Entomopathogenic bacteria isolated fromSimuliids are hematophagus insects naturally occurring in waterways throughout Brazil. Although some species have been involved in the transmission of human onchocerciasis in other regions (Gerais & Ribeiro 1986, Maia-Herzog et al. 1999) the main importance of these insects in the southeast of Brazil is socio-economic. The frequent and intensive attacks by simuliids on the transitory population in this area can reduce tourism in summer months, a fact that could negatively impact the economies of affected municipalities. To decrease the presence of these insects in these areas, a full-scale programme, based on Bacillus thuringiensis serovar israelensis, has been carried out in the north littoral zone of the State of São Paulo since 1990 (Araújo-Coutinho 1995).Recent findings of B. thuringiensis serovar oswaldocruzi and B. thuringiensis serovar braziliensis from unusual habitats (Rabinovitch et al. 1995) encourage the continued search for other endemic entomopathogens. For that purpose, Simulium larvae and adults from the north littoral zone of São Paulo and some other rivers in the State of Rio de Janeiro were evaluated for the presence of entomopathogenic bacteria. MATERIALS AND METHODSCollection of simuliids -Black fly larvae and adults were collected from breeding sites in Pau d'Alho river, State of São Paulo. This river was divided into two regions, one downstream of B. thuringiensis serovar israelensis applications and another, upstream. Another collection point was the Soberbo river, Guapimirim, State of Rio de Janeiro (Fonseca et al. 1998). This river has never been exposed to commercial formulations based on B. thuringiensis serovar israelensis. Only larvae were collected in this waterway. The third point where larvae were collected was in Rio das Pedras, Mangaratiba, State of Rio de Janeiro.Isolation of Bacillus -To eliminate external contamination, the insects were sterilized following the methodology described by Alves (1986) with a slight modification; "Superbonder" glue (cyanoacrilate ester) was used to close both oral and anal cavities. This modification was adopted because Simulium larvae are very small and the dental floss used in the original technique was not applicable. After this step, the original method was performed passing the larvae through three solutions, first in 70% alcohol for 2 sec, second in 5% sodium hypoclorite for 3 min and finally in sterile 10% sodium thiossulfate for 5 min. The specimens were then washed three times in sterile distilled water.Two different methods were used to isolate B. thuringiensis samples. In both, Simulium larvae were transferred aseptically into a sterile mortar and macerated with a sterile pestle. In the first method, the macerate was placed in tubes containing distilled water (10 ml). The suspension was heated (65ºC) for 12 min, and immediately diluted (1x10 -2

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Sixty strains of Bacillus sphaericus, including 31 insect pathogens were studied by multilocus enzyme electrophoresis and were classified into 44 zymovars (electrophoretic types). Among the entomopathogenic strains, 11 belong to the same zymovar (Z59) indicating a widespread frequent genotype. Bands of enzyme activity were not detected among the strains for the loci GPI (E.C.5.3.1.9), G6P (E.C.1.1.1.49), 6PG (E.C.1.1.1.44) and ME (E.C.1.1.1.40). The enzymatic loci NP (E.C.2.4.2.1) and ACON (E.C.4.2.1.3) were monomorphic while the other enzymes, MDH (E.C.1.1.1.37), LeDH (E.C.1.4.1.9), ADH (E.C.1.4.1.1), EST (E.C.3.1.1.1), PEP-2 (E.C.3.4.11.1), PEP-3 (E.C.3.4.11) and PEP-D (E.C. 3.4.13.9) were polymorphic. The genetic variation in the non-insect pathogenic group seemed to be greater than in the entomopathogenic group. This latter group appears to be distinct from other strains of these species. All insect pathogens were recovered in the same phenetic cluster and a diagnostic allele is reported for the identification of entomopathogenic strains.

Thirty samples of roasted ground coffee beans from 10 different commercial brands were analyzed to investigate the occurrence and levels of Bacillus cereus and Bacillus thuringiensis strains. Strains were evaluated for their genetic diversity by repetitive element sequence polymorphism PCR (Rep-PCR) and for their toxigenic profiles, i.e., the presence of hblA, hblC, hblD, nheA, nheB, nheC, cytK, ces, and entFM. Survival and multiplication of B. cereus sensu lato in the ready-to-drink coffee was determined to evaluate this beverage as a possible vehicle for B. cereus infection. B. cereus was detected in 17 (56.7%) of the 30 samples, and B. thuringiensis was detected in 8 (26.7%) of the 30 samples. Five samples did not produce any characteristic growth. The most common gene, entFM, was detected in 23 strains (92%). The NHE complex (nheA, nheB, and nheC genes) was found in 19 strains (76%). The HBL complex (hblA, hblC, and hblD) was found in 16 strains (64%). All strains were negative for ces. The cytK gene was found in 16 strains (64%). The computer-assisted cluster analysis of Rep-PCR profiles using a clustering criterion of 80% similarity revealed four main clusters. Cluster 1 was the predominant and comprised three B. thuringiensis strains with 100% similarity, cluster 2 comprised two B. cereus strains (100% similarity), cluster 3 comprised two B. thuringiensis strains (90% similarity), and cluster 4 comprised one B. thuringiensis strain and one B. cereus strain (85% similarity). The cluster analysis of fingerprints generated by Rep-PCR revealed a high genetic diversity among the B. cereus strains, suggesting that the contamination could have originated from different sources. In our experiments, when sugar was added and the beverage was kept in thermic bottles there was a significant increase in B. cereus sensu lato levels, which may increase the risk of food poisoning. These results highlight the need for additional studies on this subject to better evaluate coffee as a food poisoning vehicle.

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain Bacillus thuringiensis is a Gram-positive, spore-forming bacterium which produces a parasporal body during sporulation. The proteins contained in the parasporal crystal (delta-endotoxins) are specific insect gut toxins which have no significant effect on non-target organisms (Siegel 2001). The mode of action after ingestion involves solubilization of the protein in the alkaline conditions of the insect midgut, followed by activation of the protoxin into toxin through cleavage of specific regions by gut proteases. Then, the so called toxin binds to specific receptors in the brush border membrane vesicles of the midgut columnar cells. The binding is a two-step process, the second part being the insertion of the toxin into the cell membrane leading to pore formation, osmotic imbalance, disruption of the cell membrane and finally, death of the insect (Van Rie et al. 1990, Knowles 1994.The use of bioinsecticides based on B. thuringiensis to control insects from the orders Diptera, Coleoptera, and Lepidoptera and more recently against Hymenoptera, Orthoptera, and Mallophaga, has stimulated the search for other natural pathogens (Schenpf et al. 1998 Between 1995 to 1998, an isolation program was set up to obtain entomopathogenic Bacillus from Simulium larvae and adults in the Southeast region of Brazil. Eighteen strains of B. thuringiensis and one of B. sphaericus were isolated (Cavados et al. 2001). Preliminary bioassays were performed and one of the B. thuringiensis strains (LFB-FIOCRUZ 1035) showed promising activity against Aedes aegypti and Culex quinquefasciatus (Cavados et al. 2001).In the present study, the toxicity of the autoagglutinating strain LFB-FIOCRUZ 1035 was evaluated against two Dipteran species, S. pertinax and Ae. aegypti larvae, and compared with a standard powder formulation of the reference B. thuringiensis serovar israelensis strain IPS-82, which is used in commercially available bioinsecticides. MATERIALS AND METHODSBacterial strains -Two strains of B. thuringiensis were used, one of them IPS-82 (LFB-FIOCRUZ 584) serotype H-14, isolated from the standard powder prepared by the Pasteur Institute of Paris, and the other LFB-FIOCRUZ 1035, an autoagglutinating strain isolated from S. pertinax larvae collected in a natural breeding-site in Growth started with a pre-inoculum to reduce the duration of the lag phase of bacterial growth. After inoculation in 125 ml Erlenmeyer flasks containing 50 ml of the medium, the flasks were incubated in a New Brunswick Scientific agitator series 25D, at 175 opm and 30°C for 6 h. Subsequently, 3 ml were transferred to 500 ml Erlenmeyer flasks containing 150 ml of the soy flour and inorganic salts medium and incubated as previously described for a further 72 h period.Once sporulation had reached 95% of free spores and crystals, each culture was centrifuged (6000 g, 10°C), the supernatant removed and the remaining material was kept in an amber container with the pH adjus...

Some strains of the Gram-positive spore-forming bacterium Bacillus thuringiensis are very toxic to various insect larvae. The insecticidal activity is determined by different cytoplasmic insoluble proteins produced during sporulation which aggregate to form parasporal crystals (E Schnepf et al. 1998 Microbiol Mol Biol Rev 62: 775-806). A few strains of B. thuringiensis are used as bioinsecticides to control pests and vector insects in the orders Diptera, Lepidoptera and Coleoptera (PAW Martin & RS Travers 1989 Appl Environ Microbiol 55: 2437-2442) and more recently new isolates of B. thuringiensis have been shown to be active against Hymenoptera, Homoptera, Orthoptera and Mallophaga, as well as nematodes, mites and pro-Partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico.

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