11A that is absent in strain C24A. These data suggest that A3 actin intronic sequence variations impair virus propagation and are markers of BmMNPV-resistant populations.

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5) proximal to oriB. The helical parameters show that each bent DNA site is curved in a left-handed superhelical writhe. A 2D projection of 3D fragment trajectories revealed that oriGNAI3 is located in a relatively straight segment flanked by bent sites b1 and b2, which map in previously identified Scaffold/Matrix Attachment Region. Sites b3 and b4 are located approximately 2 kb downstream and force the fragment into a strong closed loop structure. The b5 site is also located in an S/MAR that is found just downstream of oriB. [BMB reports 2010; 43(11): 744-749]

Multiple sequence alignments of the Bombyx mori fibroin light chain gene (fib-L) from hybrids and from Chinese and Japanese strains demonstrated that 51.6% of the fib-L third intron is conserved. One of these conserved segments, 41 bp long, contains the sequence CGTTATTATACATATT, which is duplicated in the B. mori Nd-s(D) mutant. In the present work, electrophoretic mobility assays and computational analyses revealed a major peak of intrinsic bent DNA within the segment that undergoes breakage in the previously-described Nd-s(D) mutation. This result suggested that this intrinsically-curved region might mediate DNA cleavage and enhance recombination events in the third intron of the Bombyx mori fib-L gene.

The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00 degrees values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

The naturally high minimum inhibitory concentration exhibited by echinocandins against Candida parapsilosis has been known since the first introduction of these antifungal agents. Despite this awareness, clinical failures have not been reported; consequently, the resistance of C. parapsilosis to echinocandins remains unexplored. We exposed 30 isolates of C. parapsilosis to echinocandins (caspofungin, micafungin, and anidulafungin) in vitro and studied the effects of this exposure. After 60 exposures, 80, 67, and 60 % of the isolates changed from susceptible to non-susceptible to caspofungin, micafungin, and anidulafungin, respectively. In addition, four strains exhibited cross-resistance to all three echinocandins. Based on the M27-A3 (CLSI, 2008) and M27-S4 (CLSI, 2012) techniques, the susceptibility of the resistant strains to other antifungal agents was assayed. All of the tested echinocandin-resistant strains were susceptible to amphotericin B, and the resistance rate to fluconazole, voriconazole, and flucytosine was 73.3, 43.3, and 20 %, respectively. The exposure of C. parapsilosis to the three echinocandins generated cross-resistant strains and an unexpected in vitro resistance to azoles and flucytosine.

A agricultura é um dos fatores mais rentáveis no Brasil, além de ser uma das maneiras de sustento das famílias rurais e urbanas. As culturas mais cultivadas no Brasil são soja, milho e arroz. Fungos do gênero Aspergillus podem contaminar grãos de soja e milho durante seu armazenamento e pode produzir micotoxinas, como as aflatoxinas, prejudiciais a saúde de quem os consome. O objetivo desse trabalho foi identificar, através de técnicas moleculares, fungos da espécie Aspergillus flavus e estabelecer uma correlação do tempo de armazenamento dos grãos com a contaminação fúngica. Foram coletadas sementes de milho e soja em diferentes silos da região de Maringá-PR, e posteriormente realizada cultura para fungos em meio de cultivo específico. Após o isolamento e caracterização dos fungos em meio de cultura, foi realizada a extração do DNA genômico e amplificação, pela Reação em Cadeia da Polimerase (PCR), das regiões IGS e β-tubulina. Os produtos de amplificação da região IGS foram submetidos a clivagem com a enzima de restrição BgIII, para a confirmação da espécie, pela técnica de PCR-RFLP. Das amostras coletadas 68,75% estavam contaminadas por Aspergillus spp., seguindo análises microbiológicas. Destas, 31,25% resultou positivo para a espécie Aspergillus flavus. Os outros 37,5% foram dadas como negativas, por vários fatores, um deles a sensibilidade da técnica de PCR. Foi possível concluir que um pouco menos da metade das amostras encontrava-se contaminada por Aspergillus spp.

Vulvovaginal candidiasis (VVC) is a disease caused by the abnormal growth of yeast-like fungi in the mucosa of the female genital tract. Candida albicans is the principal etiological agent involved in VVC, but reports have shown an increase in the prevalence of Candida non-C. albicans (CNCA) cases, which complicates VVC treatment because CNCA does not respond well to antifungal therapy. Our group has reported the in vitro antifungal activity of extracts from Sapindus saponaria L. The present study used scanning electron microscopy and transmission electron microscopy to further evaluate the antifungal activity of hydroalcoholic extract from S. saponaria (HE) against yeast obtained from VVC and structural changes induced by HE. We observed the antifungal activity of HE against 125 vaginal yeasts that belonged to four different species of the Candida genus and S. cerevisae. The results suggest that saponins that are present in HE act on the cell wall or membrane of yeast at the first moments after contact, causing damage to these structures and cell lysis.

Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria (e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs.

We have identified bent DNA sites in the distal and proximal DNA puff BhC4-1 amplified gene promoter region of Bradysia hygida. The 2D modeling of the 3D DNA path and the ENDS ratio values calculated in this promoter region resulted in the identification of ten pronounced bent sites named BhC4Bˇ 9t oa 1. The 1847 bp fragment (ˇ 3697 toˇ 1850) in relation to the transcription start site shows multiple bending sites, BhC4Bˇ 9t oBhC4Bˇ 4, with periodicity300 bp. The analysis of the other identified bent region, starting at positionˇ 957, reveals that the BhC4B a 1 bent site colocalizes with the putative BhC4-1 minimal promoter. The sequence analysis of bent site BhC4Bˇ 4 shows a distribution of dA.dT at10 bp intervals between the middle of each tract, but intervals with more than one turn,20 bp, two helix turns, were detected in the other bent sites described here. The bent sites BhC4Bˇ 6 and BhC4B ˇ 4, contain two consensus sequences, with 60 bp each. The apparent molecular weight of fragments in the BhC4-1 promoter region were estimated in agarose gels and compared with the data obtained in polyacrylamide gels without and with ethidium bromide. The mobility reduction ratios (R-values) were determined, and a high R-value, 1.80, for a 1215 bp fragment in the distal promoter region and a 1.23 significant R-value for a 662 bp fragment in the proximal segment were found. To further analyze the predicted bent DNA sites in these fragments, the 2D trajectories of the 3D DNA path and other parameters, AT percentage, roll angle, ENDS ratio and DG, were determined. The role of these bent sites in the BhC4-1 transcription regulation is discussed. J. Cell. Biochem. 83: 1-13, 2001. fl 2001 Wiley-Liss, Inc.

O objetivo desse trabalho foi verificar os efeitos da desnutrição protéica sobre a morfologia, a morfometria e a densidade dos neurônios do plexo mientérico. Foi utilizado o íleo de 10 ratos. Para este estudo, cinco ratos com 90 dias receberam, durante 120 dias, ração hipoprotéica (grupo desnutrido) e 5 ratos (grupo de controle) receberam ração com teor protéico normal. Segmentos do íleo foram coletados e submetidos à elaboração de preparados de membrana, corados por Giemsa (Barbosa, 1978), e, para tratamento histológico de rotina, corados por HE. Os neurônios agrupavam-se formando gânglios localizados entre os estratos circular e longitudinal da túnica muscular. Com base nos comprimentos dos maiores eixos longitudinal e transversal, os neurônios foram classificados em três grupos: pequenos (14,44 a 22,32μm), médios (22,60 a 39,40) e grandes (40,70 a 63,02). A densidade neuronal no íleo de ratos em uma área de 7,08mm² foi em média 1.482 e 2.515 neurônios, respectivamente, nos grupos de controle e desnutrido. Os dados obttidos sugerem que a desnutrição protéica não provocou alteração na densidade neuronal.This paper deals with the effects of proteic desnutrition on the morphology, morphometry and density of the neurons of the myenteric plexus. The ileum of 10 rats was used. For this study five rats aging 90 days were fed during 120 days with hypoproteic ration (experimental group) and five rats (control group) received ration with normal proteic level. Segments of the ileum were obtained for the method of whole-mount preparation, stained with Giemsa (Barbosa, 1978) and for histological routine treatment stained with HE. The neurons were clustered in ganglia located between the circular and longitudinal layers of the muscular tunica. Based on the lengths of the major longitudinal and transverse axes, the neurons were selected in three groups: small, medium and large. The neuronal density on the ileum of rats corresponding to an area of 7.08mm2 was 1,482 and 2,515 neurons on the control and experimental groups respectively. The data suggested that proteic desnutrition did not cause alteration to the neuronal density.