scholarly journals PCR Detection of Escherichia coli O157:H7 Directly from Stools: Evaluation of Commercial Extraction Methods for Purifying Fecal DNA

2000 ◽  
Vol 38 (11) ◽  
pp. 4108-4113 ◽  
Author(s):  
J. L. Holland ◽  
L. Louie ◽  
A. E. Simor ◽  
M. Louie
Keyword(s):  
Escherichia Coli ◽  
False Negative ◽  
Extraction Methods ◽  
Rapid Identification ◽  
E Coli ◽  
Stool Specimens ◽  
Commercial Kits ◽  
Dna Template ◽  
Culture Positive ◽  
Culture Negative

Rapid identification of Escherichia coli O157:H7 is important for patient management and for prompt epidemiological investigations. We evaluated one in-house method and three commercially available kits for their ability to extract E. coli O157:H7 DNA directly from stool specimens for PCR. Of the 153 stool specimens tested, 107 were culture positive and 46 were culture negative. The sensitivities and specificities of the in-house enrichment method, IsoQuick kit, NucliSens kit, and QIAamp kit were comparable, as follows: 83 and 98%, 85 and 100%, 74 and 98%, and 86 and 100%, respectively. False-negative PCR results may be due to the presence of either inherent inhibitors or small numbers of organisms. The presence of large amounts of bacteria relative to the amount of the E. coli O157:H7 target may result in the lower sensitivities of the assays. All commercial kits were rapid and easy to use, although DNA extracted with the QIAamp kit did not require further dilution of the DNA template prior to PCR.

scholarly journals Performance of the ImmunoCard STAT! E. coli O157:H7 Test for Detection of Escherichia coli O157:H7 in Stools

2000 ◽  
Vol 38 (5) ◽  
pp. 1866-1868 ◽  
Author(s):  
Andrew Mackenzie ◽  
Elaine Orrbine ◽  
Lucie Hyde ◽  
Michelle Benoit ◽  
Frank Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Sensitivity ◽  
False Positives ◽  
Broth Culture ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Stool Specimens ◽  
Multicenter Prospective Study ◽  
Culture Positive ◽  
Culture Negative

ImmunoCard STAT! E. coli O157:H7 (Meridian Diagnostics, Inc., Cincinnati, Ohio) is a novel rapid (10-min) test for the presence of Escherichia coli O157:H7 in stools. The test may be performed either directly on stool specimens or on an overnight broth culture of stool. In a multicenter prospective study, 14 of 14 specimens positive by culture for E. coli O157:H7 were positive by the ImmunoCard STAT! O157:H7 test, and there were no false positives from 263 culture-negative specimens. In a retrospective study, the test was positive in 339 (81%) of 417 stored culture-positive specimens and the specificity was 95% (98 of 103 specimens). No false positives were associated with alternate stool pathogens. The ImmunoCard STAT! O157:H7 test has high sensitivity and specificity.

scholarly journals Escherichia coli O157:H7 in Environments of Culture-Positive Cattle

2005 ◽  
Vol 71 (11) ◽  
pp. 6816-6822 ◽  
Author(s):  
Margaret A. Davis ◽  
Karen A. Cloud-Hansen ◽  
John Carpenter ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Room Temperature ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Different Temperatures ◽  
Farm Structures ◽  
On Farm ◽  
Culture Positive ◽  
Culture Negative ◽  
Over Time

ABSTRACT Outbreaks of Escherichia coli O157:H7 disease associated with animal exhibits have been reported with increasing frequency. Transmission can occur through contact with contaminated haircoats, bedding, farm structures, or water. We investigated the distribution and survival of E. coli O157:H7 in the immediate environments of individually housed, experimentally inoculated cattle by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7 for 3 months. Cedar chip bedding was the most frequently culture-positive environmental sample tested (27/96 or 28.15%). Among these, 12 (44.0%) of positive bedding samples were collected when the penned animal was fecal culture negative. Survival of E. coli O157:H7 in experimentally inoculated cedar chip bedding and in grass hay feed was determined at different temperatures. Survival was longest in feed at room temperature (60 days), but bacterial counts decreased over time. The possibility that urine plays a role in the environmental survival of E. coli O157:H7 was investigated. Cedar chip bedding moistened with sterile water or bovine urine was inoculated with E. coli O157:H7. Bedding moistened with urine supported growth of E. coli O157:H7, whereas inoculated bedding moistened with only water yielded decreasing numbers of bacteria over time. The findings that environmental samples were frequently positive for E. coli O157:H7 at times when animals were culture negative and that urine provided a substrate for E. coli O157:H7 growth have implications for understanding the on-farm ecology of this pathogen and for the safety of ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens.

scholarly journals Multiplex PCR for direct identification of Campylobacter spp. in human and chicken stools

2007 ◽  
Vol 56 (10) ◽  
pp. 1350-1355 ◽  
Author(s):  
Aisha Al Amri ◽  
Abiola C. Senok ◽  
Abdulrahman Yusuf Ismaeel ◽  
Ali E. Al-Mahmeed ◽  
Giuseppe A. Botta
Keyword(s):  
Multiplex Pcr ◽  
Direct Detection ◽  
False Negative ◽  
Enteric Bacteria ◽  
Enzymic Activity ◽  
Campylobacter Coli ◽  
Biochemical Tests ◽  
Direct Evaluation ◽  
Stool Specimens ◽  
Culture Negative

Differentiation between Campylobacter jejuni and Campylobacter coli is problematic in clinical specimens due to fastidious growth requirements and limited biochemical tests. This study describes a rapid, multiplex PCR protocol for the direct detection and differentiation of C. jejuni and C. coli in stools. An evaluation was carried out of this multiplex protocol based on the detection of cadF (genus specific), and hipO (C. jejuni) and asp (C. coli) genes, using stool from patients with Campylobacter enteritis and chicken. Protocol sensitivity was assessed and specificity determined using a panel of enteric bacteria, and evaluation of 30 diarrhoeic stool specimens culture negative for Campylobacter. Of the 114 specimens (54 human and 60 chicken) evaluated by the protocol, 70 (61.4 %) were identified as C. jejuni, 35 (30.7 %) as C. coli and 9 (7.9 %) as a mixed infection/colonization with both species. All mixed infections were identified as C. jejuni by culture. Among the stool specimens that were culture negative for Campylobacter, two (6.7 %) were C. jejuni positive by multiplex PCR. The protocol sensitivity limit was 0.015–0.016 ng C. jejuni and C. coli DNA μl−1 in the specimen. There was no cross-reaction with the reference strains assessed. Comparison of hippurate test and multiplex PCR demonstrated 17 isolates with false-positive hippurate enzymic activity and 7 with false-negative activity. This rapid protocol (turnaround time 6 h) is highly sensitive and specific for direct evaluation of stool for these pathogens. It has significant application for routine clinical diagnostic and epidemiological purposes.

Molecular diversity survey on diarrheagenic Escherichia coli isolates among children with gastroenteritis in Fars, Iran

2021 ◽  
Author(s):  
Amir Emami ◽  
Neda Pirbonyeh ◽  
Fatemeh Javanmardi ◽  
Abdollah Bazargani ◽  
Afagh Moattari ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Diversity ◽  
Pediatric Patients ◽  
Watery Diarrhea ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Clinical Laboratories ◽  
Health Burden ◽  
Stool Specimens ◽  
Encoding Genes

Aim: To differentiate Escherichia coli isolates from diarrheal pediatric patients in clinical laboratories. Materials & methods: Patients with watery diarrhea were selected for sampling and tested for Diarrheagenic E. coli (DEC) by API kit. DEC isolates were tested for phylotyping, pathotyping and presence of determined virulence-encoding genes by specific molecular methods. Results: About 50% of isolates were detected as DECs (>55 and >31% were categorized B2 and D phylotypes respectively). Enterotoxigenic E. coli was the most and Enteroinvasive E. coli was the lowest prevalent pathotypes. csg and fim genes were the most present virulence factors. Conclusion: Typing of E. coli isolates from stool specimens will help to determine the diversity of diarrheal pathogens and take proper decisions to reduce the health burden of diarrheal diseases.

scholarly journals Rapid Identification of Different Escherichia coli Sequence Type 131 Clades

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yasufumi Matsumura ◽  
Johann D. D. Pitout ◽  
Gisele Peirano ◽  
Rebekah DeVinney ◽  
Taro Noguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epidemiological Studies ◽  
Rapid Identification ◽  
Sequence Type ◽  
Fluoroquinolone Resistance ◽  
Pcr Assay ◽  
Content Type ◽  
E Coli ◽  
Multiplex Pcr Assay ◽  
Clade C

ABSTRACT Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-β-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with bla CTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

Specific Identification of Escherichia coli O157 by an Immunostick Method Using Commercially Available Antibodies

1996 ◽  
Vol 59 (6) ◽  
pp. 670-673 ◽  
Author(s):  
SHIU W. HUANG ◽  
TSUNG C. CHANG
Keyword(s):  
Escherichia Coli ◽  
Rapid Identification ◽  
Escherichia Coli O157 ◽  
Commercial Preparation ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Sandwich Enzyme Immunoassay ◽  
Antigen Antibody ◽  
Phosphate Buffered Saline ◽  
Somatic Antigens

A sandwich enzyme-immunoassay performed on plastic sticks was developed to specifically identify Escherichia coli O157. Colonies of test bacteria grown on tryptic soy agar were suspended in phosphate-buffered saline, heated in a 100°C water bath for 15 min, and incubated with the plastic sticks coated with a commercial preparation of anti-E. coli O157 antibodies at 37°C for 1.5 h. After incubation, the same antibodies labeled with peroxidase were used to produce the signal of antigen-antibody reaction. For 35 strains of E. coli O157 (among them 34 were E. coli O157:H7) tested, all produced strong reactions by the immunoassay. For 162 strains of E. coli with somatic antigens other than O157 and 38 strains of other genera tested, only one strain (Salmonella bietri) produced a false-positive reaction. The specificity and sensitivity of the immunostick assay were 100% (35/35) and 99.5% (199/200), respectively. The detection limit of the assay for E. coli O157:H7 (CCRC 15991) was about 105 CFU/ml. The method, which can be carried out within 3 h, is useful for rapid identification of suspect E. coli O157 isolated on selective media.

Survival of Escherichia coli O157:H7 and Campylobacter jejuni in Bottled Purified Drinking Water under Different Storage Conditions

2011 ◽  
Vol 74 (2) ◽  
pp. 254-260 ◽  
Author(s):  
HAMZAH M. AL-QADIRI ◽  
XIAONAN LU ◽  
NIVIN I. AL-ALAMI ◽  
BARBARA A. RASCO
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Campylobacter Jejuni ◽  
Culture Media ◽  
False Negative ◽  
Storage Conditions ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Log Reduction ◽  
Negative Results

Survival of Escherichia coli O157:H7 and Campylobacter jejuni that were separately inoculated into bottled purified drinking water was investigated during storage at 22, 4, and −18°C for 5, 7, and 2 days, respectively. Two inoculation levels were used, 1 and 10 CFU/ml (102 and 103 CFU/100 ml). In samples inoculated with 102 CFU/100 ml, C. jejuni was not detectable (>2-log reduction) after storage under the conditions specified above. E. coli O157:H7 was detected on nonselective and selective media at log reductions of 1.08 to 1.25 after storage at 22°C, 1.19 to 1.56 after storage at 4°C, and 1.54 to 1.98 after storage at −18°C. When the higher inoculation level of 103 CFU/100 ml was used, C. jejuni was able to survive at 22 and 4°C, with 2.25- and 2.17-log reductions, respectively, observed on nonselective media. At these higher inoculation levels, E. coli O157:H7 was detectable at 22, 4, and −18°C, with log reductions of 0.76, 0.97, and 1.21, respectively, achieved on nonselective media. Additionally, E. coli O157:H7 showed significant differences in culturability (P < 0.05) on the nonselective and selective culture media under the different storage conditions, with storage at −18°C for 2 days being the treatment most inhibiting. The percentage of sublethal injury of E. coli O157:H7 ranged from ~33 to 75%, indicating that microbial examination of bottled water must be done carefully, otherwise false-negative results or underestimation of bacterial numbers could pose a health risk when low levels of pathogens are present.

Comparing Real-Time and Conventional PCR to Culture-Based Methods for Detecting and Quantifying Escherichia coli O157 in Cattle Feces

2014 ◽  
Vol 77 (2) ◽  
pp. 314-319 ◽  
Author(s):  
M. E. JACOB ◽  
J. BAI ◽  
D. G. RENTER ◽  
A. T. ROGERS ◽  
X. SHI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Positive Culture ◽  
Feedlot Cattle ◽  
Escherichia Coli O157 ◽  
Conventional Pcr ◽  
Culture Methods ◽  
E Coli ◽  
Cattle Feces ◽  
Culture Negative

Detection of Escherichia coli O157 in cattle feces has traditionally used culture-based methods; PCR-based methods have been suggested as an alternative. We aimed to determine if multiplex real-time (mq) or conventional PCR methods could reliably detect cattle naturally shedding high (≥104 CFU/g of feces) and low (~102 CFU/g of feces) concentrations of E. coli O157. Feces were collected from pens of feedlot cattle and evaluated for E. coli O157 by culture methods. Samples were categorized as (i) high shedders, (ii) immunomagnetic separation (IMS) positive after enrichment, or (iii) culture negative. DNA was extracted pre- and postenrichment from 100 fecal samples from each category (high shedder, IMS positive, culture negative) and subjected to mqPCR and conventional PCR assays based on detecting three genes, rfbE, stx1, and stx2. In feces from cattle determined to be E. coli O157 high shedders by culture, 37% were positive by mqPCR prior to enrichment; 85% of samples were positive after enrichment. In IMS-positive samples, 4% were positive by mqPCR prior to enrichment, while 43% were positive after enrichment. In culture-negative feces, 7% were positive by mqPCR prior to enrichment, and 40% were positive after enrichment. The proportion of high shedder–positive and culture-positive (high shedder and IMS) samples were significantly different from mqPCR-positive samples before and after enrichment (P < 0.01). Similar results were observed for conventional PCR. Our data suggest that mqPCR and conventional PCR are most useful in identifying high shedder animals and may not be an appropriate substitute to culture-based methods for detection of E. coli O157 in cattle feces.

scholarly journals Use of In Vivo-Induced Antigen Technology for Identification of Escherichia coli O157:H7 Proteins Expressed during Human Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 2665-2679 ◽  
Author(s):  
Manohar John ◽  
Indira T. Kudva ◽  
Robert W. Griffin ◽  
Allen W. Dodson ◽  
Bethany McManus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Human Infection ◽  
Escherichia Coli O157 ◽  
Standard Laboratory ◽  
E Coli ◽  
Phase Culture ◽  
Stool Specimens

ABSTRACT Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.

scholarly journals Effect of Forage or Grain Diets with or without Monensin on Ruminal Persistence and Fecal Escherichia coli O157:H7 in Cattle

2004 ◽  
Vol 70 (9) ◽  
pp. 5336-5342 ◽  
Author(s):  
M. J. Van Baale ◽  
J. M. Sargeant ◽  
D. P. Gnad ◽  
B. M. DeBey ◽  
K. F. Lechtenberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nalidixic Acid ◽  
Escherichia Coli O157 ◽  
Fecal Samples ◽  
Fecal Shedding ◽  
E Coli ◽  
Culture Positive ◽  
Forage Diets

ABSTRACT Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (1010 CFU/animal) made resistant to nalidixic acid (Nalr). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nalr E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nalr E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.

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