Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing
            <i>Escherichia coli</i>

Reischl, Udo; Youssef, Mohammad; Kilwinski, Jochen; Lehn, Norbert; Zhang, Wen Lan; Karch, Helge; Strockbine, Nancy

doi:10.1128/jcm.40.7.2555-2565.2002

Cited by 118 publications

(79 citation statements)

References 39 publications

(22 reference statements)

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“…Extraction of DNA was performed by thermal cell lysis of suspected strains (Reischl et al, 2002). The DNA was used as template for the amplification of the highly conserved region of invA gene using the primers (forward: 5´-ACA GTG CTC GTT TAC GAC CTG AAT-3´; reverse: 5´-AGA CGA CTG GTA CTG ATC GAT AAT-3´) specific for all Salmonella serovars (Chiu and Ou, 1996).…”

Section: Molecular Identificationmentioning

confidence: 99%

Prevalence and molecular characterization of Salmonella serovars in milk and cheese in Mansoura city, Egypt

El-Baz¹,

Elsherbini²,

Abdel-Khalek³

et al. 2017

J Adv Vet Anim Res

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Objective: This study was conducted to determine the prevalence of Salmonella in milk (farm bulk milk, raw market milk) and cheese (kareish, white soft cheese) samples that were collected randomly from farms, supermarkets, small vendors and shops in different districts of Mansoura city, Egypt. Materials and methods: A total of 100 farm bulk milk, raw market milk, kareish cheese and white soft cheese samples (25 of each) were screened for the prevalence of Salmonella spp. The Salmonella isolates were isolated and identified by conventional bacteriological techniques, which were further confirmed genetically by polymerase chain reaction (PCR) based on the presence of invA gene. Finally, the isolates were serotyped. Results: Salmonella could be detected in 15%(n=15/100) samples with a prevalence of 12%(n=3/25), 24%(n=6/25), 20%(n=5/25) and 4%(n=1/25) in raw market milk, raw farm bulk milk, kareish cheese and white soft cheese, respectively. The Salmonella isolates were serotyped into S. enteritidis 33.3%(n=9/27) which was the most frequent, followed by S. typhimurium 25.9%(n=7/27), S. heidelberg 14.8%(n=4/27), S. infantis 11.11%(n=3/27), S. tsevie 11.11%(n=3/27) and S. haifa 3.7%(n=1/27). Conclusion:The present study confirms the presence of Salmonella in milk and cheese samples in Mansoura, Egypt, indicating that the dairy products can act as potential sources of Salmonella infection. Thus, appropriate hygienic measures are suggestive for combating Salmonellosis in Egypt.

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Section: Molecular Identificationmentioning

confidence: 99%

Prevalence and molecular characterization of Salmonella serovars in milk and cheese in Mansoura city, Egypt

El-Baz¹,

Elsherbini²,

Abdel-Khalek³

et al. 2017

J Adv Vet Anim Res

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“…Second, real-time PCR (Light Cycler) was used to detect the following six diarrhea-associated genes (41,42): stx 1 and stx 2 , characteristic of Shiga toxin-producing E. coli (STEC); eltI and estA (Heat-Labile I and HeatStable I enterotoxin), both characteristic of enterotoxigenic E. coli (ETEC); eae, characteristic of enteropathogenic E. coli (EPEC) and attaching and effacing E. coli (A/EEC) (13); and ipaH, characteristic of both enteroinvasive E. coli (EIEC) and Shigella spp. ipaH was detected using the following primer and probes: primers IpaHF (5=-TGGAAAAACTCAGT GCCTCT-3=) and IpaHR (5=-CCAGTCCGTAAATTCATTCT-3=) and probes IpaH-HP-1 (GATAAAGTCAGAACTCTCCATTTTGT-FL) and IpaH-HP-2 ([Red 640]-GATGAGATAGAAGTCTACCTGGCCT-[Ph]).…”

Section: Clinical and Epidemiological Information The Who Collaboratingmentioning

confidence: 99%

Enteroaggregative Escherichia coli O78:H10, the Cause of an Outbreak of Urinary Tract Infection

Olesen

Scheutz

Andersen³

et al. 2012

J Clin Microbiol

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eIn 1991, multiresistant Escherichia coli O78:H10 strains caused an outbreak of urinary tract infections in Copenhagen, Denmark. The phylogenetic origin, clonal background, and virulence characteristics of the outbreak isolates, and their relationship to nonoutbreak O78:H10 strains according to these traits and resistance profiles, are unknown. Accordingly, we extensively characterized 51 archived E. coli O78:H10 isolates (48 human isolates from seven countries, including 19 Copenhagen outbreak isolates, and 1 each of calf, avian, and unknown-source isolates), collected from 1956 through 2000. E. coli O78:H10 was clonally heterogeneous, comprising one dominant clonal group (61% of isolates, including all 19 outbreak isolates) from ST10 (phylogenetic group A) plus several minor clonal groups (phylogenetic groups A and D). All ST10 isolates, versus 25% of non-ST10 isolates, were identified by molecular methods as enteroaggregative E. coli (EAEC) (P < 0.001). Genes present in >90% of outbreak isolates included fimH (type 1 fimbriae; ubiquitous in E. coli); fyuA, traT, and iutA (associated with extraintestinal pathogenic E. coli [ExPEC]); and sat, pic, aatA, aggR, aggA, ORF61, aaiC, aap, and ORF3 (associated with EAEC). An outbreak isolate was lethal in a murine subcutaneous sepsis model and exhibited characteristic EAEC "stacked brick" adherence to cultured epithelial cells. Thus, the 1991 Copenhagen outbreak was caused by a tight, non-animal-associated subset within a broadly disseminated O78:H10 clonal group (ST10; phylogenetic group A), members of which exhibit both ExPEC and EAEC characteristics, whereas O78:H10 isolates overall are phylogenetically diverse. Whether ST10 O78:H10 EAEC strains are both uropathogenic and diarrheagenic warrants further investigation.

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“…Bacterial DNA was isolated as described by Reischl et al (2002). Briefly, an overnight bacterial culture was suspended in sterile distilled water and heated at 94°C for 13 min; this was followed by centrifugation at 14 000 r·min -1 for 15 min to pellet the cell debris.…”

Section: Amplification Of Pathogenic Gene Sequencesmentioning

confidence: 99%

Gene encoding virulence markers among <i>Escherichia coli</i> isolates from diarrhoeic stool samples and river sources in rural Venda communities of South Africa

Obi¹,

Green²,

Bessong³

et al. 2004

WSA

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River water sources and diarrhoeic stools of residents in the Venda Region, Limpopo Province of South Africa were analysed for the prevalence of Escherichia coli (E. coli) and the presence of virulence genes among the isolates. A control group of 100 nondiarrhoeic stool samples was included. Escherichia coli was isolated and identified by standard cultural and biochemical methods. Pathogenicity of environmental and human isolates was determined by amplification of genes associated with virulence of E. coli, using specific primers.Of a total of 228 water and river sediment samples screened, E. coli was recovered from 200 (87.7%), and 135 (67.5%) of these had one or more genes associated with pathogenicity. The highest frequency of isolation of pathogenic strains was found in Ritavi River water and sediment (80.6%), followed by Lotanyanda River (76.9%), and the least (45.8%) in Nzhelele River 2. Escherichia coli was recovered from all of the 252 diarrhoeic stools tested (100%), and 119 (47.28%) of these had one or more genes associated with pathogenicity. The frequency of isolation of potential pathogenic E. coli from humans was highly significant (t = 6.3; pd•0.01) in comparison to water isolates. Cytotoxic necrotizing Factor 1 (cnf1) and cytotoxic necrotising Factor 2 (cnf2) coding for necrotoxigenic E. coli (NEC); bundle-forming pilus (bfpA) and enteropathogenic attachment and effacement (eaeA) coding for enteropathogenic E. coli (EPEC), occurred in 35% and 34% respectively of river isolates. Heat-stable (ST) and heat-labile (LT) toxin genes coding for enterotoxigenic (ETEC) and Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) coding for Shiga-like toxin-producing E. coli (STEC) were not encountered in the river isolates. Isolates from stool samples had 21.8% and 12.6% of EPEC and NEC strains respectively; while enterotoxigenic (ETEC), Shiga-like toxin-producing (STEC) and enteroaggregative E. coli (EAEC) had a prevalence of 5%, 5.8% and 5.8% respectively. One human isolate possessed stx2 and eaeA indicating E. coli O157: H7. No genes associated with pathogenicity were observed in human non-diarrhoeic stool isolates. Results have revealed a possibility of a recycling of pathogenic E. coli strains, particularly the EPEC and NEC strains, between the water sources and the local population.

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Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli

Cited by 118 publications

References 39 publications

Prevalence and molecular characterization of Salmonella serovars in milk and cheese in Mansoura city, Egypt

Prevalence and molecular characterization of Salmonella serovars in milk and cheese in Mansoura city, Egypt

Enteroaggregative Escherichia coli O78:H10, the Cause of an Outbreak of Urinary Tract Infection

Gene encoding virulence markers among <i>Escherichia coli</i> isolates from diarrhoeic stool samples and river sources in rural Venda communities of South Africa

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