Characterization of toxigenic genes of Bacillus cereus strains isolated from different spices sold in Algeria.

Many cooked foods are prepared with spices and dried herbs; these can be contaminated by several types of microorganisms, including aerobic spore-forming bacteria. The Bacillus cereus group is very widespread in nature and is known among the common food contaminants. They are involved in food poisoning, causing two types of syndromes, diarrheal and emetic. The aims of the present work were to determine the prevalence of toxigenic Bacillus cereus spores in spices and herbs marketed in the Laghouat area and to identify their toxigenic genes via PCR. Among the 191 samples, 14.13% were determined to be B. cereus, with concentrations ranging from 2.52 to 5.82 log cfu/g, where the highest level of contamination was observed in allspice and ginger. Moreover, entFM (100%), nhe (88.23%) and cytK (70.58%) were the most frequently identified toxin genes, whereas hbl (23.52%) was less common, and no emetic toxin-encoding gene (cesB) was found in any of the samples. Considering the results of the present study, the B. cereus microbial load and toxin gene profiles of spices show that spices have potential for public health in Algeria. In this context, it is crucial to guarantee the microbiological safety of spices by respecting good hygiene practices, eliminating bacterial spores and toxin production via sterilization and using appropriate packaging for these products.

Genetic and toxigenic diversity of Bacillus cereus group isolated from powdered foods.

Bacillus cereus is a human pathogenic bacterium that produces emetic and diarrheal foodborne diseases. This study evaluated the genetic and toxigenic diversity in B. cereus group isolates from powdered foods collected in public educational institutions, bakeries and powdered food companies located in Medellín, Colombia. B. cereus was detected in 35 of 305 (11%) powdered food samples and 52 B. cereus were isolated. The presence of ten toxin genes, hblCDAB, nheABC, cytK2, entFM and cesB, was evaluated in the isolates by multiplex PCR. The nheABC operon was found in all isolates (100%), hblCDAB in 22 (42%), hblCDA in 8 (15%) and hblCD in 3 (6%); the cytK2 gene was detected in 32 isolates (62%) and entFM in 32 (62%). Notably, the cesB gene was not detected. According to the presence of toxin genes, fifteen profiles were identified. The predominant toxigenic profile contained all toxin genes but cesB. A large genetic diversity was observed by GTG5 fingerprinting with 46 isolates grouped in seven clusters and the remaining six clustering individually. There was no relationship between toxigenic profiles and genetic clusters, but some genetic clusters seemed to be related to particular powdered food types. In general, the results evidenced high genetic and enterotoxigenic diversity among the B. cereus group isolates.

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature.

BACKGROUND: Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. METHODS: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. RESULTS: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. CONCLUSIONS: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature

Background: Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.(AU)

Gene sequence analysis of toxins from the spider Phoneutria nigriventer revealed an intronless feature

Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.(AU)

Staphylococcal toxin genes in strains isolated from cows with subclinical mastitis / Detecção de genes de toxinas em linhagens de estafilococos isolados de vacas com mastite subclínica

The present study was carried out in 11 dairy herds in four municipal districts of the rural area of the State of Pernambuco, Brazil. Out of 984 quarter milk (246 cows), 10 (1.0%) were positive for clinical mastitis, 562 (57.1%) for subclinical mastitis and 412 (41.9%) were negative. A total of 81 Staphylococcus spp. isolates were obtained from milk samples from the cows diagnosed with subclinical mastitis. From these, 53 (65.0%) were S. aureus, 16 (20.0%) coagulase-positive staphylococci (CPS) and 12 (15.0%) coagulase-negative staphylococci (CNS). The isolates were further investigated for the presence of toxin genes by multiplex and uniplex PCR. The main gene observed was seg followed by seh, sei and sej. The distribution of these observed genes among the isolates obtained from different areas showed a regional pattern for the SEs. The presence of toxin genes in the strains isolated from bovine milk demonstrates a potential problem for public health.(AU)

O presente estudo foi realizado em 11 rebanhos leiteiros de quatro municípios da área rural do estado de Pernambuco, Brasil. Dos 984 quartos mamários examinados (246 vacas), 10 (1,0%) foram positivos para a mastite clínica, 562 (57,1%) para a mastite subclínica e 412 (41,9%) foram negativos para mastite. Foram isoladas 81 linhagens de Staphylococcus spp. do leite de vacas com mastite subclínica. Destes, 53 (65,0%) foram S. aureus, 16 (20,0%) estafilococos coagulase-positivo (SCP) e 12 (15,0%) estafilococos coagulase-negativo (SCN). O principal gene observado nos estafilococos foi o seg seguido pelo seh, sei e sej. Foi constatada distribuição regional dos genes dos estafilococos isolados dos animais nos municípios estudados. A presença dos genes das toxinas nas linhagens isoladas do leite de vacas representa risco potencial para a Saúde Pública.(AU)

Detecção de Escherichia coli O157: H7 inoculada experimentalmente em amostras de leite cru por método convencional e PCR multiplex / Detection of E. coli O157: H7 experimentally inoculated in raw milk samples by conventional method and multiplex PCR

Padronizou-se um método de reação em cadeia da polimerase (PCR) multiplex para detecção de Escherichia coli O157:H7 e avaliou-se a eficiência da PCR e de um método de cultivo convencional em placas na detecção desse patógeno experimentalmente adicionado em leite estéril e em leite cru com baixa contagem bacteriana total (média de 4,01 x 10³ UFC/ml) e com alta contagem bacteriana (média de 2,10 x 10(6) UFC/ml). Foram padronizadas duas reações de PCR com o uso dos primers: "A" (RfbF; RfbR e FLICh7F/FLICh7R) e "B" (SLT-IF/SLTIR e SLT-IIF/SLT-IIR). A detecção de E. coli O157:H7 (1UFC/ml) a partir do leite estéril e do leite cru com baixa contaminação bacteriana foi possível quando se utilizou o método de contagem em placas e a PCR. A sensibilidade dos dois métodos foi menor quando se testou o leite cru com alta contaminação microbiana, sendo o método convencional mais sensível. Os resultados indicam que a presença de outros microrganismos, em alta quantidade no leite, dificulta a detecção de E. coli O157:H7 pelos métodos utilizados.(AU)

This experiment was carried out in order to evaluate the effect of the raw milk bacterial count on the efficiency of a multiplex polymerase chain reaction and a conventional plate count method for detection of Escherichia coli O157:H7. This pathogen was experimentally inoculated into sterile milk, raw milk with low bacterial count (count mean of 4.01 x 10³ cfu/ml) and, raw milk with high bacterial count (mean 2.10 x 10(6) cfu/ml). Two protocols of PCR were standardized using primers "A" (Rfbf and Rfbr and FLICh7F/FLICh7R) and "B" (SLT-IF/SLTIR and SLT-IIF/SLT-IIR). Both conventional plate count and PCR methods were able to detect the presence of E. coli O157:H7 in either sterile milk or raw milk with low bacterial count initially inoculated with 1cfu of E. coli O157:H7 per ml. The sensibility of both methods for high-contaminated raw milk samples was lower, being the conventional approach more sensitive. These results indicate that high bacterial count in raw milk can affect E. coli O157:H7 detection.(AU)

Detecção de Escherichia coli O157: H7 inoculada experimentalmente em amostras de leite cru por método convencional e PCR multiplex / Detection of E. coli O157: H7 experimentally inoculated in raw milk samples by conventional method and multiplex PCR

Padronizou-se um método de reação em cadeia da polimerase (PCR) multiplex para detecção de Escherichia coli O157:H7 e avaliou-se a eficiência da PCR e de um método de cultivo convencional em placas na detecção desse patógeno experimentalmente adicionado em leite estéril e em leite cru com baixa contagem bacteriana total (média de 4,01 x 10³ UFC/ml) e com alta contagem bacteriana (média de 2,10 x 10(6) UFC/ml). Foram padronizadas duas reações de PCR com o uso dos primers: "A" (RfbF; RfbR e FLICh7F/FLICh7R) e "B" (SLT-IF/SLTIR e SLT-IIF/SLT-IIR). A detecção de E. coli O157:H7 (1UFC/ml) a partir do leite estéril e do leite cru com baixa contaminação bacteriana foi possível quando se utilizou o método de contagem em placas e a PCR. A sensibilidade dos dois métodos foi menor quando se testou o leite cru com alta contaminação microbiana, sendo o método convencional mais sensível. Os resultados indicam que a presença de outros microrganismos, em alta quantidade no leite, dificulta a detecção de E. coli O157:H7 pelos métodos utilizados.

This experiment was carried out in order to evaluate the effect of the raw milk bacterial count on the efficiency of a multiplex polymerase chain reaction and a conventional plate count method for detection of Escherichia coli O157:H7. This pathogen was experimentally inoculated into sterile milk, raw milk with low bacterial count (count mean of 4.01 x 10³ cfu/ml) and, raw milk with high bacterial count (mean 2.10 x 10(6) cfu/ml). Two protocols of PCR were standardized using primers "A" (Rfbf and Rfbr and FLICh7F/FLICh7R) and "B" (SLT-IF/SLTIR and SLT-IIF/SLT-IIR). Both conventional plate count and PCR methods were able to detect the presence of E. coli O157:H7 in either sterile milk or raw milk with low bacterial count initially inoculated with 1cfu of E. coli O157:H7 per ml. The sensibility of both methods for high-contaminated raw milk samples was lower, being the conventional approach more sensitive. These results indicate that high bacterial count in raw milk can affect E. coli O157:H7 detection.