Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Evaluation of in vitro and in vivo effects of semipurified proteinase inhibitors from theobroma seeds on midgut protease activity of lepidopteran pest insects

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.     

Peat-assisted phytoremediation of waste foundry sands: plant growth, metal accumulation and fertility aspects

We investigated the potential of peat additions to improve plant growth and fertility and to reduce plant metal uptake in waste foundry sands (WFS) landfills. The WFS contains 78211 mg kg(-1) and 371 mg kg(-1) concentrations of Cr and Ni, respectively, and varied metal concentrations. The experiment investigated the growth of Brassica juncea plants on fertilized WFS mixed with peat at concentrations of 0, 2.5, 5, and 10% (w/w). The highest peat treatment allowed substantial plant growth and increased Ni mass in shoots, which was positively correlated to shoot biomass increments. On a concentration basis, peat additions did not increase shoot Ni values, thus suggesting that plants grown on peat-treated WFS may not increase risks to human and ecological receptors. Chromium was below detection levels in shoots for all peat treatments. Peat-treated substrates also promoted increased CEC values and higher water holding capacity, therefore improving the WFS agronomical properties. These results indicate that peat can be used as an amendment to assist in the phytoremediation of WFS landfill areas. However, there was evidence for increased mobilization of Cr and Ni in the substrate solution which can pose a threat to local groundwater.     

Leptospira interrogans biofilm formation in Rattus norvegicus (Norway rats) natural reservoirs

Rattus norvegicus (Norway rat) is the main reservoir host of pathogenic Leptospira, the causative agent of leptospirosis, in urban environments. Pathogenic Leptospira forms biofilms in the environment, possibly contributing for bacterial survival and maintenance. Nonetheless, biofilms have not yet been studied in natural animal reservoirs presenting leptospiral renal carriage. Here, we described biofilm formation by pathogenic Leptospira inside the renal tubules of R. norvegicus naturally infected and captured in an urban slum endemic for leptospirosis. From the 65 rats carrying Leptospira in their kidneys, 24 (37%) presented biofilms inside the renal tubules. The intensity of leptospiral colonization in the renal tubules (OR: 1.00; 95% CI 1.05–1.1) and the type of occlusion pattern of the colonized renal tubules (OR: 3.46; 95% CI 1.20–9.98) were independently associated with the presence of Leptospira biofilm. Our data showed that Leptospira interrogans produce biofilms during renal chronic colonization in rat reservoirs, suggesting a possible role for leptospiral biofilms in the pathogenesis of leptospirosis and bacterial carriage in host reservoirs.     

Discovery of a high molecular weight complex of calcium, phosphate, fetuin, and matrix gamma-carboxyglutamic acid protein in the serum of etidronate-treated rats.

In the present study we report the discovery of a novel protein-mineral complex in the serum of rats treated with doses of the bone-active bisphosphonate etidronate that inhibit normal bone mineralization. The composition of this high molecular mass protein-mineral complex consists of about 18% mineral, 80% fetuin, and 2% matrix Gla protein (MGP) by weight, and the presence of the complex in serum after an injection of 8 mg etidronate/100 g of body weight elevates calcium by 1.8-fold (to 4.3 mm), phosphate by 1.6-fold (to 5.6 mm), and MGP by 25-fold (to 12 microg/ml). The serum mineral complex reaches maximal levels at 6 h after subcutaneous injection of etidronate and is subsequently cleared from serum by 24 h. This highly specific complex of fetuin, MGP, and mineral prevents the growth, aggregation, and precipitation of the mineral component, which indicates that the previously reported calcification inhibitory activities of fetuin and MGP may be related to their ability to form stable complexes with nascent mineral nuclei. Treatment with the vitamin K-antagonist warfarin prevents the increase in serum MGP after etidronate injection, which shows that the increase in serum MGP is due to new synthesis and that the gamma-carboxylation of MGP is necessary for its binding to the serum mineral complex.     

Expression of the Theobroma cacao Bax‐inhibitor‐1 gene in tomato reduces infection by the hemibiotrophic pathogen Moniliophthora perniciosa

Programmed cell death (PCD) plays a key role in plant responses to pathogens, determining the success of infection depending on the pathogen lifestyle and on which participant of the interaction triggers cell death. The hemibiotrophic basidiomycete Moniliophthora perniciosa is the causal agent of witches' broom disease of Theobroma cacao L. (cacao), a serious constraint for production in South America and the Caribbean. It has been hypothesized that M. perniciosa pathogenesis involves PCD, initially as a plant defence mechanism, which is diverted by the fungus to induce necrosis during the dikaryotic phase of the mycelia. Here, we evaluated whether the expression of a cacao anti‐apoptotic gene would affect the incidence and severity of M. perniciosa infection using the ‘Micro‐Tom’ (MT) tomato as a model. The cacao Bax‐inhibitor‐1 (TcBI‐1) gene, encoding a putative basal attenuator of PCD, was constitutively expressed in MT to evaluate function. Transformants expressing TcBI‐1, when treated with tunicamycin, an inducer of endoplasmic reticulum stress, showed a decrease in cell peroxidation. When the same transformants were inoculated with the necrotrophic fungal pathogens Sclerotinia sclerotiorum, Sclerotium rolfsii and Botrytis cinerea, a significant reduction in infection severity was observed, confirming TcBI‐1 function. After inoculation with M. perniciosa, TcBI‐1 transformant lines showed a significant reduction in disease incidence compared with MT. The overexpression of TcBI‐1 appears to affect the ability of germinating spores to penetrate susceptible tissues, restoring part of the non‐host resistance in MT against the S‐biotype of M. perniciosa.     

Incidence and diversity of arbuscular mycorrhizal fungi and successor herbaceous plants in an agro-system irrigated with produced water

The purpose of this study was to evaluate the diversity of herbaceous plants and arbuscular mycorrhizal fungi following the cultivation of sunflower (Helianthus annuus L., cv. BRS 321) irrigated with produced water. The sunflower plants were irrigated during three successive cycles with different types of water: produced water obtained through simple filtration (PWSF), and secondly, produced water treated by reverse osmosis (PWRO), and the control with groundwater from the aquifer Açu (WCA). In June 2014, five months after the final harvest, the treatments were evaluated in terms of the diversity of successor plants and their roots colonized by arbuscular mycorrhiza (AM); and samples of soil, in which the following were measured: the spore abundance of AM fungi, the levels of glomalin in easily extracted glomalin and total glomalin. Of a total of eighteen species of herbaceous plants which were identified in the experimental field, Dactyloctenium aegyptium was related with the use of PWSF, Panicum sp. and Diodella apiculata with the use of PWRO, and Trianthema portulacastrum and Eragrostis tenella with the control WCA. The diversity of AM fungi was affected by irrigation with PWSF, in which two species of Acaulospora, one species of Gigaspora and species of Paraglomus were absent, compared to the treatment with PWRO. Acaulospora sp.1 was related with the WCA control as an indicator species. The use of produced water which had undergone reverse osmosis had a short-term effect on the content of glomalin which is easily extractable from the soil but did not change the mycorrhization rates of plants. These results enable us to infer that irrigation with produced water leads to a reduction in the diversity of herbaceous plants and of arbuscular mycorrhizal fungi in the soil, confirming the importance of monitoring agro-systems irrigated with residual water.     

Resource‐directed foraging of the Neotropical mistletoe Struthanthus flexicaulis (Loranthaceae)

• Some parasitic plants are capable of vegetative propagation, which allows them access to new hosts and improves nutrient availability. We aimed to determine what factors positively influence this propagation in the xylem‐tapping Struthanthus flexicaulis, focusing on the use of directed foraging as a means of optimising access to resources.
• The study site was a rupicolous plant community in southeast Brazil. We evaluated how the success of branch propagation (by contagion) of the mistletoe is influenced by the crown height and density of its main host, the legume Mimosa calodendron. Oriented foraging was tested through comparing the response of mistletoe branches toward a potential host (resources) or a plastic net (no resources).
• Successful contagion was related to high density and the departure height of a branch from a host crown. In a high‐density area, 89% of branches reached a new host versus 21% in a low‐density area. Formation of an appressorium on either the plastic nets or hosts elicited branch growth reorientation towards it, as well as development of new branches. These responses were significantly stronger towards hosts than nets, and attributed to effective xylem tapping.
• The foraging strategy of S. flexicaulis benefits from branch response (growth and reorientation) to the development of appressoria and their resulting haustoria. The growth of appressoria on non‐resource substrates and the ensuing response optimises the mistletoe spread by allowing these substrates to act as physical support for spreading branches and to infect a host that was previously out of reach.