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)

Colonization rate of Clostridium Difficile in healthy children / 中华儿科杂志

Objective@#To learn the colonization of Clostridium difficile in local healthy children and to investigate the colonization rate and toxin types of Clostridium difficile at different ages.@*Method@#From September 2014 to January 2015 in a case observational study, healthy children′s fecal specimens from the health care department of Beijing Children′s Hospital were collected. The children were divided into four groups according to age: <1 year old(n=53), 1-<3 years old(n=50), 3-<6 years old(n=50) and 6-<14 years old(n=50) respectively. Polymerase chain reaction (PCR) was used to detect Clostridium difficile toxin genes including tcdA, tcdB, binary toxin CDT (cdtA and cdtB), and toxin regulatory genes including tcdC, tcdD and tcdE. And then the positive samples were sequenced. Measurement data were compared by using t test and rank sum test, while, enumeration data were compared using chi-square test.@*Result@#Fifteen (7.4%) specimens were positive for Clostridium difficile toxin genes in 203 stool specimens. Of the 15 positive specimens, eight(53.3%) were tcdA+ tcdB-(A+ B-), four were A+ B+ , 3(20.0%) were A-B+ , the binary toxin-positive specimens were not detected. TcdC, tcdD, tcdE positive specimens were 8, 6 and 11, respectively. Gene mutations were not found in positive samples by DNA sequencing. In the 15 positive samples, four (7.5%) specimens were in <1 year old group; four (8.0%) specimens were in 1-<3 years old group; one(2.0%) specimen was in 3-<6 years old group; and 6(12.0%) specimens were in 6-<14 years old group. The colonization rate had no significance in different age groups.@*Conclusion@#The colonization rate of Clostridium difficile in healthy children was 7.4%. And toxigenic Clostridium difficile can be detected in all age groups.

Campylobacter spp.isolation, its toxin genes detection and molecular subtyping in diarrhea patients in Shanghai in 2014 / 上海预防医学

[ Objective] To investigate the status quo of Campylobacter spp.infection in Shanghai and study its molecular characteristics and virulence and toxin genes. [ Methods ] Stool samples collected from diarrheal patients were cultured for bacterial pathogens using membrane filter method.The strains were identified by biochemical tests and PCR.PCR was applied to detect six virulence and toxin genes including flaA,cdtA,cdtB,cdtC,virB11,cadF.Pulsed-field gel electrophoresis ( PFGE) was carried out for subtyping. [Results] A total of 43 Campylobacter spp.(1.9%) were collected from 2 235 stool samples in Shanghai in 2014 including 41 Campylobacter jejuni isolates(95.3%) and 2 Campylobacter coli isolates(4.7%) .The data showed 100.0%(43/43) of the isolates were positive for flaA and cadF, and 93.0%(40/43) of the isolates positive for cdtA and cdtB.And 88.4%(38/43) of the isolates were posi-tive for cdtC.Only 7.0%(3/43) of the isolates were positive for virB11.Using PFGE, 43 Campylobacter jejuni and Campylobacter coli strains were subtyped into 6 clusters. [ Conclusion] The genes of flaA and cadF are ubiquitous on Campylobacter spp.isolates.The distribution of cdt gene cluster in Campylobacter spp.is high, while that of virB11 is low.The PFGE types of Campylobacter spp.isolated in Shanghai are of diversified and complicated features, which causes mainly sporatic diarrhea.

Prevalence of seg, seh and sei Genes among Clinical and Nasal Staphylococcus aureus Isolates in Palestine.

Aims: To investigate the presence of the staphylococcal enterotoxin genes seg, seh and sei among clinical and nasal isolates. Place and Duration of Study: Department of Biology and Biotechnology, An-Najah N. University, Palestine, in 2011. Methodology: A total 124 S. aureus isolates were collected, forty three were nasal and 81 were clinical isolates. PCR technique was used to detect enterotoxin genes seg, seh and sei, mecA gene and analysis of SCCmec types. Enterotoxigenic strains were also typed using coagulase typing kit. Results: Fifty two (41.9%) isolates were positive for one or more of these enterotoxin genes. The prevalence of toxin genes among S. aureus isolated from nasal swabs 25/43 (58.1%) was higher than those isolated from clinical samples 27/81 (33.3%). Combination of the toxin genes was noted only in MSSA isolate from both nasal swabs and clinical samples. Distribution of toxin genes in MSSA isolates was higher (49.5%) than those in MRSA isolates (21.2%). SCCmec typing showed that the MRSA enterotoxigenic strain were belonged to types II, III and IVa. MRSA strains were found to belong to coagulase serotypes II, III and VII, while MSSA strains were belonged to serotypes II-VII. In nasal samples, 16/25 (64.0%) of enterotoxigenic strains showed the genotype seg+/sei+, while in clinical samples 1/27 (3.7%), 1/27 (3.7%) and 3/27 (11.1%) of enterotoxigenic strains showed the genotypes seg+/seh+, seg+/sei+ and seg+/seh+/sei+, respectively. This study showed that the majority of the isolates 42/124 (33.9%) were seg+, while none of nasal strains harbored seh gene. Conclusion: The prevalence of seg, seh and sei genes in the S. aureus isolated from nasal swabs differed significantly from those obtained from clinical samples, as well as the prevalence of the same genes in MSSA differed significantly from those in MRSA. In addition, S. aureus isolates from clinical and nasal swabs could serve as a possible reservoir of newly described seg, seh and sei genes.

PCR detection of ‘STX 1’ and ‘STX 2’ toxigenic genes in multiple antibiotic resistant escherichia coli population and phenotypic detection of ESBL producing escherichia coli. isolates from local variety of poultry.

PCR detection of ‘stx 1’ and ‘stx 2’ toxigenic genes in multiple antibiotic resistant Escherichia coli population and Phenotypic detection of ESBL producing Escherichia coli isolated from traditional local variety of poultry was conducted The study revealed that out of 30 samples collected 20 were positive for E.coli All the 20 isolates were subjected for the detection of 2 toxin genes (stx1 & stx2 genes) by PCR. Only 3 isolates (L3, L4 & L5) gave rise to PCR product against its specific primers of stx1. All the isolates were negative for the presence of stx2 gene. Thus 15% of E.coli population under this study is potential isolates and indicates that healthy retail poultry birds are the carrier of E.coli pathogenic strains. The poultry birds may therefore, be considered as a major reservoir of E.coli.

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.

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.