scholarly journals Isolation and characterization E. coli O157:H7 from meat &cheese and detection of Stx1, Stx2, hlyA, eaeA by using multiplex PCR

2011 ◽  
Vol 5 (2) ◽  
pp. 15-24
Author(s):  
Shatha T. Ahmed ◽  
Amina N. Jasim ◽  
Ayad M. Ali
Keyword(s):  
Escherichia Coli ◽  
Diagnostic System ◽  
Latex Agglutination ◽  
Diagnostic Methods ◽  
Bacterial Isolates ◽  
Biochemical Tests ◽  
E Coli ◽  
Isolation And Characterization ◽  
Minced Beef ◽  
Polymerase Chain

total of 189 samples were collected from 74 raw uncooked minced beef meat, 115 local white cheeses from 3 different areas in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identified by using morphological diagnostic methods; Samples were cultured on liquid enrichment medium, incubated at 41.5Cº for 6 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 66 non-sorbitol fermenting bacterial isolates were obtained of which 13 were identified as Escherichia coli from (6 meat and 7 cheese samples). By using traditional biochemical tests and Api20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates. Four specific biochemical tests (Cellobiose fermentation, β-Glocuronidase production, KCN and Enterohemolysin production) were done to differentiate serotype O157 differentiation from other NSF bacteria. Only 2 isolates belonging to the serotype O157 were obtained of which one isolate from meat and other isolate from cheese. Latex agglutination test for O157 and H7 showed that the 5 isolates gave positive results with both kits. The Bacterial isolates were identified by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated from various sources by using specific primers in mPCR. The result showed that gene content variety in two E. coli O157:H7 isolates, 1 from meat contain all 4 genes and other isolate from cheese contains 2 genes: Stx1 and hlyA .

scholarly journals Molecular diagnosis of E.coliO157:H7 Which Isolated from Children with Diarrhea by using Multiplex PCR

2010 ◽  
Vol 7 (1) ◽  
pp. 207-215
Author(s):  
Baghdad Science Journal
Keyword(s):  
Escherichia Coli ◽  
Diagnostic System ◽  
Latex Agglutination ◽  
Diagnostic Methods ◽  
Bacterial Isolates ◽  
Biochemical Tests ◽  
E Coli ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Positive Results

A total of 96 stool samples were collected from children with bloody diarrhea from two hospitals in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identifed by using morphological diagnostic methods, Samples were cultured on liquid enrichment medium, incubated at 37C? for 24 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 32 non-sorbitol fermenting bacterial isolates were obtained of which 11 were identified as Escherichia coli by using traditional biochemical tests and API20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates . Four special biochemical tests were done for serotype O157:H7 differentiation from other NSF bacteria. Only 3 isolates belonging to the serotype O157:H7 were obtained . Latex agglutination test for O157 and H7 showed that the 3 isolates gave positive results with both tests. The Bacterial isolates were identifed by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated By using specific primers in MPCR . The result showed that one E. coli O157:H7 isolates contain all 4 genes , other isolates contain 3 genes: Stx2, hlyA & eaeA.

An Improved Rapid Technique for Isolation of Escherichia coli O157:H7 from Foods

1995 ◽  
Vol 58 (1) ◽  
pp. 7-12 ◽  
Author(s):  
STEPHEN D. WEAGANT ◽  
JAMES L. BRYANT ◽  
KAREN G. JINNEMAN
Keyword(s):  
Escherichia Coli ◽  
Comparative Method ◽  
Immunomagnetic Separation ◽  
Latex Agglutination ◽  
Escherichia Coli O157 ◽  
Biochemical Tests ◽  
Isolation Technique ◽  
Selective Enrichment ◽  
E Coli ◽  
Polymerase Chain

A newly revised enrichment and agar-plating system was tested for selectivity and sensitivity in recovery of unstressed and cold-stressed Escherichia coli O157:H7 from foods. Various foods inoculated with known levels of enterohemorrhagic E. coli O157:H7 (EHEC) were tested by enrichment for 6 h at 37°C in modified tryptic soy broth (mTSB) base supplemented with vancomycin, cefsulodin and cefixime, referred to as EHEC enrichment broth (EEB). Subsequently, portions were spread-plated on sorbitol–MacConkey agar supplemented with tellurite and cefixime (TCSMAC). Further selective enrichment was also examined using immunomagnetic separation (IMS) from the EEB prior to spread-plating on TCSMAC agar. These methods were compared to a procedure of enrichment in mTSB (supplemented with novobiocin) at 37°C for 24 h followed by spread-plating of decimal dilutions on hemorrhagic colitis 4–methylumbelliferyl–B–D–glucuronide (HC–MUG) agar. The new enrichment isolation technique was found to be sensitive at a level of one EHEC organism per 10 g of food in four food types. This represents an approximate l00-fold to 1,000-fold enhancement in sensitivity over the comparative method for foods with high levels of competitive microflora. These enrichment-isolation protocols also were compared in analysis of naturally contaminated raw or undercooked ground beef samples implicated in foodborne illness. EEB-TCSMAC with and without IMS were combined with rapid biochemical tests, and with O157 latex agglutination and confirmation of toxin genes by polymerase chain reaction (PCR) to provide a completed test within 30 h of initiating testing. The new system was successful in 15 of 17 samples, where only 6 of 17 were found positive by the comparative technique.

Occurrence of Escherichia coli O157 and Other Verocytotoxin-Producing E. coli in Retail Raw Meats in the Netherlands

1996 ◽  
Vol 59 (12) ◽  
pp. 1267-1272 ◽  
Author(s):  
ANNET E. HEUVELINK ◽  
KAREL WERNARS ◽  
ENNE de BOER
Keyword(s):  
Escherichia Coli ◽  
The Netherlands ◽  
Selective Enrichment ◽  
E Coli ◽  
Enrichment Cultures ◽  
Poultry Products ◽  
Minced Beef ◽  
Test Kit ◽  
Polymerase Chain ◽  
Raw Meats

Raw meats obtained from retail outlets in the Netherlands were examined for the presence of Escherichia coli of serogroup O157 and other verocytotoxin (VT)-producing E. coli (VTEC), in three different surveys. In the first survey O157 VTEC were detected and isolated by selective plating onto sorbitol MacConkey agar following selective enrichment in modified tryptone soy broth with acriflavin. The organisms were isolated from 2 (0.3%) of 770 samples of minced mixed beef and pork, but not detected in samples of raw minced beef (n = 1,000), minced pork (n = 260), or poultry products (n = 300). In the second survey an additional 360 raw meats were examined with the 3M Petrifilm™ Test Kit-HEC, after selective enrichment in modified E. coli broth containing novobiocin. VT-negative E. coli O157 strains were isolated from 22 (6.1%) samples. In the third survey 180 enrichment cultures of the first survey were screened for the presence of VT1 and VT2 genes with a polymerase chain reaction (PCR). Twenty-nine (16.1%) of the 180 enrichment cultures showed a positive PCR: one for the VT1 gene only, 17 for the VT2 gene only, and 11 for both the VT1 and VT2 gene. A total of 46 VTEC strains were isolated from 10 randomly selected PCR-positive samples. Serotyping revealed that 41 of the 46 VTEC isolates belonged to nine different O serogroups; the remaining five were unidentifiable. A number of the serogroups recovered have been associated with human disease.

scholarly journals PHYLOGENETIC GROUP OF ESCHERICHIA COLI IN URINE OF CHILDREN ATTENDING A MATERNAL AND CHILD CENTRE, LAGOS

2020 ◽  
Vol 7 ◽  
pp. 76
Author(s):  
Anotu Mopelola Deji-Agboola ◽  
Esther Abieyuwa Utomwen ◽  
Olubunmi Adetokunbo Osinupebi
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Phylogenetic Group ◽  
Bacterial Isolates ◽  
Chain Reaction ◽  
Phylogenetic Groups ◽  
E Coli ◽  
Antibiotic Susceptibility Test ◽  
Polymerase Chain ◽  
Very High

Urinary tract infection (UTI) is one of the most common bacterial infections causing significant morbidity in children. This study determines the phylogenetic group of Escherichia coli  isolated from the urine of children attending Maternal and Child Centre,  Amuwo-Odofin, Lagos State. Clean voided midstream urine sample collected  from 215 children aged five years and below . The urine samples were culture on Mac Conkey and blood agar, bacterial 5counts greater than or equal to 1 x 10CFU/ml were regarded as positive for UTI. Identification of isolates and antibiotic susceptibility test was performed using standard methods. Polymerase Chain Reaction was used to classify the isolated E. coli into A, B1, B2 and D phylogenetic groups using presence or absence of ChuA, Yja A and TspE4C2. The prevalence of UTI was 51.2% with preponderance in male 62.7% aged 24 – 35months 75.0%. Staphylococcus aureus 19.1% followed by Pseudomonas aeruginosa 12.7% and Escherichia coli10.0% were common bacterial isolates. The isolates were highly resistant to Augmentin (71.8%) and Ampicillin 91.8%; the Escherichia coli were resistant to Augmentin 54.5% and Ampicillin 100%. The E. coli were classed into B1,A, and D phylogenetic groups with percentages of 54.5%27.3% and 18.2% respectively. , The prevalence of bacteria UTI among children in this study was very high, the isolates were highly resistant to the antibiotics tested, the E.coli belong to phylogenetic groups A, B1 and D and all were resistant to Ampicillin.

scholarly journals Isolation, Molecular Detection and Antimicrobial Susceptibility Profile of ‎Escherichia coli O157:H7 in Household - reared Small Ruminants in Zaria ‎Metropolis, Kaduna State, Nigeria

2020 ◽  
Vol 17 (4) ◽  
pp. 16-23
Author(s):  
R. O. Yakubu ◽  
M. K. Lawan ◽  
J. K. P. Kwaga ◽  
J. Kabir
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Susceptibility ◽  
Small Ruminants ◽  
Resistance Index ◽  
Latex Agglutination ◽  
Escherichia Coli O157 ◽  
Biochemical Tests ◽  
Cross Sectional ◽  
E Coli ◽  
Test Kit

Escherichia coli O157:H7 is a zoonotic enteric pathogen of public health significance worldwide. A cross-sectional study was carried out during which 384 faecal samples of household-reared small ruminants and water used in the various houses where the animals are reared were collected. The samples were enriched on tryptone soya broth and cultured on EMB and CT-SMAC to isolate E. coli and E. coli O157:H7 respectively; subjected to conventional biochemical tests and E. coliO157:H7 was confirmed using Wellcolex latex agglutination test kit. E. coli O157:H7 isolates were subjected to antimicrobial susceptibility test and multiplex PCR was carried out to detect the presence of virulence genes stx1, stx2, eaeA and hlyA. The results of the isolation showed isolation rate of E. coli O157:H7 of 4.69% (9/192), 0.52% (1/192) which were obtained from faeces and water samples respectively. The results of the characterisation showed that one of the E. coli O157:H7 isolated harboured the eaeA and hlyA genes but was negative for stx1 and stx2 genes. The highest number of isolates showed resistance to erythromycin (90.9%) while the least was to gentamicin (6.3%). About 97.7% (43/44) of the isolates had multiple antibiotic resistance index greater than 0.2. In conclusion, household-reared small ruminants in the study area were found to be reservoirs of E. coli O157:H7 and humans living within these households are at risk of infection. The multiple antibioticresistance recorded in this study suggests widespread use of antimicrobial drugs in the study area.

scholarly journals Detection of shiga toxin producing Escherichia coli isolated from children and cattle using PCR technique

2010 ◽  
Vol 9 (3) ◽  
pp. 76
Author(s):  
H. N. A'aiz, And F. A. Abdulla A. H. Al- Hama
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Dna Marker ◽  
Hospitalized Children ◽  
Bacterial Isolates ◽  
Macconkey Agar ◽  
Biochemical Tests ◽  
Fecal Samples ◽  
E Coli ◽  
Stx2 Gene

This study was undertaken to detect STEC isolates, gene(Stx2) in Escherichia coli isolatesand characterize them by biochemical tests , enterohemolysin production and PCR.During aperiod of seven months (November 2007 to May 2008), a total of 280 fecal samples werecollected from 120 hospitalized children suffering from diarrhea and 160 cattle fecal samples .Feces specimens were screened for the presence of NSF E. coli and STEC by cultured onsorbitol MacConkey agar (SMAC).A total of 209 (74.6%) non-sorbitol fermenting (NSF)bacterial isolates were obtained , 69 (57.5%) from children fecal samples and 140 (87.5%) fromcattle feces . Of which 5 (4.16%) NSF E. coli isolated from children fecal samples and 38(23.75%) from cattle feces. NSF isolates were identified as Shiga toxin producing E. coli(STEC), but only 16 (10%) isolates of cattle and 2 (1.6%)isolates of children were PCR-positivefor (Stx2) gene which gave amplification bands at 346 bp using DNA marker in the interpretationof the results. Among 18 STEC studied, a total of 16 (88.8%) isolates expressed enterohemolysinon washing sheep blood agar plates.On the other hand, the study was showed that the sensitivityand specificity of PCR technique in diagnosis of STEC were 41.8% , 100% respectively, incomparison with other tests like biochemical tests, sensitivity and specificity of these tests were(100% , 86.9%) respectively.

scholarly journals High prevalence of qnrA and qnrB genes among fluoroquinolone-resistant Escherichia coli isolates from a tertiary hospital in Southern Nigeria

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Chibuzor M. Nsofor ◽  
Mirabeau Y. Tattfeng ◽  
Chijioke A. Nsofor
Keyword(s):  
Escherichia Coli ◽  
Susceptibility Testing ◽  
Tertiary Hospital ◽  
Vaginal Swab ◽  
Fluoroquinolone Resistance ◽  
E Coli ◽  
Diffusion Technique ◽  
High Prevalence ◽  
Polymerase Chain ◽  
And Control

Abstract Background This study was aimed to determine the prevalence of qnr genes among fluoroquinolone-resistant Escherichia coli (FREC) isolates from Nigeria. Antimicrobial susceptibility testing was performed by disc diffusion technique. Polymerase chain reaction was used to identify Escherichia coli (E. coli) and for the detection of qnr genes. Results A total of 206 non-duplicate E. coli were isolated from 300 clinical specimens analyzed. In all, 30 (14.6%) of these isolates were FREC; the resistance to fluoroquinolones among these 30 FREC showed 80% (24), 86.7% (26), 86.7% (26), 100% (30), 86.7% (26), 93.3% (28) and 86.7% (26) were resistant to pefloxacin, ciprofloxacin, sparfloxacin, levofloxacin, nalidixic acid, ofloxacin and moxifloxacin, respectively. The distribution of FREC among the various sample sources analyzed showed that 14%, 10%, 13.3%, 16.7% and 20% of the isolates came from urine, stool, high vaginal swab, endo cervical swab and wound swab specimens, respectively. More FREC were isolated from female samples 73.3% (22) compared to male samples 26.7% (8) and were more prevalent among the age group 26–35 years (40%). Twenty eight out of the 30 (93.3%) FREC isolates possessed at least one fluoroquinolone resistance gene in the form of qnrA 10 (33.3%) and qnrB 18 (60%), respectively; qnrS was not detected among the FREC isolates analyzed and 13.5% of the isolates possessed both the qnrA and qnrB genes. Phylogenetic analysis showed that these isolates were genetically diverse. Conclusions These findings suggest a possible resistance to fluoroquinolone is of high interest for better management of patients and control of antimicrobial resistance in Nigeria.

scholarly journals Molecular surveillance of shiga toxigenic Escherichia coli in selected beef abattoirs in Osun State Nigeria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Femi Ayoade ◽  
Judith Oguzie ◽  
Philomena Eromon ◽  
Omolola E. Omotosho ◽  
Tosin Ogunbiyi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Block Design ◽  
Latex Agglutination ◽  
Pcr Analysis ◽  
Accession Number ◽  
E Coli ◽  
Representative Isolate ◽  
Randomized Block Design ◽  
Toxigenic Strains

AbstractShiga toxigenic strains of E. coli (STEC) known to be etiological agents for diarrhea were screened for their incidence/occurrence in selected abattoirs sources in Osogbo metropolis of Osun State, Nigeria using a randomized block design. Samples were plated directly on selective and differential media and E. coli isolates. Multiplex PCR analysis was used to screen for the presence of specific virulence factors. These were confirmed serologically as non-O157 STEC using latex agglutination serotyping kit. Sequence analysis of PCR products was performed on a representative isolate showing the highest combination of virulence genes using the 16S gene for identification purposes only. Results showed that the average cfu/cm2 was significantly lower in the samples collected at Sekona-2 slaughter slab compared with those collected at Al-maleek batch abattoir and Sekona-1 slaughter slab in ascending order at P = 0.03. Moreover, the average cfu/cm2E. coli in samples collected from butchering knife was significantly lower when compared with that of the workers’ hand (P = 0.047) and slaughtering floor (P = 0.047) but not with the slaughter table (P = 0.98) and effluent water from the abattoir house (P = 0.39). These data suggest that the abattoir type may not be as important in the prevalence and spread of STEC as the hygiene practices of the workers. Sequence analysis of a representative isolate showed 100% coverage and 96.46% percentage identity with Escherichia coli O113:H21 (GenBank Accession number: CP031892.1) strain from Canada. This sequence was subsequently submitted to GenBank with accession number MW463885. From evolutionary analyses, the strain from Nigeria, sequenced in this study, is evolutionarily distant when compared with the publicly available sequences from Nigeria. Although no case of E. coli O157 was found within the study area, percent occurrence of non-O157 STEC as high as 46.3% at some of the sampled sites is worrisome and requires regulatory interventions in ensuring hygienic practices at the abattoirs within the study area.

scholarly journals On-column Refolding of an Insoluble His6-tagged Recombinant EC-SOD Overexpressed in Escherichia coli

2005 ◽  
Vol 37 (4) ◽  
pp. 265-269 ◽  
Author(s):  
Xi-Qiang Zhu ◽  
Su-Xia Li ◽  
Hua-Jun He ◽  
Qin-Sheng Yuan
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Mass Ratio ◽  
Chain Reaction ◽  
Expression Plasmid ◽  
Protein Subunits ◽  
E Coli ◽  
Metal Affinity ◽  
Polymerase Chain ◽  
Escherichia Coli Expression

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

Prevalence of non-O157 Shiga Toxin-producing E. Coli in Children and Calves in Al- Muthanna Province, Iraq

2021 ◽  
Vol 14 (2) ◽  
pp. 78-90
Author(s):  
Ahmed Jarad ◽  
Kh. Al- Jeboori
Keyword(s):  
Shiga Toxin ◽  
Latex Agglutination ◽  
Macconkey Agar ◽  
Biochemical Tests ◽  
Bacteriological Study ◽  
E Coli ◽  
Additional Information ◽  
Single Colony ◽  
Big Six ◽  
Reliable Isolation

The present study focus on non-O157 Shiga toxin-producing E. Coli (STEC), included a bacteriological study was subjected to provide additional information for non-O157 STEC prevalence in children and calves. Isolation by using selective culturing media (CHROMagar STEC and CHROMagar O157) from 127 children suffering from diarrhea and 133 calves in Al- Muthanna province. Characterization depends on culturing positive colony on MacConkey agar and Levin’s Eosin Methylene blue agar, staining single colony from the growth by gram stain, biochemical tests; Indole, the Methyl Red, Voges-Proskauer, Citrate test, Oxidase, Catalase, Urease, Motility, Kligler Iron and Api-20E, were done to confirm a diagnosis of non-O157 STEC, The reliable isolation as non-O157 STEC serotyping by specific latex agglutination test for the target non-O157 STEC (big six) serogroup (O26, O45, O103, O111, O121 and O145). The current study showed the prevalence of non-O157 STEC was 20 of out 127 (15.73%) in samples collected from children and 27 / 133 (20.30%) in calves samples in conclusion the Non-O157 STEC is an important cause of diarrhea in children, and calves; finally, the calves play an important reservoir for Non-O157 STEC.

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