Andrew Macrae

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

Aim: To evaluate the rpoB gene as a biomarker for PCR‐DGGE microbial analyses using soil DNA from the Cerrado, Brazil. 
Methods: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. 
Results: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. 
Conclusions, Significance and Impact of the Study: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.

Antibacterial activity of GUAVA, Psidium guajava Linnaeus, leaf extracts on diarrhea-causing enteric bacteria isolated from Seabob shrimp, Xiphopenaeus kroyeri (Heller)

O cha de folhas de goiaba Psidium guajava Linnaeus e comumente usado como remedio nas gastrenterites e diarreias infantis por aqueles que nao tem acesso a antibioticos. Esta pesquisa estudou o efeito antibacteriano sobre bacterias causadoras de diarreias, do oleo essencial e do extrato de folhas de goiabeira usando como diluente: metanol, hexano e acetato de etila. Os extratos foram testados sobre Staphylococcus aureus, Salmonella spp. e Escherichia coli. As bacterias testadas foram isoladas de camarao sete-barbas Xiphopenaeus kroyeri (Heller) usando-se como controle cepas padrao, de cada especie. Das bacterias testadas, o melhor efeito inibitorio foi observado sobre Staphylococcus aureus. O extrato de metanol apresentou maior inibicao bacteriana. O oleo essencial mostrou acao inibitoria contra S. aureus e Salmonella spp. Foi demonstrada alguma resistencia das cepas isoladas de camarao aos antibioticos comerciais testados. Estes dados confirmam que remedios a base de folhas de goiabeira podem ser utilizados em casos de diarreia provocada por essas bacterias, quando o uso de antibiotico for restrito. Conclui-se que extratos de folhas de goiaba e o seu oleo essencial sao muito ativos contra S. aureus, tornando-os importantes fontes em potencial de novos compostos antimicrobianos.Guava leaf tea of Psidium guajava Linnaeus is commonly used as a medicine against gastroenteritis and child diarrhea by those who cannot afford or do not have access to antibiotics. This study screened the antimicrobial effect of essential oils and methanol, hexane, ethyl acetate extracts from guava leaves. The extracts were tested against diarrhea-causing bacteria: Staphylococcus aureus, Salmonella spp. and Escherichia coli. Strains that were screened included isolates from seabob shrimp, Xiphopenaeus kroyeri (Heller) and laboratory-type strains. Of the bacteria tested, Staphylococcus aureus strains were most inhibited by the extracts. The methanol extract showed greatest bacterial inhibition. No statistically significant differences were observed between the tested extract concentrations and their effect. The essential oil extract showed inhibitory activity against S. aureus and Salmonella spp. The strains isolated from the shrimp showed some resistance to commercially available antibiotics. These data support the use of guava leaf-made medicines in diarrhea cases where access to commercial antibiotics is restricted. In conclusion, guava leaf extracts and essential oil are very active against S. aureus, thus making up important potential sources of new antimicrobial compounds.

Chemical and microbiological characterization of mangrove sediments after a large oil-spill in Guanabara Bay - RJ - Brazil

Seventeen months after a 1,3 million L oil spill into Guanabara Bay, analyses of mangrove sediments showed that the three sites closest to the spill remain highly polluted (>10 µg-g-1 polyaromatic hydrocarbons). A fourth site was less polluted, from which most hydrocarbon degrading bacteria were isolated.

Multiple drug resistant Staphylococcus aureus strains isolated from a fish market and from fish handlers

The aim of this study was to investigate the presence of antibiotic resistant Staphylococcus aureus strains in fish stalls and in hands and nasal and oral cavities of fish handlers of the Mucuripe Fish Market, Fortaleza, Ceara, Brazil. All S. aureus isolates were resistant to Ampicillin and 44 % were multi-drug resistant.

Microbial diversity in Brazilian mangrove sediments: a mini review

The importance and protection of mangrove ecosystems has been recognized in Brazilian Federal law since 1965. Being protected in law, however, has not always guaranteed their protection in practice. Mangroves are found in coastal and estuarine locations, which are prime real estate for the growth of cities, ports and other economic activities important for Brazilian development. In this mini-review we introduce what mangroves are and why they are so important. We give a brief overview of the microbial diversity found in mangrove sediments and then focus on diversity studies from Brazilian mangroves. We highlight the breadth and depth of knowledge about mangrove microbial communities gained from studying Brazilian mangroves. We report on the exciting findings of molecular microbial ecology methods that have been very successfully applied to study bacterial communities. We note that there have been fewer studies that focus on fungal communities and that fungal diversity studies deserve more attention. The review ends with a look at how a combination of new molecular biology methods and isolation studies are being developed to monitor and conserve mangrove ecosystems and their associated microbial communities. These recent studies are having a global impact and we hope they will help to protect and re-establish mangrove ecosystems.

Streptomyces misionensis PESB-25 Produces a Thermoacidophilic Endoglucanase Using Sugarcane Bagasse and Corn Steep Liquor as the Sole Organic Substrates

Streptomyces misionensis strain PESB-25 was screened and selected for its ability to secrete cellulases. Cells were grown in a liquid medium containing sugarcane bagasse (SCB) as carbon source and corn steep liquor (CSL) as nitrogen source, whose concentrations were optimized using response surface methodology (RSM). A peak of endoglucanase accumulation (1.01 U·mL−1) was observed in a medium with SCB 1.0% (w/v) and CSL 1.2% (w/v) within three days of cultivation. S. misionensis PESB-25 endoglucanase activity was thermoacidophilic with optimum pH and temperature range of 3.0 to 3.6 and 62° to 70°C, respectively. In these conditions, values of 1.54 U mL−1 of endoglucanase activity were observed. Moreover, Mn2+ was demonstrated to have a hyperactivating effect on the enzyme. In the presence of MnSO4 (8 mM), the enzyme activity increased threefold, up to 4.34 U·mL−1. Mn2+ also improved endoglucanase stability as the catalyst retained almost full activity upon incubation at 50°C for 4 h, while in the absence of Mn2+, enzyme activity decreased by 50% in this same period. Three protein bands with endoglucanase activity and apparent molecular masses of 12, 48.5 and 119.5 kDa were detected by zymogram.

Streptomyces lunalinharesii sp. nov., a chitinolytic streptomycete isolated from cerrado soil in Brazil

A novel chitinolytic actinomycete isolated from a Brazilian cerrado soil, designated strain RCQ1071(T), was assigned to the genus Streptomyces on the basis of chemical and morphological characteristics. The almost-complete nucleotide sequence of the 16S rRNA gene of strain RCQ1071(T) was determined and also placed this strain in the genus Streptomyces. Phylogenetic analyses of 16S rRNA gene sequences showed that strain RCQ1071(T) formed a long branch in a group related to Streptomyces albulus, sharing approximately 98 % sequence similarity. Levels of DNA-DNA relatedness between strain RCQ1071(T) and members of this group, namely S. albulus DSM 40492(T), Streptomyces noursei DSM 40635(T) and Streptomyces yunnanensis DSM 41793(T), were 38.3, 27.8 and 46 %, respectively, strongly indicating that strain RCQ1071(T) was not a member of any of these species. The relatively long branch length within a stable clade together with the phenotypic data strongly supported that strain RCQ1071(T) represented a novel species. Based on the combination of physiological, phylogenetic and genomic data, strain RCQ1071(T) is suggested to represent a novel species of the genus Streptomyces, for which the name Streptomyces lunalinharesii sp. nov. is proposed. The type strain is RCQ1071(T) (=ATCC BAA-1231(T) =CIP 108852(T) =DSM 41876(T)).

Killer yeasts inhibit the growth of the phytopathogen Moniliophthora perniciosa, the causal agent of Witches' Broom disease

Fruit and soil yeasts isolated from the Amazon, Atlantic Rainforests and an organic farm were screened for killer activity against yeasts. Killer yeasts were then tested against the phytopathogen Moniliophthora perniciosa (syn. Crinipellis perniciosa) and a Dipodascus capitatus strain and a Candida sp strain inhibited its growth.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Selection of a Streptomyces strain able to produce cell wall degrading enzymes and active against Sclerotinia sclerotiorum

Control of plant pathogen Sclerotinia sclerotiorum is an ongoing challenge because of its wide host range and the persistence of its sclerotia in soil. Fungicides are the most commonly used method to control this fungus but these can have ecotoxicity impacts. Chitinolytic Streptomyces strains isolated from Brazilian tropical soils were capable of inhibiting S. sclerotiorum growth in vitro, offering new possibilities for integrated pest management and biocontrol, with a new approach to dealing with an old problem. Strain Streptomyces sp. 80 was capable of irreversibly inhibiting fungal growth. Compared to other strains, its crude enzymes had the highest chitinolytic levels when measured at 25°C and strongly inhibited sclerotia from S. sclerotiorum. It produced four hydrolytic enzymes involved in fungal cell wall degradation when cultured in presence of the fungal mycelium. The best production, obtained after three days, was 0.75 U/ml for exochitinase, 0.9 U/ml for endochitinase, 0.16 U/ml for glucanase, and 1.78 U/ml for peptidase. Zymogram analysis confirmed two hydrolytic bands of chitinolytic activity with apparent molecular masses of 45.8 and 206.8 kDa. One glucanase activity with an apparent molecular mass of 55 kDa was also recorded, as well as seven bands of peptidase activity with apparent molecular masses ranging from 15.5 to 108.4 kDa. Differential interference contrast microscopy also showed alterations of hyphal morphology after co-culture. Streptomyces sp. 80 seems to be promising as a biocontrol agent against S. sclerotiorum, contributing to the development of new methods for controlling plant diseases and reducing the negative impact of using fungicides.