Jeane Q. Chaves

Genetic diversity, antimicrobial resistance and toxigenic profiles of Bacillus cereus isolated from food in Brazil over three decades

Bacillus cereus is an ever-present problem. It is widely distributed in several environments such as soil and plants and is commonly isolated from food and additives. In this study we analyzed 97 foodborne B. cereus sensu stricto strains isolated in Brazil in the 1980s, 1990s and 2000s in order to investigate the genetic diversity (assessed by Rep-PCR), antimicrobial resistance and toxigenic profiles (presence of hblA, hblC and hblD; nheA, nheB and nheC as well as cytK, ces and entFM genes) of such strains. The majority of the strains (79, 81.4%) were β-hemolytic. The NHE complex was found in 82 strains (84.5%) and HBL complex was found in 61 (62.9%) strains. All strains were negative to ces. The cytK-2 gene was found in 44 (45.4%) strains. The predominant toxigenic pattern was type I (32, 33%) which included strains positive for all toxin genes but ces. Computer assisted cluster analysis of Rep-PCR profiles showed a high genetic diversity. Seven major clusters comprising two or more strains were found and cluster 1 was predominant (ten strains, nine of them showing 100% similarity). This cluster included strains isolated in the 1980s and the 1990s. Cluster analysis of Rep-PCR profiles based on decade of isolation, source, hemolytic pattern, toxigenic and antibiotic resistance patterns revealed a similar clustering pattern as found in the analysis including all strains. The inability to observe a predominant band pattern when Rep-PCR cluster analysis was based on decade of isolation suggests that this diversity has been maintained over time. All strains were susceptible to gentamicin. We detected resistance to tetracycline (11 strains showing intermediate resistance and nine completely resistant strains), clindamycin (ten intermediate strains) and vancomycin (one strain). Clindamycin resistance showed statistical association with strains isolated in 2000s. The predominant resistance pattern was type A (72, 72.2%) which included strains susceptible to all drugs tested. Our results suggest that the majority of the strains present in several types of food in Brazil pose a potential risk to cause food poisoning due to the high prevalence of toxin genes found in these strains. However, additional studies involving cytotoxicity tests and affiliation of these strains to phylogenetic groups based on molecular data would be useful to better evaluate this potential and could provide a more accurate indication of the risk.

Keratinolytic activity of Bacillus subtilis AMR using human hair.

Aims:  To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides.

Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae)

The bacterium Bacillus thuringiensis (Bt) produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584), serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.

Molecular and toxigenic characterization of Bacillus cereus and Bacillus thuringiensis strains isolated from commercial ground roasted coffee.

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.

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

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 autoagglutinating 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.

A new strain of Bacillus thuringiensis serovar israelensis very active against blackfly larvae.

Laboratorio de Fisiologia Bacteriana, Departamentode Bacteriologia **Departamento de Bioquimica eBiologia Molecular, Instituto Oswaldo Cruz, Av.Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil*Superintendencia de Controle de Endemias,Secretaria de Estado de Saude de Sao Paulo, SP, Brasil***Instituto de Microbiologia Prof. Paulo de Goes,Centro de Ciencias da Saude, Universidade Federal doRio de Janeiro, Rio de Janeiro, RJ, BrasilKey words: Bacillus thuringiensis - Aedes aegypti -Aedes albopictus - Anopheles - blackfly - mosquitocontrol

Biological activity of Bacillus thuringiensis strains against larvae of the Blowfly Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae)

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, slightly 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.

Molecular Characterization and Risk Assessment of Bacillus cereus Sensu Lato Isolated from Ultrahigh-Temperature and Pasteurized Milk Marketed in Rio de Janeiro, Brazil

The presence of Bacillus cereus in milk is a major concern in the dairy industry. In this study 27 Bacillus cereus sensu lato isolates from pasteurized and ultrahigh-temperature (UHT) milk (24 whole UHT and 4 pasteurized samples) collected at supermarket chains in Rio de Janeiro, Brazil, were evaluated to assess the potential risk for food poisoning. Toxigenic and virulence profiles were defined by gene-specific PCR. Affiliation to phylogenetic groups was assigned by panC sequencing. Microbiological analysis revealed the presence of B. cereus s.l. in eight (33.3%) brands (six brands of UHT and two brands of pasteurized milk). Twenty-seven isolates were recovered (13 B. cereus and 14 Bacillus thuringiensis ). Predominant toxigenic patterns were type I (contains all toxin genes except ces) and type II (does not contain cytK and ces), with seven (25.9%) isolates each. Predominant virulence patterns were type 2 (does not contain hlyII or shp) and type 3 (contains all virulence genes), with five (18.5%) isolates each. All isolates belonged to phylogenetic groups III and IV. Presence of hbl, piplc, and sph was associated with group IV isolates. Our results suggest that B. thuringiensis and B. cereus sensu stricto should be considered potential foodborne pathogens. Because the majority of the milk isolates studied have the potential to cause food poisoning because of the high prevalence of toxin and virulence genes and the specific phylogenetic group affiliations, these milk products can be potentially hazardous for human consumption.

Isolation and genetic characterization of Lysinibacillus sphaericus strains found in mosquito larvae (Diptera: Culicidae)

Introduction Lysinibacillus sphaericus is a highly effective and specific bioinsecticide used for the control of Culicidae larvae. Objective This study aimed to identify and characterize L. sphaericus strains isolated from Culex quinquefasciatus larvae in Brazil. Methods C. quinquefasciatus larvae were collected from streams in the urban area of São Paulo state. L. sphaericus strains were identified through cytomorphology, biochemical, and physiological analyses. Qualitative bioassays were performed to evaluate the toxicity of the strains against C. quinquefasciatus. The crystal compound protein pattern of L. sphaericus strains was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Five reference strains were used as standards in all tests performed. Repetitive extragenic palindromic elements-polymerase chain reaction (REP-PCR) was utilized in an attempt to differentiate pathogenic and nonpathogenic isolates. Results Twenty-one strains were isolated. Only one presented toxic activity against C. quinquefasciatus. REP-PCR results identified 23 patterns among the 26 strains used in the study, and the fragment analysis showed low similarity (16%) between L. sphaericus isolates and the five reference strains. Conclusion Comparison of strains isolated in this study using REP-PCR showed a low similarity to other strains, demonstrating the high intraspecific variability for L. sphaericus.