Detection of Enterotoxigenic Clostridium perfringens in Spices Used in Mexico by Dot Blotting Using a DNA Probe

Several reports on the microbiology of spices and herbs indicate the presence of Clostridium perfringens, a spore-forming foodborne pathogen responsible for gastrointestinal disease. In the present study, a total of 380 samples of spices and herbs (cumin seed, black pepper, oregano, garlic powder, and bay leaves) widely used in Mexico were analyzed for the presence of C. perfringens, and the enterotoxigenicity of the isolates was determined by a dot-blot technique using an enterotoxin digoxigenin-labeled DNA probe. C. perfringens counts varied from <100 to 500 CFU/g in garlic powder, from <100 to 200 CFU/g in black pepper, from <100 to 433 CFU/g in cumin seed, from <100 to 340 CFU/g in oregano, and from <100 to 450 CFU/g in bay leaves. The dot-blot technique detected the enterotoxin gene in 8 (4.25%) of 188 confirmed isolates of C. perfringens. dot-blot

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toxic effects of aflatoxin microorganisms in plants used as spices

Acta Agraria Debreceniensis ◽

10.34101/actaagrar/i/8375 ◽

2010 ◽

pp. 59-62

Author(s):

Camelia Bara

Keyword(s):

Black Pepper ◽

Toxic Effects ◽

Garlic Powder ◽

Methods Of Analysis ◽

Low Concentrations ◽

Celery Seed ◽

Cumin Seed

As an extension of the analysis of black, white and capsicum peppers for aflatoxins , we have examined an additional 11 types of spices and4 herbs for these mycotoxins. The investigations consisted of assessment of the applicability of available methods of analysis and modifications ofthese, where necessary together, with a limited survey of each spice and herb for aflatoxins. The analysis of 13 types of ground spices reportedthe presence of low concentrations of aflatoxins in some samples of black pepper, celery seed, and nutmeg. We decided to include in our study 5of the spices examined by these workers (cinnamon, celery seed, coriander, nutmeg, and turmeric) for a comparison purpose. In addition weexamined ginger, mace, cumin seed, dill seed, garlic powder, onion powder, and the herbs marjoram, rosemary, thyme, and sage.

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Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.800-810.1992 ◽

1992 ◽

Vol 12 (2) ◽

pp. 800-810

Author(s):

K S Chang ◽

S A Stass ◽

D T Chu ◽

L L Deaven ◽

J M Trujillo ◽

...

Keyword(s):

Cdna Library ◽

Acute Promyelocytic Leukemia ◽

Blot Analysis ◽

Blot Hybridization ◽

Promyelocytic Leukemia ◽

Dna Probe ◽

Translocation Breakpoint ◽

Chromosome 15 ◽

Dot Blot ◽

Mrna Species

A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.

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Development and standardization of the Dot Blot test for serological diagnosis of bovine leptospirosis

Research Society and Development ◽

10.33448/rsd-v10i6.15091 ◽

2021 ◽

Vol 10 (6) ◽

pp. e22710615091

Author(s):

Higor Oliveira Silva ◽

Mariana Assunção de Souza ◽

Tatiane Cristina Fernandes Tavares ◽

Pollyanna Mafra Soares ◽

Anna Monteiro Correia Lima

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Predictive Value ◽

Bovine Serum ◽

Low Cost ◽

High Sensitivity ◽

Dot Blot ◽

Blot Technique ◽

Bovine Leptospirosis

Leptospirosis a public health problem and an endemic zoonosis in Brazil, is diagnosed by serological methods. Therefore, low-cost and easy to execute methodologies with good/high sensitivity, such as Dot Blot, are an important diagnostic tool. The aim of this study was to standardize and validation the dot-blot technique for the serological diagnosis of bovine leptospirosis. Several concentrations of antigens applied to nitrocellulose membranes, and different dilutions of conjugated bovine serum were evaluated to develop and standardize the test. The best distinction/contrast between positive and negative samples was observed for 1μL antigen (0.11μg/μL outer membrane protein of the Hardjo serovar (OMPH) and 0.08 μg/μL outer membrane protein of the Wolffi serovar (OMPW)), 1:500 and 1:10000 bovine serum dilution and conjugate, respectively. The Dot Blot test efficiency was 71.87% and kappa index, 0.46 (p<0.0001). The other parameters measured were: sensitivity 91.89%; specificity 59.32%; positive predictive value 58.62%; and, negative predictive value 59.32%. In addition to high sensitivity, other advantages of the Dot Blot technique have been identified, such as practicality, low cost since it does not require sophisticated devices and the fact that the Hardjo and Wolffi OMP also reacted with serovars from other pathogenic serogroups. The results provided positive expectations for the use of Dot Blot as support in the diagnosis of bovine leptospirosis, especially if used as a screening test, stimulating further research for the future development of kits for diagnostic purposes.

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First identification and Limited Dissemination of mcr-1 Colistin Resistance in Salmonella Isolates in Jiaxing

Journal of Food Protection ◽

10.4315/jfp-21-069 ◽

2021 ◽

Author(s):

Ping Li ◽

Li Zhan ◽

Henghui Wang ◽

Wenjie Gao ◽

Lei Gao ◽

...

Keyword(s):

Public Health ◽

Multidrug Resistance ◽

Antimicrobial Resistance ◽

Gastrointestinal Disease ◽

Foodborne Pathogen ◽

Multidrug Resistant ◽

Colistin Resistance ◽

Antimicrobial Resistance Genes ◽

Genetic Features ◽

Multidrug Resistant Strain

Salmonella , a major foodborne pathogen, causes severe gastrointestinal disease in people and animals worldwide. Plasmid-borne mcr-1 , which confers colistin resistance in Salmonella, has significant epidemiological interest for public health safety. Here, we report the first evidence of mcr-1 -mediated colistin resistance in one multidrug-resistant strain，namely 16062 in this study, from 355 Salmonella isolates collected for Jiaxing foodborne pathogen monitoring in Zhejiang Province in 2015–2019. In addition to colistin, 16062 displayed multidrug resistance to various antimicrobials (β-lactams, quinolone, sulfonamide, florfenicol, ampicillin, streptomycin, nalidixic acid, aminoglycoside, and trimethoprim-sulfamethox). The mcr-1 -carrying IncX4 plasmid (p16062-MCR) in this study shares a conserved structure with other mcr -IncX4 plasmids. We found that other antimicrobial-resistance genes ( aac(6')-Ib-cr , aadA1 , aadA2 , aph(3')-Ia , oqxA , oqxB , sul1 , and cmlA1 ) are located on p16062-cmlA, an atypical IncHI2 plasmid, in isolate 16062. This is the first identification of transferable colistin resistance in foodborne Salmonella isolate collected in Jiaxing city, the 5-year monitoring of which revealed limited dissemination. By determining the genetic features of the plasmid vehicle, the characteristics of transferable mcr genes circulating in isolates from Jiaxing are now clearer.

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Clostridium perfringens as Foodborne Pathogen in Broiler Production: Pathophysiology and Potential Strategies for Controlling Necrotic Enteritis

Animals ◽

10.3390/ani10091718 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1718

Author(s):

Zuamí Villagrán-de la Mora ◽

María Esther Macías-Rodríguez ◽

Jenny Arratia-Quijada ◽

Yesica Sughey Gonzalez-Torres ◽

Karla Nuño ◽

...

Keyword(s):

Clostridium Perfringens ◽

Current Knowledge ◽

Foodborne Pathogen ◽

Dietary Factors ◽

Necrotic Enteritis ◽

Growth Promoters ◽

Immunological Responses ◽

Poultry Products ◽

Source Of Infection ◽

Antibiotic Growth Promoters

Clostridium perfringens (Cp.) is the cause of human foodborne desease. Meat and poultry products are identified as the main source of infection for humans. Cp. can be found in poultry litter, feces, soil, dust, and healthy birds’ intestinal contents. Cp. strains are known to secrete over 20 identified toxins and enzymes that could potentially be the principal virulence factors, capable of degrading mucin, affecting enterocytes, and the small intestine epithelium, involved in necrotic enteritis (NE) pathophysiology, also leading to immunological responses, microbiota modification and anatomical changes. Different environmental and dietary factors can determine the colonization of this microorganism. It has been observed that the incidence of Cp-associated to NE in broilers has increased in countries that have stopped using antibiotic growth promoters. Since the banning of such antibiotic growth promoters, several strategies for Cp. control have been proposed, including dietary modifications, probiotics, prebiotics, synbiotics, phytogenics, organic acids, and vaccines. However, there are aspects of the pathology that still need to be clarified to establish better actions to control and prevention. This paper reviews the current knowledge about Cp. as foodborne pathogen, the pathophysiology of NE, and recent findings on potential strategies for its control.

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Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site

Thrombosis and Haemostasis ◽

10.1160/th12-08-0561 ◽

2013 ◽

Vol 109 (02) ◽

pp. 187-198 ◽

Cited By ~ 4

Author(s):

Masahiro Takeyama ◽

Hironao Wakabayashi ◽

Philip Fay

Keyword(s):

Light Chain ◽

Protein C ◽

Activated Protein C ◽

Fluid Phase ◽

Fold Increase ◽

Binding Assays ◽

Dot Blot ◽

Binding Studies ◽

Dot Blot Assay ◽

Dot Blotting

SummaryAlthough factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg336) and A2 subunits (Arg562), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGRAPC) (apparent K d ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalysed cleavage at Arg336 (K i = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007–2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed reductions in the rates of APC-catalysed inactivation of the cofactor and cleavage at the primary inactivation site (Arg336), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a >4-fold increase in K m. These results show a contribution of a number of residues within the 2007–2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.

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Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid

Journal of Bacteriology ◽

10.1128/jb.00325-08 ◽

2008 ◽

Vol 190 (13) ◽

pp. 4648-4659 ◽

Cited By ~ 61

Author(s):

Daniel Paredes-Sabja ◽

Barbara Setlow ◽

Peter Setlow ◽

Mahfuzur R. Sarker

Keyword(s):

Water Content ◽

Uv Radiation ◽

Clostridium Perfringens ◽

Spore Germination ◽

Gastrointestinal Disease ◽

Dipicolinic Acid ◽

High Heat ◽

Wild Type ◽

Active Growth ◽

Moist Heat

ABSTRACT Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca2+ and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca2+ and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca2+ and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 103- to 104-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective α/β-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.

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Microbiological Survey of Retail Herbs and Spices from Mexican Markets

Journal of Food Protection ◽

10.4315/0362-028x-64.1.99 ◽

2001 ◽

Vol 64 (1) ◽

pp. 99-103 ◽

Cited By ~ 38

Author(s):

SANTOS GARCÍA ◽

FABIOLA IRACHETA ◽

FERNANDO GALVÁN ◽

NORMA HEREDIA

Keyword(s):

Pathogenic Bacteria ◽

Salmonella Typhi ◽

Black Pepper ◽

Shigella Dysenteriae ◽

Fecal Coliforms ◽

Garlic Powder ◽

Glass Containers ◽

Polyethylene Bags ◽

Rhizopus Sp ◽

Herbs And Spices

In the present study, 304 samples of herbs and spices (garlic powder, cumin seeds, black pepper, oregano, and bay leaves) widely used in Mexico were analyzed for the presence of Bacillus cereus, Salmonella Typhi, Shigella dysenteriae, Escherichia coli, total and fecal coliforms, total mesophilic aerobic organisms, and fungi. Samples were nonpackaged or packaged in polyethylene bags or glass containers. High levels (105 to 107 CFU/g) of mesophilic aerobic microorganisms were found in most of the samples of garlic powder, cumin seed, and black pepper. Lower levels (<102 CFU/g) were found in oregano and bay leaves. Total and fecal coliforms counts were dependent on the type of packaging. More than 70% of the polyethylene-packaged samples had less than 103 CFU/g of microorganisms. Glass and nonpackaged spices showed lower levels of these microorganisms. B. cereus was present in 32 samples of which most were polyethylene packaged. The other pathogenic bacteria were not detected. Aspergillus niger was detected in 29% of the samples, Rhizopus sp. in 19%, and Penicillum sp. and Cunninghamella in 8%.

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