Viviane Zahner

Molecular Characterization of Brevibacillus laterosporus and Its Potential Use in Biological Control

ABSTRACT Thirty-three strains of Brevibacillus laterosporus, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods. Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated. In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk Biomphalaria glabrata. Among the targets tested, Biomphalaria glabrata demonstrated the highest degree of sensitivity to B. laterosporus, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion. Larvae of the coleopteron Anthonomus grandis were also susceptible, presenting mortality levels of between 33 and 63%. Toxicity was also noted towards the lepidopteron Anticarsia gemmatalis. In contrast, no mortality was recorded among test populations of Tenebrio molitor or Spodoptera frugiperda. The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively. None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype. However, a 1,078-bp amplicon was detected for all strains of B. laterosporus when a primer for amplification of the BOXA1R region was used. Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis. These amplicons were not detected for other phenotypically related Brevibacillus species, indicating that they represent markers that are specific for B. laterosporus, which may prove useful for the isolation and identification of new strains of this species.

Study on the bacterial midgut microbiota associated to different Brazilian populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae)

The bacterial community associated with the midgut of three Brazilian Lutzomyia longipalpis (Lutz & Neiva) populations, two from endemic areas for visceral leishmaniasis (Jacobina, Bahia State and São Luís, Maranhão State) and one from a non-endemic area (Lapinha Cave, Minas Gerais State), was identified. Five groups, 35 females each, from each population were separated; a total of 175 females per collecting area were analyzed. The species identification was based on molecular and traditional bacteriological methods. All bacteria were either affiliated to non-Enterobacteriaceae, such as Acinetobacter, Burkholderia, Flavimonas, Pseudomonas and Stenotrophomonas, or and to Enterobacteriaceae, such as Citrobacter, Enterobacter, Escherichia, Klebsiella, Serratia, Pantoea, Morganella and Weeksella. Stenotrophomonas was found to be associated with all three populations studied. In addition, Serratia spp., which are well documented as laboratory contaminant of insects, were detected only in the Jacobina population. We also discuss the impact of the colonization of insect gut by bacteria on the development and transmission of pathogens.

Study on morphology, pathogenicity, and genetic variability of Beauveria bassiana isolates obtained from Boophilus microplus tick

Fifty isolates of Beauveria bassiana (Balsamo) Vuillemin, 1912 (Ascomycota: Clavicipitaceae) were analyzed by morphology, for their pathogenic potential to Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) larvae, and by Random Amplified Polymorphic DNA-Polymerase Chain Reaction technique. Morphological analysis demonstrated that isolates present characteristics compatible to those described for B. bassiana in the literature. Virulence test demonstrated that all isolates present lethal effect on larvae and that the lethal concentration varies among isolates. The most virulent isolate was the only one obtained from human infection, which was also the only isolate presenting synnemata. The study on genetic variability among the isolates allowed the identification of 23 electrophoretic profiles. The established groupings suggest that most of the isolates obtained from B. microplus of the same locality present low genetic variation. In this way, the data in the present study will contribute to a meticulous characterization of these B. bassiana isolates.

Characterization of nitrogen‐fixing Paenibacillus species by polymerase chain reaction–restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.

Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli (ETEC) strains isolated in Rio de Janeiro city, Brazil

Enterotoxigenic Escherichia coli (ETEC) strains have been implicated as important etiological agents of diarrheal disease, especially in developing countries. This group of microorganisms has been associated with a diverse range of genotypic and phenotypic markers. In the present study, 21 ETEC isolates previously defined according to the toxigenic genotypes, were characterized on the basis of O:H typing, cell adherence patterns, and colonization factors (CFs) antigens. Genetic diversity was investigated by random amplification polymorphic DNA (RAPD-PCR), pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE). LT-I probe-positive isolates belonged to serotypes ONT:HNT, O7:H24, O48:H21, O88:H25, O148:H28, O159:H17 and O159:H21. ST-h probe-positive isolates belonged to serotypes O159:H17, O148:H28 and O6:H-. Serotypes O148:H28, O159:H17 and O6:H- were associated with the CS6, CFA/I and CS1 CS3 antigens, respectively. Most ETEC strains exhibited a diffuse pattern of adherence to cultured epithelial cells. In general, phenotypic and genotypic characteristics correlated well. RAPD-PCR, PFGE and MLEE showed reproducibility and good discriminatory potential. The application of molecular typing systems allowed the detection of significant diversity among the isolates, indicating a non-clonal origin and revealing intra-serotype variation overlooked by classical epidemiological approaches. The phenotypic and genotypic diversity observed lead us to recommend the use of different typing systems in order to elucidate the epidemiology of ETEC infection.

Distribution of Genes Encoding Putative Virulence Factors and Fragment Length Polymorphisms in the vrrA Gene among Brazilian Isolates of Bacillus cereus and Bacillus thuringiensis

ABSTRACT One hundred twenty-one strains of the Bacillus cereus complex, of which 80 were isolated from a variety of sources in Brazil, were screened by PCR for the presence of sequences (bceT, hblA, nheBC, plc, sph, and vip3A) encoding putative virulence factors and for polymorphisms in variable-number tandem repeats (VNTR), using a variable region of the vrrA open reading frame as the target. Amplicons were generated from isolates of B. cereus and Bacillus thuringiensis for each of the sequences encoding factors suggested to play a role in infections of mammals. Intriguingly, the majority of these sequences were detected more frequently in Bacillus thuringiensis than in B. cereus. The vip3A sequence, which encodes an insecticidal toxin, was detected exclusively in B. thuringiensis. VNTR analysis demonstrated the presence of five different fragment length categories in both species, with two of these being widely distributed throughout both taxa. In common with data generated from previous studies examining European, Asian, or North American populations, our investigation of Brazilian isolates supports the notion that B. cereus and B. thuringiensis should be considered to represent a single species.

Serotype H5a5b is a major clone within mosquito-pathogenic strains of Bacillus sphaericus

Seventy six mosquito pathogenic strains of Bacillus sphaericus and 10 non-pathogens were examined by pulsed field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA. Non-pathogenic strains were clearly distinguished from the entomopathogenic types which were assigned to 21 groups (SmaI restriction patterns; SRPs). Some agreement between SRP based on PFGE and serotyping was noted, in particular all 39 strains of serotype 5a5b examined revealed identical SRPs indicating total conservation of the SmaI restriction site in these bacteria. Serotype 5a5b (SRP 12) strains comprise a widely distributed and abundant clonal lineage. Most serotypes, however, were divided into several SRPs. Seven strains from serotype 2a2b were covered in five SRPs in which toxin synthesis was correlated with chromosomal structure. Similarly, toxicity correlated with SRP in strains from serotypes 3 and 6.

Application of 16S rDNA-DGGE and Plate Culture to Characterization of Bacterial Communities Associated with the Sawfly, Acantholyda erythrocephala (Hymenoptera, Pamphiliidae)

Culture-based analysis was employed in parallel with PCR amplification of 16S rDNA, coupled with denaturing gradient gel electrophoresis (DGGE), to profile bacterial species associated with different developmental stages of the pine false webworm (PFW), Acantholyda erythrocephala, a sawfly pest responsible for incidents of severe defoliation in commercially important tree plantations in North America. Culture-based analysis revealed that Pseudomonas spp. along with Bacillus sphaericus and Arthrobacter sp. were the predominant components of the microflora of the internal organs and identified life-stage-specific associations including Photorhabdus temperata with egg and larval samples and a Janthinobacterium sp. with eonymphs. PCR-DGGE confirmed the predominance of Pseudomonas spp. and B. sphaericus in the majority of samples but did not detect Arthrobacter sp., P. temperate, or Janthinobacterium sp. In contrast, DGGE revealed the presence of a Chryseobacterium sp. as the predominant component of the PFW micoflora at all life stages, with the exception of adults. This species had been infrequently cultured, at low levels, from a limited number of samples and the existence of a possible relationship between this bacterium and the PFW had gone unnoticed using the culture-based approach. Our findings highlight the advantages of applying a dual approach to the study of microbe-insect associations and demonstrate that the benefits of one system can be used to overcome some of the limitations of the other.

Detection of Carbapenemase Genes in Aquatic Environments in Rio de Janeiro, Brazil

ABSTRACT This study reveals the presence of different carbapenemase genes (blaKPC, blaNDM, blaGES, and blaOXA48-like genes) detected directly from water samples and clonal dispersion (by pulsed-field gel electrophoresis [PFGE] and multilocus sequence typing [MLST]) of KPC-2-producing Enterobacteriaceae in two important urban aquatic matrixes from Rio de Janeiro, Brazil, highlighting the role of aquatic environments as gene pools and the possibility of community spreading.

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