Development of a rapid screening method for the detection of pathogenic Escherichia coli using a combination of Colilert® Quanti-Trays/2000 and PCR

2010 ◽  
Vol 10 (1) ◽  
pp. 7-13 ◽  
Author(s):  
K. B. Omar ◽  
N. Potgieter ◽  
T. G. Barnard
Keyword(s):  
Escherichia Coli ◽  
Internal Control ◽  
Screening Method ◽  
Rapid Screening ◽  
E Coli ◽  
Toilet Seat ◽  
Pathogenic Escherichia Coli ◽  
Wide Range ◽  
Polymerase Chain ◽  
Pcr Targeting

The use of culture based methods for the detection of Escherichia coli (E. coli) only gives information on the occurrence of E. coli and not pathogenicity of the organisms detected. To detect the both indicator and diarrhoeagenic E. coli (DEC) a method was developed using the Colilert® Quanti-Tray/2000 system and polymerase chain reaction (PCR). A total of 619 samples were collected from springs (33), boreholes (24), taps (5), rivers (36), streams (8), domestic storage containers (253), raw sewage (1), final effluents (5), stool (149), soil (5) and toilet seat swab (100) samples from various provinces in South Africa. E. coli was enumerated using the Colilert® Quanti-Tray's/2000 and DNA extracted from E. coli positive wells and used as template for a multiplex-PCR targeting genes specific to entero-pathogenic- (EPEC), entero-haemorrhagic- (EHEC), entero-invasive- (EIEC), entero-toxigenic (ETEC) and entero-aggregative E. coli (EAEC). The internal control was detected in 99% of positive E. coli samples. EAEC was detected in 17%, EIEC in 4%, ETEC in 11%, EHEC in 9% and EPEC in 21% of the E. coli positive samples. It is shown that this method can be used for the detection of DEC from a wide range of samples enriched with Colilert® Quanti-Tray's/2000.

scholarly journals Virulence gene profiles of avian pathogenic Escherichia coli isolated from chickens with colibacillosis in Bulawayo, Zimbabwe

2015 ◽  
Vol 82 (1) ◽  
Author(s):  
Joshua Mbanga ◽  
Yvonne O. Nyararai
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Economic Losses ◽  
Avian Pathogenic Escherichia Coli ◽  
Pcr Assay ◽  
E Coli ◽  
Gene Profiles ◽  
Pathogenic Escherichia Coli ◽  
Polymerase Chain

Colibacillosis, a disease caused by avian pathogenic Escherichia coli (APEC), is one of the main causes of economic losses in the poultry industry worldwide. This study was carried out in order to determine the APEC-associated virulence genes contained by E. coli isolates causing colibacillosis in chickens. A total of 45 E. coli isolates were obtained from the diagnostics and research branch of the Central Veterinary Laboratories, Bulawayo, Zimbabwe. These isolates were obtained from chickens with confirmed cases of colibacillosis after postmortem examination. The presence of the iutA, hlyF, ompT, frz, sitD, fimH, kpsM, sitA, sopB, uvrY, pstB and vat genes were investigated by multiplex polymerase chain reaction (PCR) assay. Of the 45 isolates, 93% were positive for the presence of at least one virulence gene. The three most prevalent virulence genes were iutA (80%), fimH (33.3%) and hlyF (24.4%). The kpsM, pstB and ompT genes had the lowest prevalence, having been detected in only 2.2% of the isolates. All 12 virulence genes studied were detected in the 45 APEC isolates. Virulence gene profiles were constructed for each APEC isolate from the multiplex data. The APEC isolates were profiled as 62.2% fitting profile A, 31.1% profile B and 6.7% profile C. None of the isolates had more than seven virulence genes. Virulence profiles of Zimbabwean APEC isolates are different from those previously reported. Zimbabwean APEC isolates appear to be less pathogenic and may rely on environmental factors and stress in hosts to establish infection.

scholarly journals Evaluation of multiplex SYBR green real-time PCR assay for detection of pathogenic Escherichia coli

2020 ◽  
Vol 9 (2) ◽  
pp. 448
Author(s):  
Ema Komalasari ◽  
Winiati P. Rahayu ◽  
Siti Nurjanah
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Rt Pcr ◽  
E Coli ◽  
Melting Curves ◽  
Pathogenic Escherichia Coli ◽  
Wide Range ◽  
Pcr Method

Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.

scholarly journals PCR detection of Vibrio cholerae, Escherichia coli, and Salmonella sp. from bottled drinking water in Iran

2018 ◽  
Vol 12 (09) ◽  
pp. 700-705
Author(s):  
Mojtaba Bonyadian ◽  
Hamdallah Moshtaghi ◽  
Hanie Nadi
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Vibrio Cholerae ◽  
Screening Method ◽  
Chain Reaction ◽  
Culture Methods ◽  
E Coli ◽  
Drinking Waters ◽  
Polymerase Chain

Introduction: The quality of drinking water has an important role in human health. This study was aimed to detect Escherichia. coli, Salmonella sp. and Vibrio cholerae from bottled drinking waters produced in Iran. Methodology: A total of 240 samples of bottled water of different brands were collected for testing between March 2015 to December 2015 in Shahrekord-Iran. Samples were examined by polymerase chain reaction (PCR) combined with culture methods for the detection of E. coli, Salmonella sp., and V. cholerae. Results: The results of PCR revealed that the uidA gene from E. coli, IpaB gene from Salmonella sp, and epsM gene from V. cholerae were detected in 6 (2.5%), 1 (0.4 %), 0 (0%) of the samples, respectively. But in culture methods, only E. coli 5 (2.1%) were isolated from the samples. The contamination with E. coli was significantly higher (P < 0.05) in water produced during the hot seasons than the cold seasons. Conclusions: This study confirmed the presence of Escherichia coli as the main microorganism in bottle drinking water in Iran. Also, our study showed that PCR can be used as a screening method for monitoring the enteric pathogens in drinking water.

scholarly journals Characterization and antimicrobial resistance patterns of Escherichia coli isolated from feces of healthy broiler chickens

2016 ◽  
Vol 83 (0) ◽  
Author(s):  
Ariel Eurides Stella ◽  
Maria Cristina De Oliveira ◽  
Vera Lúcia Dias da Silva Fontana ◽  
Renato Paris Maluta ◽  
Clarissa Araújo Borges ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Broiler Chickens ◽  
Clinical Signs ◽  
Avian Pathogenic Escherichia Coli ◽  
E Coli ◽  
The Past ◽  
Pathogenic Escherichia Coli ◽  
Resistance Patterns ◽  
Resistant Strains ◽  
Polymerase Chain

ABSTRACT: Avian pathogenic Escherichia coli (APEC) strains are isolated from lesions of poultry presenting colibacillosis, which is a disease that causes either systemic or localized clinical signs. Such strains share many characteristics with E. coli strains that cause extra-intestinal illness in humans. There is not a consensus on how to define the APEC pathotype with regard to the presence of virulence traits. On the other hand, in the past few years, five minimal predictors for APEC detection were proposed. The E. coli isolates in this work were tested through polymerase chain reaction (PCR) to the five proposed minimal predictors and cva C. The strains presenting them were categorized as potential APEC. The APEC and non-APEC categories showed high resistance (> 50%) to cephalotin, erythromycin, streptomycin, sulphametoxazol/trimethoprim, ampicillin, and amoxicillin. Potential APEC strains were significantly more resistant to cephalotin (p < 0.05) and neomcycin (p < 0.01) than non-APEC. These latter were significantly more resistant to tetracycline (p < 0.01) than the potential APEC strains. These results demonstrate that feces of poultry present E. coli strains with resistant features, showing or not the potential of causing colibacillosis in poultry. Because APEC and extra-intestinal illness in humans may be similar, these resistant strains are of interest to public health.

scholarly journals Extraintestinal Pathogenic Escherichia Coli-Induced Pneumonia in Three Kittens and Fecal Prevalence in a Clinically Healthy Cohort Population

2009 ◽  
Vol 21 (5) ◽  
pp. 609-615 ◽  
Author(s):  
Margaret A. Highland ◽  
Barbara A. Byrne ◽  
Chitrita DebRoy ◽  
Eileen M. Samitz ◽  
Tracy S. Peterson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pulsed Field Gel Electrophoresis ◽  
Genetic Profile ◽  
Chain Reaction ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Hemorrhagic Pneumonia ◽  
Acute Necrotizing ◽  
Healthy Cohort ◽  
Polymerase Chain

Three kittens, ages 5, 9, and 17 weeks, were found dead by separate caregivers and were submitted for necropsy. At gross necropsy, each kitten had hemorrhagic or bloody fibrinoserous thoracic fluid and differing distributions of pulmonary consolidation. On histologic examination, the pulmonary lesion in each kitten was similar and was characterized by acute necrotizing and hemorrhagic pneumonia and pleuritis, with numerous intralesional small Gram-negative rods. A pure culture of a distinct serotype of Escherichia coli was identified in lung tissue from each kitten (O4:H5, O6:H7, O6:H5). Lung isolates, genotyped by polymerase chain reaction, carried genes that are characteristic of extraintestinal pathogenic E. coli (ExPEC), including cnf-1, papG allele I, papA, papC, sfa, fim, hlyD, malX, iroN, fyuA, kpsMII, and ompT. Escherichia coli isolates from the intestines of 2 of the kittens were 100% related to the respective lung isolate, as determined by pulsed-field gel electrophoresis. Cultures of fecal samples collected from a clinically healthy cohort population of kittens revealed 16 of 19 tested kittens (84%) to be shedding hemolytic E. coli. Ten different serotypes were identified from 43 hemolytic E. coli fecal isolates from the cohort population, each of which had a genetic profile consistent with that typical of ExPEC. To the authors’ knowledge, this is the first report to describe a cluster of isolated cases of pneumonia in kittens caused by distinct serotypes of ExPEC and to evaluate the prevalence of hemolytic E. coli carrying ExPEC-associated genes in the feces of a cohort population of kittens.

scholarly journals DELETION AND AMBER MUTANTS OF fla LOCI IN ESCHERICHIA COLI K-12

Genetics ◽  
1976 ◽  
Vol 84 (3) ◽  
pp. 403-421
Author(s):  
Hisato Kondoh ◽  
Haruo Ozeki
Keyword(s):  
Escherichia Coli ◽  
High Temperature ◽  
Genetic Map ◽  
Screening Method ◽  
The Other ◽  
Rapid Screening ◽  
E Coli ◽  
Flagellar Filament ◽  
Rapid Screening Method ◽  
K 12

ABSTRACT A rapid screening method for amber fla mutants of E. coli was devised and many mutants were obtained. In addition, strains with deletions of the fla genes in the his-uvrC region were isolated from high-temperature survivors of a λcI857 lysogen in which the prophage is located between his and fla. Utilizing these mutants, eleven fla genes (I-XI) and one hag gene were identified in the his-uvrc region, in the following order: his-supD-I-II-(III, IV)-V-(VI, VII)-VIII-IX-hag-(X, XI)-uvrC. The fla genes X and XI and hag are located at about 42.5 min and the other fla genes at about 43.0 min on the E. coli genetic map (Bachmann, Low and Taylor 1976). Mutants of fla gene X showed a slight sensitivity to chi phage, although they lack the flagellar filament.

scholarly journals High diversity and variability of pipolins among a wide range of pathogenic Escherichia coli strains

2020 ◽  
Author(s):  
Saskia-Camille Flament-Simon ◽  
María de Toro ◽  
Liubov Chuprikova ◽  
Miguel Blanco ◽  
Juan Moreno-González ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Phylogenetic Groups ◽  
Dna Viruses ◽  
E Coli ◽  
Host Interaction ◽  
Attachment Sites ◽  
Pathogenic Escherichia Coli ◽  
Wide Range ◽  
Recent Addition

AbstractSelf-synthesizing transposons are integrative mobile genetic elements (MGEs) that encode their own B-family DNA polymerase (PolB). Discovered a few years ago, they are proposed as key players in the evolution of several groups of DNA viruses and virus-host interaction machinery. Pipolins are the most recent addition to the group, are integrated in the genomes of bacteria from diverse phyla and also present as circular plasmids in mitochondria. Remarkably, pipolins-encoded PolBs are proficient DNA polymerases endowed with DNA priming capacity, hence the name, primer-independent PolB (piPolB).We have now surveyed the presence of pipolins in a collection of 2238 human and animal pathogenic Escherichia coli strains and found that, although detected in only 25 new isolates (1.1%), they are present in E. coli strains from a wide variety of pathotypes, serotypes, phylogenetic groups and sequence types. Overall, the pangenome of strains carrying pipolins is highly diverse, despite the fact that a considerable number of strains belongs to only three clonal complexes (CC10, CC23 and CC32). Comparative analysis with a set of 67 additional pipolin-harboring strains from GenBank further confirmed these results. The genetic structure of pipolins shows great flexibility and variability, with the piPolB gene and the attachment sites being the only common features. Most pipolins contain one or more recombinases that would be involved in excision/integration of the element in the same conserved tRNA gene. This mobilization mechanism might explain the apparent incompatibility of pipolins with other integrative MGEs such as integrons.In addition, analysis of cophylogeny between pipolins and pipolin-harboring strains showed a lack of congruence between several pipolins and their host strains, in agreement with horizontal transfer between hosts. Overall, these results indicate that pipolins can serve as a vehicle for genetic transfer among circulating E. coli and possibly also among other pathogenic bacteria.

scholarly journals Co-relationship between Escherichia coli in broiler cellulitis and liver lesions

2020 ◽  
Author(s):  
R. M. Silva ◽  
I. M. M Silva ◽  
M. C. Jesus ◽  
M. D. B. Fernandes ◽  
F. S. Oliveira ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Subcutaneous Tissue ◽  
Systemic Infection ◽  
Fast Method ◽  
Liver Lesions ◽  
Internal Organs ◽  
Production Chain ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Polymerase Chain

Abstract Pathogenic strains of Escherichia coli may invade the subcutaneous tissue of poultry and cause cellulitis, whilst the pathogen may also cause lesions in internal organs such as the liver. Current paper co-relates Escherichia coli and virulence genes characteristic of Avian Pathogenic Escherichia coli (APEC) in broilers´ cellulitis and liver lesions. One hundred carcasses were retrieved from the production chain in an avian abattoir in the state of Bahia, Brazil, between August 2013 and January 2014, due to detection of cellulitis lesions. Cellulitis and liver samples were retrieved aseptically to quantify E. coli by Petrifilm™ count fast method (3M Company) (AOAC 998.8). Virulent genes iss and iutA were removed from E. coli isolates by Polymerase Chain Reaction (PCR). Escherichia coli was isolated from 82.0% of broilers removed from the production chain and the bacterium was concomitantly detected in cellulitis and liver lesions in 40.0% of broilers. E. coli counts ranged between 1.00 and 4.73 log CFU/g in liver lesions and between 2.00 and 9.00 log UFC/g in cellulitis lesions. Virulent genes iutA and iss were detected in 97.56% and 89.02% of E. coli isolates, respectively. Genotype analysis demonstrated the concomitant amplification of genes iutA and iss in 60.0% (n=40) of samples of cellulitis and liver lesions in which the simultaneous isolation of E. coli occurred. There was a positive and significant co-relationship (r=0.22; p<0.05) between the variables occurrence of E. coli isolated from liver samples and the occurrence of E. coli isolated from cellulitis lesions. There were also positive and significant co-relationships between populations of E. coli from liver isolates and cellulitis lesions (r=0.46; p<0.05) when E. coli isolated in the liver and in cellulitis lesions was detected. Since results showed a relationship between E. coli in cellulitis and liver lesions and possible systemic infection, the occurrence of cellulitis lesions as a criterion for total discarding of carcass may be suggested.

scholarly journals Insertion Element IS3-Based PCR Method for Subtyping Escherichia coli O157:H7

1998 ◽  
Vol 36 (5) ◽  
pp. 1180-1184 ◽  
Author(s):  
Curt J. Thompson ◽  
Claire Daly ◽  
Timothy J. Barrett ◽  
Jane P. Getchell ◽  
Mary J. R. Gilchrist ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Screening Method ◽  
Pcr Amplification ◽  
Rapid Screening ◽  
Insertion Element ◽  
E Coli ◽  
Pcr Method ◽  
Template Dna ◽  
Single Primer

An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E. coli O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS3 PCR subtypes were identified. While not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak-related isolates. IS3 PCR banding patterns were reproducible between amplifications and between subcultures. IS3 PCR could serve as a simple, rapid screening method for the identification of unrelated E. coli O157:H7 isolates.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

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