BACKGROUND Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Resumo B. amyloliquefaciens é um bastonete Gram-positivo, aeróbio e móvel, e é encontrado, frequentemente, no solo. Este bastonete foi descrito como um promotor de crescimento de plantas e é usado em diversos processos industriais. Este estudo relata um episódio de deterioração gasosa de molho de salada causada por B. amyloliquefaciens, que ocorreu em uma unidade de produção localizada no Rio de Janeiro, Brasil. Foram isoladas nove cepas de B. amyloliquefaciens de molhos de salada deteriorados, do açúcar utilizado como matéria-prima na fabricação e da planta de produção. A análise genotípica dos isolados por Rep-PCR gerou oito perfis de bandas, agrupados em cinco clusters Rep-PCR. Quando reinoculados em molho de salada fresco, três cepas de B. amyloliquefaciens, pertencentes aos clusters Rep-PCR A, D e E, foram capazes de reproduzir o processo de deterioração gasosa, enquanto as cepas pertencentes aos clusters Rep-PCR B e C não produziram deterioração visível, o que sugere que essas cepas não estavam diretamente envolvidas no processo de deterioração. O grupo predominante, Rep-PCR cluster A, incluiu cepas isoladas de molhos de semente de maracujá e barbecue, e do açúcar (matéria-prima), sugerindo uma fonte comum de contaminação para esses molhos.

Abstract B. amyloliquefaciens is a Gram-positive, aerobic, motile rod, often found in soil, which has been described as a plant growth promoter and is used in several industrial processes. This study reports an episode involving the gassy spoilage of salad dressing caused by B. amyloliquefaciens in a production facility located in Rio de Janeiro, Brazil. Nine B. amyloliquefaciens strains were isolated from spoiled salad dressings, the sugar used as a raw material in the manufacture and from the production plant. A genotypic analysis of the isolates by Rep-PCR generated eight band profiles grouped in five Rep-PCR clusters. When re-inoculated into fresh salad dressing three B. amyloliquefaciens isolates belonging to the Rep-PCR clusters A, D and E were able to reproduce the gassy spoilage process, whereas the isolates belonging to the Rep-PCR clusters B and C did not produce any visible spoilage, suggesting that these isolates were not directly involved in the spoilage process. The predominant Rep-PCR cluster, cluster A, included strains isolated from barbecue and passion fruit seed salad dressings and from sugar (raw material), suggesting it is a common source of contamination for such salad dressings.

Diferentes linhagens de Bacillus thuringiensis Berliner têm provado ser poderoso inseticida biológico contra larvas de várias ordens de insetos. Dada a importância epidemiológica das moscas do gênero Chrysomya Robineau-Desvoidy na produção de miíases cutâneas secundárias e transmissão mecânica de agentes patogênicos, avaliou-se a atividade de duas linhagens de B. thuringiensis (LFB-FIOCRUZ 907 e LFB-FIOCRUZ 856) sobre larvas de Chrysomya putoria (Wiedemann). A linhagem LFB-FIOCRUZ 907 foi testada em quatro diferentes concentrações misturadas à dieta e a linhagem LFB-FIOCRUZ 856 em três concentrações. As larvas de C. putoria apresentaram sensibilidade ao tratamento com a linhagem LFB-FIOCRUZ 907, nas concentrações testadas, sendo a concentração mais alta a de maior eficiência, causando maior mortalidade e reduzindo mais intensamente o peso larval e a taxa de emergência dos adultos. A linhagem LFB-FIOCRUZ 856 apresentou toxicidade muito baixa, redizindo ligeiramente a emergência dos adultos na concentração de 326 mg/ 25 g e o peso larval nas concentrações de 326 mg/ 25 g e 86 mg/ 25 g.

Different strains of Bacillus thuringiensis Berliner were proved to be a powerful biologic insecticide against larvae of several insect orders. Due to the epidemiological importance of blowflies of the Chrysomya Robineau-Desvoidy genus in the production of secondary cutaneous myiasis and mechanic transmission of pathogenic agents, the performance of two strains of B. thuringiensis (LFB-FIOCRUZ 907 and LFB-FIOCRUZ 856) was tested against larvae of Chrysomya putoria (Wiedemann). The LFB-FIOCRUZ 907 strain was tested in four different concentrations, added to the diet; the LFB-FIOCRUZ 856 strain was tested in three concentrations. C. putoria larvae showed sensibility to the treatment with the LFB-FIOCRUZ 907 strain at the tested concentrations. The higher concentration presented the best efficiency, causing higher mortality and reducing larval weight and adult emergence more intensely. The LFB-FIOCRUZ 856 strain showed low toxicity, sliglity reducing emergence time of adults at 326 mg/ 25 g concentration and larval weight at 326 mg/ 25 g and 86 mg/ 25 g concentrations.

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinanting B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.

Foi feito um estudo comparativo da susceptibilidade de larvas de terceiro estádio de Lutzomyia longipalpis (L. & N.) (Diptera: Psychodidae: Phlebotominae), vetor da leishmaniose visceral americana, a duas estirpes de Bacillus thuringiensis sorovar israelensis e uma de Bacillus sphaericus, todas patogênicas a dípteros Culicidae, além de uma de B. thuringiensis sorovar morrisoni, patogênica a larvas de Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae). Larvas de L. longipalpis mostraram-se susceptíveis às duas amostras de B. thuringiensis sorovar israelensis, mas com baixa e nenhuma susceptibilidade às estirpes de B. sphaericus e de B. thuringiensis sorovar morrisoni, respectivamente.

A study was conducted to compare the susceptibility of third instar larvae of Lutzomyia longipalpis (L. & N.) (Diptera: Psychodidae: Phlebotominae), the vector of the American visceral leishmaniasis to two strains of Bacillus thuringiensis serovar israelensis, one strain of Bacillus sphaericus (all pathogenic to Diptera Culicidae) and a strain of B. thuringiensis ser. morrisoni (pathogenic to larvae of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)). Larvae of L. longipalpis showed similar susceptibility to the two strains of B. thruringiensis ser. israelensis, while B. sphaericus and B. thuringiensis ser. morrisoni showed low and no larvicidal effect to this vector, respectively.

The objective of this research is to present a method to bioassay entomopathogenic Bacillus species against sand fly (Diptera, Psychodidae, Phlebotominae) larvae. The vector Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera, Psychodidae, Phlebotominae) and some Bacillus strains were used in laboratory to develop this method. Some characteristics of this method are commented and the importance to develop a standard method to test pathogenicity of Bacillus species against phlebotomine larvae is also discussed.

Os efeitos ovicida e larvicida de quatro linhagens de Bacillus thuringiensis var. kurstaki foram estudados em bioensaio onde fezes de ovinos contendo ovos de Haemonchus contortus foram incubadas com cultivo em caldo nutritivo constituído por mais de 90% de esporos da bactéria. A cultura foi adicionada a fezes frescas e fezes incubadas por 24 e 48 horas. Após adição da cultura, as fezes eram homogeneizadas e incubadas a 27°C por sete dias. Após este período as larvas eram retiradas através da baermanização e preservadas em formalina para serem quantificadas posteriormente. Duas cepas ocasionaram reduções significativas quando o caldo foi adicionado a fezes frescas ou a fezes incubadas por 24 horas, enquanto uma delas reduziu significativamente o número de larvas recolhidas dos cultivos quando o caldo foi adicionado a fezes incubadas por 24 ou 48 horas. Uma das cepas ocasionou redução significativa apenas quando o caldo foi adicionado em fezes cultivadas por 48 horas.

Ovicidal and larvicidal effect of Bacillus thuringiensis var. kurstaki on free living stages of trichostrongylid nematodes is being surveyed using a bioassay in which 48-72 hours culture broth (>90% of spores) of the bacteria is added to fresh feces or feces incubated previously for 24 or 48 hours. Treated feces were incubated at 27°C for seven days after which the larvae were recovered through baermanization and formalized for a later counting. Ovicidal effect was observed in two strains and larvacidal effect in four strains.

Cento e quatorze amostras de Bacilus cereus foram isoladas durante contagens presuntivas em placas a partir de dezoito grupos de alimentos, industrializados não, crus ou cuzidos, pertencentes a dez classes. As contagens presuntivas para a espécie variam entre 10 [ao quadrado] a 6 x 10 [ao cubo]/g ou ml. Dentre estes isolamentos, treze amostras provieram de três casos de toxinfecção alimentar (envolvendo um mínimo de 57 indivíduos), e pareceram estar relacionadas com a toxinfecção em razão dos ensaios da qualidade bacteriológica dos alimentos ingeridos. Como procedimento adotado para correlacionar toxicidade e produção de doença no homem., os fluidos de cultivo de todas as amostras foram ensaiados quanto à capacidade de provocar aumento da permeabilidade capilar (APC) e necrose na pele de coelhos, assim como,morte de camundongos albinos. APC foi positivo em 86,85% das 114 amostras, morte de camundongos ocorreu em 65,79% e a combinação do APC e morte foi observada em 59,65% APC mais necrose ou somente necrose ocorreram com 34,21% dos líquidos de cultivo. Morte, APC e necrose,associados,foram observados em 28,07% das amostras. APC,APC e morte com ou sem necrose, foram também evidenciadas nas amostras originárias de alimentos causadores de doença, o que confirma a conhecida individualidade de ação de alguns dos fatores promotores de intoxicação alimentar. As baixas contagens presuntivas de B. cereus, como 10 [ao quadrado]- 10 [ao cubo]/g ou ml, encontradas nos alimentos implicados ou não com toxinfecção alimentar, conduzem à recomendaçãode que o número de B. cereus por g ou ml de amostra de alimento deve ser reavalido, aliando-se a isto a ampliação das classes de alimentos a serem conduzidas para o controle bacteriológico da espécie.

One hundred and fourteen strains of Bacillus cereus were isolated during presumptive plate-counts from 18 groups of industrialized, non-industrialized, crude or cooked food, belonging to 10 separate classes. Specific presumptive counts ranged from 10[raised to the power of 2] to 6 X 10[raised to the power of 3]/g or ml. Among these isolates, 13 strains were derived from 3 outbreaks of food poisoning (involving a minimum of 57 people), as determined by the assayed bacteriological quality of the ingested foods. As an adopted procedure to correlate toxicity and ability to promote illness in man, culture fluids of all strains were assayed to determine their ability to increase vascular permeability (APC) to cause necrosis in rabbits skin and to kill albino mice. APC was positive in 86.85% of the 114 strains, death of albino mice occurred in 65.79% and a combination of APC and death was observed in 59.65%. APC plus necrosis, or only necrosis, occurred with 34.21% of the culture fluids. Death, APC and death with or without necrosis, were demonstrated in the strains implicated with illness. This confirmed the known individuality of action exhibited by certain B. cereus food-borne toxigenic factors. The low presumptive counts of this bacterium in the order of 10[raised to the power of 2]-10[raised to the power of 3]/g or ml found in food, implicated or not with illness, suggests that the recommended number of B. cereus per g or ml of food sample should be reevaluated in our country. Furthermore, a wider range of food should be brought under bacteriological sanitary control for this species.

Um mutante espontâneo de Bacillus licheniformis, derivado da amostra esporogênica 2390, foi estudado com vistas ao reconhecimento do estágio da evolução para esporo em que o mesmo se encontrava bloqueado. Eletronmicrografias sugeriram que as células desse mutante, colhidas durante a fase estacionária da curva de crescimento, não ultrapassaram o estágio I da esporogênese (i.e., permaneceram com o nucleóide disposto como filamento axial), enquanto a produção de antibiótico (bacitracina) e a atividade proteolítica foram francamente detectadas. A linhagem mutante, designada Spolp-72, nas condições experimentais empregadas não biossintetizou esporos por estarvação em solução de sais inorgãnicos, mas evidenciou uma frequência de esporulação menor que 10*-7, após crescimento vegetativo em meio de cultura favorável á esporogênese. A amostra Spolp-72 externa um crescimento vegetativo inicial restringido, quando comparada com a amostra 2390, enquanto que, inversamente, sua atividade respiratória é significativamente mais elevada. Este último comportamento foi confirmado no presente trabalho, contrastando, nesse particular, com outros tipos de mutantes de esporulação já descritos, os quais se encontram bloqueados nos primeiros estágios da via esporogenética.

A spontaneous mutant strain derived from the sporogenic B. licheniformis 2390 was studied with a view to determining at what developmental stage toward sporulation it was blocked. Electronmicrographs suggested that the mutant cells harvested during the stationary phase of the growth curve were unable to go beyond stage I of sporogenesis (i. e., their nucleoid remained as an axial filament). On the other hand, antibiotic production (bacitracin) and proteolytic activity were easily detected. Under the present experimental conditions the mutant strain, named Spolp-72, did not synthesize spores by starvation in a solution of inorganic salts, in contrast with the parental strain. However, it showed a sporulation frequency under 10*-7 when cultivated in media favorable to sporogenesis. This strain shows a restricted initial vegetative growth when compared withstrain 2390, whereas, inversely, its respiratory metabolism (oxigen consumption) is signicantly higher. The last-mentioned behavior was confirmed in the present study, contrasting with other described types of sporulation mutants which are blocked at early stages of the sporogenetic path.