Andréa M. A. Nascimento

Molecular approaches: advantages and artifacts in assessing bacterial diversity

Bacteria account for a major proportion of Earth’s biological diversity. They play essential roles in quite diverse environments and there has been an increasing interest in bacterial biodiversity. Research using novel and efficient tools to identify and characterize bacterial communities has been the key for elucidating biological activities with potential for industrial application. The current approach used for defining bacterial species is based on phenotypic and genomic properties. Traditional and novel DNA-based molecular methods are improving our knowledge of bacterial diversity in nature. Advances in molecular biology have been important for studies of diversity, considerably improving our knowledge of morphological, physiological, and ecological features of bacterial taxa. DNA–DNA hybridization, which has been used for many years, is still considered the golden standard for bacteria species identification. PCR-based methods investigating 16S rRNA gene sequences, and other approaches, such as the metagenome, have been used to study the physiology and diversity of bacteria and to identify novel genes with potential pharmaceutical and other biotechnological applications. We examined the advantages and limitations of molecular methods currently used to analyze bacterial diversity; these are mainly based on the 16S rRNA gene. These methods have allowed us to examine microorganisms that cannot be cultivated by routine methods and have also been useful for phylogenetic studies. We also considered the importance of improvements in microbe culture techniques and how we can combine different methods to allow a more appropriate assessment of bacterial diversity and to determine their real potential for industrial applications.

Exoproducts of the Escherichia coli strain H22 inhibiting some enteric pathogens both in vitro and in vivo

Aims:  The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo.

Prokaryotic diversity and dynamics in a full-scale municipal solid waste anaerobic reactor from start-up to steady-state conditions.

The prokaryotic diversity of an anaerobic reactor for the treatment of municipal solid waste was investigated over the course of 2 years with the use of 16S rDNA-targeted molecular approaches. The fermentative Bacteroidetes and Firmicutes predominated, and Proteobacteria, Actinobacteria, Tenericutes and the candidate division WWE1 were also identified. Methane production was dominated by the hydrogenotrophic Methanomicrobiales (Methanoculleus sp.) and their syntrophic association with acetate-utilizing and propionate-oxidizing bacteria. qPCR demonstrated the predominance of the hydrogenotrophic over aceticlastic Methanosarcinaceae (Methanosarcina sp. and Methanimicrococcus sp.), and Methanosaetaceae (Methanosaeta sp.) were measured in low numbers in the reactor. According to the FISH and CARD-FISH analyses, Bacteria and Archaea accounted for 85% and 15% of the cells, respectively. Different cell counts for these domains were obtained by qPCR versus FISH analyses. The use of several molecular tools increases our knowledge of the prokaryotic community dynamics from start-up to steady-state conditions in a full-scale MSW reactor.

Synergistic bactericidal activity of Eremanthus erythropappus oil or β-bisabolene with ampicillin against Staphylococcus aureus

The activity of Eremanthus erythropappus oil (EO) and some of its compounds and their potential synergistic interaction with ampicillin against different strains of Staphylococcus aureus were investigated. Determination of chemical composition of EO by gas chromatography-mass spectrometry (GC/MS) and bioguided chemical fractionation led to the identification of β-bisabolene as the main active compound. A synergistic bactericidal activity of EO or β-bisabolene with ampicillin against Staphylococcus aureus was observed in a time-kill assay. EO and β- bisabolene have the potential to restore the effectiveness of ampicillin against resistant S. aureus.

Metagenome of a microbial community inhabiting a metal-rich tropical stream sediment.

Here, we describe the metagenome and functional composition of a microbial community in a historically metal-contaminated tropical freshwater stream sediment. The sediment was collected from the Mina Stream located in the Iron Quadrangle (Brazil), one of the world’s largest mining regions. Environmental DNA was extracted and was sequenced using SOLiD technology, and a total of 7.9 Gbp was produced. A taxonomic profile that was obtained by comparison to the Greengenes database revealed a complex microbial community with a dominance of Proteobacteria and Parvarcheota. Contigs were recruited by bacterial and archaeal genomes, especially Candidatus Nitrospira defluvii and Nitrosopumilus maritimus, and their presence implicated them in the process of N cycling in the Mina Stream sediment (MSS). Functional reconstruction revealed a large, diverse set of genes for ammonium assimilation and ammonification. These processes have been implicated in the maintenance of the N cycle and the health of the sediment. SEED subsystems functional annotation unveiled a high degree of diversity of metal resistance genes, suggesting that the prokaryotic community is adapted to metal contamination. Furthermore, a high metabolic diversity was detected in the MSS, suggesting that the historical arsenic contamination is no longer affecting the prokaryotic community. These results expand the current knowledge of the microbial taxonomic and functional composition of tropical metal-contaminated freshwater sediments.

Antibiotic-resistant gram-negative bacteria in birds from the Brazilian Atlantic forest

Abstract We evaluated the antibiotic resistance of bacteria isolated from cloacal swabs of wild birds collected with mist nets in the Jequitinhonha river valley, in the state of Minas Gerais, Brazil. A total of 191 isolates from 19 individuals of 16 species was obtained and tested for resistance to five antibiotics. At Salto da Divisa 97% of the isolates exhibited a resistant phenotype, and resistance to more than one antibiotic was frequent (71%). At Jequitinhonha 36% of isolates were resistant, but 94% showed resistance to only one antibiotic. Of the five antibiotics tested, resistance to ampicillin was most frequent (in both areas), whereas kanamycin resistance was found in only one isolate. The data here obtained and other data reported in the literature show that the general premise that antibiotic-resistant bacteria arise primarily in hospitals or animal farms should be reconsidered. Bactérias Gram-Negativas Resistentes a Antibióticos em Aves da Mata Atlântica Brasileira Resumo. Avaliamos a resistência a antibióticos de bactérias isoladas por swab cloacal em aves selvagens capturadas com redes de neblina em duas regiões do Vale do Rio Jequitinhonha, Minas Gerais, Brasil. Foram obtidos 191 isolados de 19 indivíduos de 16 espécies e foi testada a resistência desses isolados a cinco antibióticos. Em Salto da Divisa, 97% dos isolados exibiram fenótipo resistente e foi freqüente (71%) a resistência a mais de um antibiótico. Em Jequitinhonha, 36% dos isolados exibiram fenótipo resistente, dos quais 94% apresentaram resistência a apenas um antibiótico. Em ambas as áreas, a maioria dos isolados apresentou resistência à ampicilina, enquanto somente um único isolado foi resistente à canamicina. Os dados aqui obtidos e outros relatados na literatura mostram que a premissa geral de que bactérias resistentes a antibióticos surgem principalmente em hospitais ou fazendas de animais deve ser reconsiderada.

Recent updated aspects of colicins of Enterobacteriaceae

The colicins are protein compounds produced by, and active against, Escherichia coli and others members of Enterobacteriaceae family. At least 34 different colicins have been described and found to share an interesting number of features. In the present review we focus on the major characteristics of colicins of gram-negative bacteria and explore their production and practical applications.The colicins are protein compounds produced by, and active against, Escherichia coli and others members of Enterobacteriaceae family. At least 34 different colicins have been described and found to share an interesting number of features. In the present review we focus on the major characteristics of colicins of gram-negative bacteria and explore their production and practical applications.

Capacity of mercury volatilization by mer (from Escherichia coli) and glutathione S-transferase (from Schistosoma mansoni) genes cloned in Escherichia coli.

A study was carried out to evaluate the capacity for mercury volatilization by genetically engineered strains that express the mer and glutathione S-transferase genes from Escherichia coli and Schistosoma mansoni, respectively. This method enabled strains containing simultaneously mer and glutathione S-transferase genes to grow in high concentrations of mercuric chloride (30 microg/ml) and to volatilize part of the mercury (248 microg/g cell dry wt.) present in the culture medium, while strains bearing only a single gene, did not have the same behavior. Up to 70% of the total mercury of bacterial volatilization occurred in the first 4 h. Although the findings were preliminary, the genetically engineered strain containing simultaneously the mer and glutathione S-transferase genes show a great potential for bioremediation. It may be used in a closed system to remove by volatilization, and recover mercury (Hg0) from contaminated effluents, such as industrial effluent, for instance.

Bacteria and Genes Involved in Arsenic Speciation in Sediment Impacted by Long-Term Gold Mining

The bacterial community and genes involved in geobiocycling of arsenic (As) from sediment impacted by long-term gold mining were characterized through culture-based analysis of As-transforming bacteria and metagenomic studies of the arsC, arrA, and aioA genes. Sediment was collected from the historically gold mining impacted Mina stream, located in one of the world’s largest mining regions known as the “Iron Quadrangle”. A total of 123 As-resistant bacteria were recovered from the enrichment cultures, which were phenotypically and genotypically characterized for As-transformation. A diverse As-resistant bacteria community was found through phylogenetic analyses of the 16S rRNA gene. Bacterial isolates were affiliated with Proteobacteria, Firmicutes, and Actinobacteria and were represented by 20 genera. Most were AsV-reducing (72%), whereas AsIII-oxidizing accounted for 20%. Bacteria harboring the arsC gene predominated (85%), followed by aioA (20%) and arrA (7%). Additionally, we identified two novel As-transforming genera, Thermomonas and Pannonibacter. Metagenomic analysis of arsC, aioA, and arrA sequences confirmed the presence of these genes, with arrA sequences being more closely related to uncultured organisms. Evolutionary analyses revealed high genetic similarity between some arsC and aioA sequences obtained from isolates and clone libraries, suggesting that those isolates may represent environmentally important bacteria acting in As speciation. In addition, our findings show that the diversity of arrA genes is wider than earlier described, once none arrA-OTUs were affiliated with known reference strains. Therefore, the molecular diversity of arrA genes is far from being fully explored deserving further attention.

Synergic interaction between ascorbic acid and antibiotics against Pseudomonas aeruginosa

Investigou-se in vitro o efeito da combinacao do acido ascorbico (AA) com seis antibioticos frente a 12 isolados multirresistentes de Pseudomonas aeruginosa. As concentracoes inibitorias minimas (CIM) foram determinadas pelo metodo de diluicao em caldo. Foi estudado o efeito do AA nas CIM pelo calculo das concentracoes inibitorias fracionais (CIF). Para quase todas as combinacoes AA-antibiotico foi detectado efeito sinergico, exceto para ampicilina e tobramicina. Indiferenca foi observada na interacao com todos os antibioticos, porem antagonismo foi somente observado para cloranfenicol. Os resultados deste estudo indicam que o sinergismo contra P. aeruginosa resistentes pode ocorrer entre AA e cloranfenicol, canamicina, estreptomicina e tetraciclina, ainda que as linhagens sejam resistentes aos antibioticos individualmente. Alem disso, estes resultados encorajam futuros trabalhos in vivo a respeito da interacao AA-antimicrobianos na incessante busca de novas alternativas para o controle de linhagens multirresistentes de P.aeruginosa.