scholarly journals Glutamate Decarboxylase Genes as a Prescreening Marker for Detection of Pathogenic Escherichia coliGroups

2001 ◽  
Vol 67 (7) ◽  
pp. 3110-3114 ◽  
Author(s):  
Michael A. Grant ◽  
Stephen D. Weagant ◽  
Peter Feng
Keyword(s):  
Shiga Toxin ◽  
Glutamate Decarboxylase ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
Food Samples ◽  
E Coli ◽  
Virulence Markers ◽  
Multiplex Pcr Assay ◽  
Lactose Fermentation ◽  
Genes Encoding

ABSTRACT The enzyme glutamate decarboxylase (GAD) is prevalent inEscherichia coli but few strains in the various pathogenicE. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA andgadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagicE. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using agadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR togadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx 1 andstx 2 was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenicE. coli groups.

A Multiplex PCR Assay Mediated by Universal Primers for the Detection of Adulterated Meat in Mutton

2019 ◽  
Vol 82 (2) ◽  
pp. 325-330 ◽  
Author(s):  
WANWAN LIU ◽  
XIAONAN WANG ◽  
JING TAO ◽  
BANGSHENG XI ◽  
MAN XUE ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Detection System ◽  
Meat Products ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Food Samples ◽  
Detection Sensitivity ◽  
Universal Primers ◽  
Multiplex Pcr Assay ◽  
Multiple Pcr

ABSTRACT This study aimed to establish a multiplex PCR detection system mediated by “universal primers,” which would be able to determine whether mutton meat contained nonmutton ingredients from rats, foxes, and ducks. Based on the sequence variation of specific mitochondrial genes, nine different multiplex PCR primers were designed, and four kinds of meat products were rapidly identified by electrophoresis using an optimized multiplex PCR system based on the molecular weight differences of the amplified products. Multiplex PCR applications optimized for meat food source from food samples for testing was used to verify the accuracy of the identification method. The results showed that the primers in multiple PCR system mediated by universal primers could be used for the rapid identification of rat, fox, duck, and sheep meat in mutton products, and the detection sensitivity could reach 0.05 ng/μL. The identification of food samples validated the practical value of this method. Therefore, a multiplex PCR system mediated by universal primers was established, which can be used to quickly identify the origin of animal ingredients from rats, foxes, and ducks in mutton products.

scholarly journals The characterization of ESBL genes in Escherichia coli and Klebsiella pneumoniae causing nosocomial infections in Vietnam

2013 ◽  
Vol 7 (12) ◽  
pp. 922-928 ◽  
Author(s):  
Nguyen Hoang Thu Trang ◽  
Tran Vu Thieu Nga ◽  
James I Campbell ◽  
Nguyen Trong Hiep ◽  
Jeremy Farrar ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Nosocomial Infections ◽  
Enteric Bacteria ◽  
E Coli ◽  
Plasmid Analysis ◽  
Resistance Plasmids ◽  
Genes Encoding ◽  
Third Generation Cephalosporins

Background: Extended-spectrum β-lactamases (ESBLs) are enzymes capable of hydrolyzing oxyimino-β-lactams and inducing resistance to third generation cephalosporins. The genes encoding ESBLs are widespread and generally located on highly transmissible resistance plasmids. We aimed to investigate the complement of ESBL genes in E. coli and Klebsiella pneumoniae causing nosocomial infections in hospitals in Ho Chi Minh City, Vietnam. Methodology: Thirty-two non-duplicate isolates of E. coli and Klebsiella pneumoniae causing nosocomial infections, isolated between March and June 2010, were subjected to antimicrobial susceptibility testing. All isolates were PCR-amplified to detect the blaSHV, blaTEM and blaCTX-M ESBL genes and subjected to plasmid analysis. Results: We found that co-resistance to multiple antimicrobials was highly prevalent, and we report the predominance of the blaCTX-M-15 and blaCTX-M-27 genes, located on highly transmissible plasmids ranging from 50 to 170 kb in size. Conclusions: Our study represents a snap shot of ESBL-producing enteric bacteria causing nosocomial infections in this setting. We suggest that antimicrobial resistance in nosocomial E. coli and Klebsiella pneumoniae is rampant in Vietnam and ESBL organisms are widespread. In view of these data and the dramatic levels of antimicrobial resistance reported in Vietnam we advocate an urgent review of antimicrobial use in the Vietnamese healthcare system.

scholarly journals Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

2002 ◽  
Vol 128 (3) ◽  
pp. 357-362 ◽  
Author(s):  
N. FEGAN ◽  
P. DESMARCHELIER
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Human Infection ◽  
Pulsed Field Gel Electrophoresis ◽  
Toxin Gene ◽  
Escherichia Coli O157 ◽  
Animal Population ◽  
E Coli ◽  
Virulence Markers ◽  
Animal Isolates

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%). PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.

Simultaneous Enrichment of Shiga Toxin–Producing Escherichia coli O157 and O26 and Salmonella in Food Samples Using Universal Preenrichment Broth

2009 ◽  
Vol 72 (10) ◽  
pp. 2065-2070 ◽  
Author(s):  
MASASHI KANKI ◽  
KAZUKO SETO ◽  
JUNKO SAKATA ◽  
TETSUYA HARADA ◽  
YUKO KUMEDA
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Food Samples ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Significant Difference ◽  
Peptone Water ◽  
Radish Sprouts ◽  
Pork Samples ◽  
Better Than

Universal preenrichment broth (UPB) was compared with modified Escherichia coli broth with novobiocin (mEC+n) for enrichment of Shiga toxin–producing E. coli O157 and O26, and with buffered peptone water (BPW) for preenrichment of Salmonella enterica. Ten strains each of the three pathogens were inoculated into beef and radish sprouts following thermal, freezing, or no treatment. With regard to O157 and O26, UPB incubated at 42°C recovered significantly more cells from inoculated beef than UPB at 35°C and from radish sprout samples than UPB at 35°C and mEC+n. With regard to Salmonella, UPB incubated at 42°C was as effective as UPB at 35°C and BPW at recovering cells from beef and radish sprout samples. No significant difference was noted between the effectiveness of UPB at 42°C and UPB at 35°C or BPW in the recovery of Salmonella from 205 naturally contaminated poultry samples. By using UPB at 42°C, one O157:H7 strain was isolated from the mixed offal of 53 beef samples, 6 cattle offal samples, and 50 pork samples all contaminated naturally, with no pathogen inoculation. The present study found that UPB incubated at 42°C was as effective as, or better than, mEC+n for enrichment of O157 and O26 and comparable to BPW for preenrichment of Salmonella. These findings suggest that a great deal of labor, time, samples, and space may be saved if O157, O26, and Salmonella are enriched simultaneously with UPB at 42°C.

scholarly journals 449 Late-Breaking: Impact of feeding a propriety yeast-based synbiotic product on fecal shedding of top-7 Shiga toxin-producing Escherichia coli in feedlot cattle

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 141-141
Author(s):  
Raghavendra Amachawadi ◽  
Xiaorong Shi ◽  
LeighAnn George ◽  
Miles Theurer ◽  
Twig Marston ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Virulence Genes ◽  
Multivariable Analysis ◽  
Feedlot Cattle ◽  
Control Group ◽  
Fecal Samples ◽  
Fecal Shedding ◽  
E Coli ◽  
Multiplex Pcr Assay ◽  
The Impact

Abstract Shiga toxin-producing E. coli (STEC) belonging to serogroups O26, O45, O111, O103, O121, O145, and O157, called ‘top-7’, are major foodborne pathogens. Cattle are a major reservoir, in which STEC colonize the hindgut and are shed in the feces, which is a major source of contamination of food. Our objective was to evaluate the impact of a proprietary yeast-based synbiotic product (prebiotic and probiotic; Alltech, Inc., Nicholasville, KY) on fecal shedding of top-7 STEC in feedlot cattle. Twenty existing pens, housing 40–112 steers per pen, with an estimated 60 to 90 days to slaughter, were randomly assigned to a control group or a treatment group that received 22 g of the synbiotic product per steer per day, as a top dress, in a finishing diet. Twenty pen-floor fecal samples were collected from each pen on days 0, 21, 42, and 54. Fecal samples were enriched and subjected to a multiplex PCR assay targeting serogroup-specific genes for the top-7 STEC and three major virulence genes, stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), and eae (intimin). Bivariate descriptive statistics for the major serogroups and virulence genes were assessed prior to multivariable analysis using mixed effects logistic regression. The overall prevalence of the top-7 serogroups were 44.5% of O26, 41.3% of O157, 15.1% of O103, 13.7% of O45, 7.8% of O121, and 0.6% of O111. The overall prevalence of stx1, stx2, and eae were 43.9%, 70.8%, and 49%, respectively. E. coli O26, O157, and O45 had a significant treatment and sampling day interaction (P < 0.0001). On d 42, fecal samples from treated group had lower prevalence (P < 0.01) of O26, O103, and O45 compared to the control group. In conclusion, the in-feed administration of the synbiotic product appears to reduce fecal shedding of certain top-7 STEC serogroups in the feedlot cattle.

scholarly journals Characterization of Shiga Toxin-ProducingEscherichia coliIsolated from Ground Beef Collected in Different Socioeconomic Strata Markets in Buenos Aires, Argentina

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Patricia Llorente ◽  
Laura Barnech ◽  
Kinue Irino ◽  
María Valeria Rumi ◽  
Adriana Bentancor
Keyword(s):  
Shiga Toxin ◽  
Buenos Aires ◽  
Ground Beef ◽  
Contamination Level ◽  
Contamination Rate ◽  
E Coli ◽  
Virulence Markers ◽  
Route Of Transmission ◽  
Socioeconomic Strata

Consumption of raw/undercooked ground beef is the most common route of transmission of Shiga toxin-producingE. coli(STEC). The aim of the study was to determine the STEC contamination level of the ground beef samples collected in 36 markets of different socioeconomic strata in Buenos Aires, Argentina, and the characterization of the isolated strains. Ninety-one out of 252 (36.1%) samples werestx+. Fifty-seven STEC strains were recovered. Eleven STEC strains belonged to O157 serogroup, and 46 to non-O157 serogroups. Virulence markers of the 57 STEC werestx1, 5.3% (3/57);stx2, 86.0% (49/57);stx1/stx2, 8.8% (5/57);ehxA, 61.4% (35/57);eae, 26.3% (15/57);saa, 24.6% (14/57). Shiga toxin subtypes werestx2, 31.5% (17/54);stx2c-vhb, 24.1% (13/54);stx2c-vha, 20.4% (11/54);stx2/stx2c-vha, 14.8% (8/54);stx2/stx2c-vhb, 5.6% (3/54);stx2c-vha/vhb, 3.7% (2/54). Serotypes O178:H19 and O157:H7 were prevalent. Contamination rate of STEC in all strata was high, and the highest O157 contamination was observed at low strata at several sampling rounds. Persistence of STEC was not detected. Sixteen strains (28.1%) were resistant to ampicillin, streptomycin, amikacin, or tetracycline. The STEC contamination level of ground beef could vary according to the sociocultural characteristics of the population.

scholarly journals Assessment of Shiga Toxin-Producing Escherichia coli Isolates from Wildlife Meat as Potential Pathogens for Humans

2009 ◽  
Vol 75 (20) ◽  
pp. 6462-6470 ◽  
Author(s):  
Angelika Miko ◽  
Karin Pries ◽  
Sabine Haby ◽  
Katja Steege ◽  
Nadine Albrecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Farm Animals ◽  
Clonal Lineage ◽  
E Coli ◽  
Virulence Markers ◽  
Potential Source ◽  
Human Illness ◽  
High Level ◽  
Game Animals

ABSTRACT A total of 140 Shiga toxin-producing Escherichia coli (STEC) strains from wildlife meat (deer, wild boar, and hare) isolated in Germany between 1998 and 2006 were characterized with respect to their serotypes and virulence markers associated with human pathogenicity. The strains grouped into 38 serotypes, but eight O groups (21, 146, 128, 113, 22, 88, 6, and 91) and four H types (21, 28, 2, and 8) accounted for 71.4% and 75.7% of all STEC strains from game, respectively. Eighteen of the serotypes, including enterohemorrhagic E. coli (EHEC) O26:[H11] and O103:H2, were previously found to be associated with human illness. Genes linked to high-level virulence for humans (stx 2, stx 2d, and eae) were present in 46 (32.8%) STEC strains from game. Fifty-four STEC isolates from game belonged to serotypes which are frequently found in human patients (O103:H2, O26:H11, O113:H21, O91:H21, O128:H2, O146:H21, and O146:H28). These 54 STEC isolates were compared with 101 STEC isolates belonging to the same serotypes isolated from farm animals, from their food products, and from human patients. Within a given serotype, most STEC strains were similar with respect to their stx genotypes and other virulence attributes, regardless of origin. The 155 STEC strains were analyzed for genetic similarity by XbaI pulsed-field gel electrophoresis. O103:H2, O26:H11, O113:H21, O128:H2, and O146:H28 STEC isolates from game were 85 to 100% similar to STEC isolates of the same strains from human patients. By multilocus sequence typing, game EHEC O103:H2 strains were attributed to a clonal lineage associated with hemorrhagic diseases in humans. The results from our study indicate that game animals represent a reservoir for and a potential source of human pathogenic STEC and EHEC strains.

scholarly journals Detection of a Streptomycin/Spectinomycin Adenylyltransferase Gene (aadA) in Enterococcus faecalis

1999 ◽  
Vol 43 (1) ◽  
pp. 157-160 ◽  
Author(s):  
Nancye C. Clark ◽  
Ørjan Olsvik ◽  
Jana M. Swenson ◽  
Carol A. Spiegel ◽  
Fred C. Tenover
Keyword(s):  
Enterococcus Faecalis ◽  
Pcr Primers ◽  
Binding Assays ◽  
Aminoglycoside Resistance ◽  
Cycle Sequencing ◽  
E Coli ◽  
Genes Encoding ◽  
Gram Negative Organisms ◽  
High Level ◽  
Rhodamine Dye

Genes encoding streptomycin/spectinomycin adenylyltransferases [ANT(3")(9)] have been reported to exist in gram-negative organisms and Staphylococcus aureus. During a study of high-level aminoglycoside resistance in enterococci, we encountered an isolate ofEnterococcus faecalis that was streptomycin resistant but did not appear to contain the 6′-adenylyltransferase gene (aadE) when examined by PCR with specific primers. Phosphocellulose paper binding assays indicated the presence of an ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and 8,000 μg/ml, respectively, were observed for the isolate. PCR primers corresponding to a highly conserved region of the aadA gene were used to amplify a specific 284-bp product. The product hybridized with a digoxigenin-labeled PCR product from E. coliC600(pHP45Ω) known to contain the aadA gene. TheaadA gene was transferred via filter matings from theE. faecalis donor to E. faecalisJH2-2. PCR primers designed for analysis of integrons were used to amplify a 1-kb product containing the aadA gene, which was cloned into the vector pCRII and transformed into Escherichia coli DH5-α competent cells. d-Rhodamine dye terminator cycle sequencing was used to determine the gene sequence, which was compared to previously reported sequences of aadAgenes. We found the aadA gene in E. faecalis to be identical to the aadA genes reported by Sundström et al. for E. coli plasmid R6-5 (L. Sundström, P. Rådström, G. Swedberg, and O. Sköld, Mol. Gen. Genet. 213:191–201, 1988), by Fling et al. for theaadA within transposon Tn7 (M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095–7106, 1985), and by Hollingshead and Vapnek for E. coliR538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17–30, 1985). Previous reports of the presence of the aadA gene in enterococci appear to be erroneous and probably describe anaadE gene, since the isolates were reported to be susceptible to spectinomycin.

scholarly journals The Streptomyces coelicolor genome encodes a type I ribosome-inactivating protein

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 3021-3030 ◽  
Author(s):  
Ana G. Reyes ◽  
Nick Geukens ◽  
Philip Gutschoven ◽  
Stijn De Graeve ◽  
René De Mot ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Streptomyces Coelicolor ◽  
Protein Translation ◽  
Type I ◽  
Free System ◽  
Ribosome Inactivating Proteins ◽  
E Coli ◽  
Genes Encoding ◽  
A Cell ◽  
A Subunit

Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in numerous plants, but also constitute a subunit of the bacterial Shiga toxin. Classification of plant RIPs is based on the absence (type I) or presence (type II) of an additional lectin module. In Shiga toxin, sugar binding is mediated by a distinct RIP-associated homopentamer. In the genome of two actinomycetes, we identified RIP-like proteins that resemble plant type I RIPs rather than the RIP subunit (StxA) of Shiga toxin. Some representatives of β- and γ-proteobacteria also contain genes encoding RIP-like proteins, but these are homologous to StxA. Here, we describe the isolation and initial characterization of the RIP-like gene product SCO7092 (RIPsc) from the Gram-positive soil bacterium Streptomyces coelicolor. The ripsc gene was expressed in Escherichia coli as a recombinant protein of about 30 kDa, and displayed the characteristic N-glycosidase activity causing specific rRNA depurination. In Streptomyces lividans and E. coli, RIPsc overproduction resulted in a dramatic decrease in the growth rate. In addition, intracellular production was deleterious for Saccharomyces cerevisiae. However, when applied externally to microbial cells, purified RIPsc did not display antibacterial or antifungal activity, suggesting that it cannot enter these cells. In a cell-free system, however, purified S. coelicolor RIPsc protein displayed strong inhibitory activity towards protein translation.

scholarly journals Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

2000 ◽  
Vol 66 (2) ◽  
pp. 844-849 ◽  
Author(s):  
G. Sabat ◽  
P. Rose ◽  
W. J. Hickey ◽  
J. M. Harkin
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Selective Amplification ◽  
E Coli

ABSTRACT A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminatingE. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl2 concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detectingE. coli DNA in heterogeneous DNA samples, such as those extracted from soil.

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