RESUMO A presente pesquisa avaliou o potencial microbiano de uma biopilha na biorremediação de solos contaminados por hidrocarbonetos, montada em uma base de distribuição de combustíveis na região metropolitana de Porto Alegre, Rio Grande do Sul. Desta biopilha, foram avaliadas as concentrações dos hidrocarbonetos totais de petróleo (HTP) e de compostos benzeno, tolueno, etilbenzeno e xilenos (BTEX), em nove pontos, durante quatro etapas de operação e em três profundidades por ponto. De cada coleta, também foram reservadas amostras de solo para realização das análises microbiológicas. A partir dessas amostras, buscou-se identificar fungos e leveduras pela análise de suas estruturas reprodutivas em lâminas de microcultivo, e bactérias pela técnica da Reação em Cadeira da Polimerase (PCR) e sequenciamento do gene que codifica o RNAr 16S. Ainda, avaliou-se a capacidade dos microrganismos isolados em degradar óleo diesel comercial, utilizando o indicador redox 2,6-diclorofenol indofenol (DCPIP). Como resultado deste estudo, observaram-se elevados percentuais de redução nas concentrações de HTP e BTEX na biopilha, como 92 e 100%, respectivamente. Das amostras de solo da biopilha, foram isoladas 101 cepas de microrganismos, das quais foram identificadas 19 cepas de fungos filamentosos, 34 de bactérias e 1 de levedura. Os resultados evidenciaram a capacidade de alguns gêneros de fungos, como Aspergillus, Trichoderma, Penicillium, Cladosporium e Verticillium, e bactérias, como Bacillus spp. e Streptomyces sp., em degradar hidrocarbonetos constituintes do óleo diesel comercial.

ABSTRACT This research evaluated the microbial potential of a biopile in bioremediation of soils contaminated by hydrocarbons, mounted on a fuel distribution base in the metropolitan region of Porto Alegre, Rio Grande do Sul. Of this biopile were evaluated concentrations of total petroleum hydrocarbons (TPH) and benzene compounds, toluene, ethylbenzene and xylenes (BTEX) at nine points, for four stages of operation and a point in three depths. Of each collection were also reserved sampled soil to perform the microbiological testing. From the samples, we sought to identify fungi and yeasts by analyzing their reproductive structures microcultivation blades, and bacteria by the technique of Polymerase Reaction Chair (PCR) and gene sequencing encoding the 16S rRNA. Still, we evaluated the ability of microorganisms to degrade commercial diesel oil, using the redox indicator 2,6-dichlorophenol indophenol (DCPIP). As result of this study, there was a high percentage reduction in the concentration of TPH and BTEX in biopilha as 92 and 100%, respectively. Of soil samples were isolated from biopilha 101 strains of microorganisms, of which 19 were identified strains of filamentous fungi and 34 bacterial yeast. The results showed the ability of some genera of fungi such as Aspergillus, Trichoderma, Penicillium, Cladosporium and Verticillium and bacteria such as Bacillus spp. and Streptomyces sp. to degrade hydrocarbons constituents of commercial diesel oil.

Abstract: INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

O objetivo deste estudo foi avaliar potenciais danos no DNA e citotoxicidade em técnicos de laboratório de patologia expostos a solventes orgânicos, principalmente ao xileno. Amostras de sangue periférico e células bucais foram coletadas de 18 técnicos ocupacionalmente expostos a solventes orgânicos e de 11 indivíduos não expostos aos solventes. Os técnicos foram avaliados em dois momentos: segunda-feira e sexta-feira. Danos no DNA e citotoxicidade foram avaliados usando o Ensaio Cometa e o Teste de Micronúcleos e Anomalias Nucleares. Quinze indivíduos (83,5%) do grupo exposto aos solventes queixaram-se de algum sintoma com provável relação com o contato com vapores de solventes orgânicos. Os danos no DNA no grupo exposto aos solventes foram aproximadamente 2 vezes maiores na sexta-feira do que na segunda-feira, e em ambos momentos os indivíduos desse grupo apresentaram maiores níveis de danos no DNA em relação aos controles. Não foi encontrada diferença significativa na frequência de micronúcleos nas células bucais entre os técnicos de laboratório e os indivíduos do grupo controle. Entretanto, na análise realizada na sexta-feira, os técnicos apresentaram maior frequência (aproximadamente 3 vezes) de células cariolíticas e células indicativas de apoptose (cariorréticas e picnóticas) em relação ao grupo controle. Considerando a frequência de dano e o tempo de trabalho, uma correlação positiva foi encontrada entre o grupo de indivíduos expostos aos solventes (r=0,468; p=0,05). Os resultados sugerem que técnicos de laboratório de patologia expostos de forma inadequada a solventes orgânicos possuem aumento dos níveis de danos no DNA.

The aim of this study was to evaluate potential DNA damage and cytotoxicity in pathology laboratory technicians exposed to organic solvents, mainly xylene. Peripheral blood and buccal cells samples were collected from 18 technicians occupationally exposed to organic solvents and 11 non-exposed individuals. The technicians were sampled at two moments: Monday and Friday. DNA damage and cytotoxicity were evaluated using the Comet Assay and the Buccal Micronucleus Cytome assay. Fifteen subjects (83.5%) of the exposed group to solvents complained about some symptom probably related to contact with vapours of organic solvents. DNA damage in the exposed group to solvents was nearly 2-fold higher on Friday than on Monday, and in both moments the individuals of this group showed higher levels of DNA damage in relation to controls. No statistical difference was detected in buccal cell micronucleus frequency between the laboratory technicians and the control group. However, in the analysis performed on Friday, technicians presented higher frequency (about 3-fold) of karyolytic and apoptotic-like cells (karyorrhectic and pyknotic) in relation to control group. Considering the damage frequency and the working time, a positive correlation was found in the exposed group to solvents (r=0.468; p=0.05). The results suggest that pathology laboratory workers inappropriately exposed to organic solvents have increased levels of DNA damage.

Atualmente, para extrair o DNA bacteriano, existem diversos métodos baseados em diferentes princípios. Entretanto, a quantidade e qualidade do DNA obtido por cada um destes métodos é variável e depende do tipo de micro-organismo em questão; os estafilococos coagulase-negativos (CoNS), por exemplo, possuem parede celular espessa difícil de lisar. O objetivo deste estudo foi comparar a quantidade e a qualidade do DNA extraído de isolados clínicos de CoNS utilizando quatro metodologias diferentes: dois protocolos caseiros e dois kits comerciais. A determinação da quantidade e da qualidade do DNA foi realizada por espectrofotometria. O DNA extraído também foi analisado em eletroforese em gel de agarose e por PCR. A concentração média de DNA foi mais alta no método de lise térmica (). Entretanto, com relação à qualidade do DNA, o kit comercial que utiliza um método de extração baseado em uma coluna de separação apresentou melhor resultado (média da relação A260/280 = 1,95). As quatro técnicas testadas forneceram DNA passível de amplificação por PCR, porém com diferentes rendimentos. A qualidade do DNA extraído de bactérias é importante, pois possibilita a realização de maior número de técnicas de biologia molecular e também armazenamento do material por maior período de tempo. Neste sentido, a técnica de extração por coluna de separação apresentou melhor desempenho frente aos CoNS.

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

The emergence of Extended-Spectrum Beta-Lactamase (ESBL)-producing microorganisms in Brazilian hospitals is a challenge that concerns scientists, clinicians and healthcare institutions due to the serious risk they pose to confined patients. The goal of this study was the detection of ESBL production by clinical strains of Escherichia coli and Klebsiella sp. isolated from pus, urine and blood of patients at Hospital Universitário Santa Maria, Rio Grande Sul, RS, Brazil and the genotyping of the isolates based on bla SHV genes. The ESBL study was carried out using the Combined Disc Method, while Polymerase Chain Reaction (PCR) was used to study the bla SHV genes. Of the 90 tested isolates, 55 (61.1%) were identified as ESBL-producing by the combined disk method. The bla SHV genes were found in 67.8% of these microorganisms. K. pneumoniae predominated in the samples, presenting the highest frequency of positive results from the combined disk and PCR.

Neste estudo estimou-se a distribuição e prevalência de β-lactamases de espectro estendido pertencentes às famílias TEM, SHV e CTX-M entre amostras de Escherichia coli e Klebsiella spp. no Hospital Universitário de Santa Maria, Rio Grande do Sul. Durante 14 meses, 90 microrganismos foram selecionados como prováveis produtores de ESBL. Os isolados foram submetidos a testes fenotípicos confirmatórios para a presença de ESBL. A seguir, os tipos de ESBLs presentes em cada microrganismo foram determinados através da pesquisa dos respectivos genes através da reação em cadeia da polimerase. Empregando-se o método do disco combinado, a presença de ESBLs foi confirmada em 55 (61,1%) amostras; quando o método do duplo disco foi utilizado, 57 (63,3%) amostras foramprodutoras de ESBLs. Com base na PCR, as ESBLs do tipo TEM e SHV foram mais presentes em Klebsiella pneumoniae enquanto que ESBL do tipo CTX-M foram mais presentes em Klebsiella oxytoca.

In this study, the distribution and prevalence of extended-spectrum β-lactamases belonging to the TEM, SHV and CTX-M families were estimated among samples of Escherichia coli and Klebsiella spp. at the university hospital of Santa Maria, Rio Grande do Sul. Over a 14-month period, 90 microorganisms were selected as likely ESBL producers. The isolates were subjected to confirmatory phenotype tests for the presence of ESBL. Through investigating the respective genes using the polymerase chain reaction, the ESBL types present in each microorganism were then determined. Fifty-five samples (61.1%) were confirmed as ESBL-positive by means of the combined disc method, and 57 (63.3%) were found to be ESBL producers by means of the double disc method. From the polymerase chain reaction, ESBLs of TEM and SHV types were more frequently present in Klebsiella pneumoniae, while ESBL of CTX-M type was more frequently present in Klebsiella oxytoca.