Sensitive Detection of Escherichia coli O157:H7 by Conventional Plating Techniques

2006 ◽  
Vol 69 (3) ◽  
pp. 689-692 ◽  
Author(s):  
KIERAN N. JORDAN ◽  
MATTHEW M. MAHER
Keyword(s):  
Escherichia Coli ◽  
Direct Detection ◽  
Latex Agglutination ◽  
Escherichia Coli O157 ◽  
Macconkey Agar ◽  
Plate Method ◽  
Potassium Tellurite ◽  
E Coli ◽  
Cheese Ripening ◽  
Unpasteurized Milk

The direct detection and estimation of concentration of Escherichia coli O157:H7 down to 1 CFU/g of cheese was achieved by conventional plating techniques. Cheese was manufactured with unpasteurized milk inoculated with E. coli O157: H7 at 34 ± 3 CFU/ml. The numbers of E. coli O157:H7 were monitored during cheese ripening by plating on sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) and on CT-O157:H7 ID medium. Using the pour plate method, E. coli O157:H7 colonies could easily be distinguished from non-O157:H7 colonies on CT-O157:H7 ID medium but not on CT-SMAC. Higher numbers of E. coli O157:H7 were detectable with O157:H7 ID medium. Latex agglutination and PCR were used to confirm the identification of typical E. coli O157:H7 colonies, and nontypical colonies as not being E. coli O157:H7. As few as 1 CFU/g of cheese could be detected. E. coli O157:H7 also was detected in deliberately contaminated milk at concentrations as low as 4 CFU/10 ml.

Survival of a Five-Strain Cocktail of Escherichia coli O157:H7 during the 60-Day Aging Period of Cheddar Cheese Made from Unpasteurized Milk†

2006 ◽  
Vol 69 (5) ◽  
pp. 990-998 ◽  
Author(s):  
J. E. SCHLESSER ◽  
R. GERDES ◽  
S. RAVISHANKAR ◽  
K. MADSEN ◽  
J. MOWBRAY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Treatment ◽  
Cheddar Cheese ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Cheese Ripening ◽  
Aging Period ◽  
Plastic Bags ◽  
Unpasteurized Milk ◽  
Acid Tolerant

The U.S. Food and Drug Administration Standard of Identity for Cheddar cheeses requires pasteurization of the milk, or as an alternative treatment, a minimum 60-day aging at ≥2°C for cheeses made from unpasteurized milk, to reduce the number of viable pathogens that may be present to an acceptable risk. The objective of this study was to investigate the adequacy of the 60-day minimum aging to reduce the numbers of viable pathogens and evaluate milk subpasteurization heat treatment as a process to improve the safety of Cheddar cheeses made from unpasteurized milk. Cheddar cheese was made from unpasteurized milk inoculated with 101 to 105 CFU/ml of a five-strain cocktail of acid-tolerant Escherichia coli O157:H7. Samples were collected during the cheese manufacturing process. After pressing, the cheese blocks were packaged into plastic bags, vacuum sealed, and aged at 7°C. After 1 week, the cheese blocks were cut into smaller-size uniform pieces and then vacuum sealed in clear plastic pouches. Samples were plated and enumerated for E. coli O157:H7. Populations of E. coli O157:H7 increased during the cheese-making operations. Population of E. coli O157:H7 in cheese aged for 60 and 120 days at 7°C decreased less than 1 and 2 log, respectively. These studies confirm previous reports that show 60-day aging is inadequate to eliminate E. coli O157:H7 during cheese ripening. Subpasteurization heat-treatment runs were conducted at 148°F (64.4°C) for 17.5 s on milk inoculated with E. coli O157:H7 at 105 CFU/ml. These heat-treatment runs resulted in a 5-log E. coli O157: H7 reduction.

Survival and Recovery of Escherichia coli O157: H7 in Inoculated Bottled Water

1998 ◽  
Vol 61 (8) ◽  
pp. 948-952 ◽  
Author(s):  
D. W. WARBURTON ◽  
J. W. AUSTIN ◽  
B. H. HARRISON ◽  
G. SANDERS
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Macconkey Agar ◽  
Potassium Tellurite ◽  
Direct Plating ◽  
Selective Media ◽  
E Coli ◽  
Selective Agents ◽  
Selective Plating ◽  
Scanning Electron

A methodology used to isolate Escherichia coli O157:H7 from water and survival of this pathogen in inoculated water is described. The methodology used in the isolation of E. coli O157:H7 included the use of selective plating on Sorbitol MacConkey agar (supplemented with potassium tellurite [2.5 mg/liter], cefixime [0.05 mg/liter], and cefsulodin [10 mg/liter], and modified hemorrhagic colitis agar (also supplemented with potassium tellurite [2.5 mg/liter] and cefsulodin [10 mg/liter]). There were no significant differences (P < 0.05) between the recoveries of E. coli O157:H7 on these two selective media. Direct plating on these selective agars was used to determine the length of time that E. coli O157:H7 was able to grow, remain viable, and be resistant to the selective agents. E. coli O157:H7 survived in inoculated water for up to >300 days, depending on the type of water. Observation by scanning electron microscopy indicated that E. coli O157:H7 cells attached to, and multiplied on, the container walls.

Behavior of Escherichia coli O157:H7 during the Manufacture and Aging of Gouda and Stirred-Curd Cheddar Cheeses Manufactured from Raw Milk

2010 ◽  
Vol 73 (12) ◽  
pp. 2217-2224 ◽  
Author(s):  
DENNIS J. D'AMICO ◽  
MARC J. DRUART ◽  
CATHERINE W. DONNELLY
Keyword(s):  
Escherichia Coli ◽  
Population Level ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
Milk Whey ◽  
Selective Enrichment ◽  
Point Counts ◽  
E Coli ◽  
Unpasteurized Milk ◽  
Low Levels

This study was conducted to examine the fate of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses made from raw milk. Cheeses were manufactured from unpasteurized milk experimentally contaminated with one of three strains of E. coli O157:H7 at an approximate population level of 20 CFU/ml. Samples of milk, whey, curd, and cheese were collected for enumeration of bacteria throughout the manufacturing and aging process. Overall, bacterial counts in both cheese types increased almost 10-fold from initial inoculation levels in milk to approximately 145 CFU/g found in cheeses on day 1. From this point, counts dropped significantly over 60 days to mean levels of 25 and 5 CFU/g in Cheddar and Gouda, respectively. Levels of E. coli O157:H7 fell and stayed below 5 CFU/g after an average of 94 and 108 days in Gouda and Cheddar, respectively, yet remained detectable after selective enrichment for more than 270 days in both cheese types. Changes in pathogen levels observed throughout manufacture and aging did not significantly differ by cheese type. In agreement with results of previous studies, our results suggest that the 60-day aging requirement alone is insufficient to completely eliminate levels of viable E. coli O157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.

Virulence Characterization and Molecular Subtyping of Typical and Atypical Escherichia coli O157:H7 and O157:H(−) Isolated from Fecal Samples and Beef Carcasses in Mexico

2015 ◽  
Vol 78 (2) ◽  
pp. 264-272 ◽  
Author(s):  
CLAUDIA NARVÁEZ-BRAVO ◽  
ALEJANDRO ECHEVERRY ◽  
MARKUS F. MILLER ◽  
ARGENIS RODAS-GONZÁLEZ ◽  
M. TODD BRASHEARS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Immunomagnetic Separation ◽  
Latex Agglutination ◽  
Escherichia Coli O157 ◽  
High Genetic Diversity ◽  
E Coli ◽  
System A ◽  
Metabolic Genes ◽  
Beef Carcasses

The objective of the study was to characterize virulence genes and subtype Escherichia coli O157:H7 and O157:H(−) isolates obtained from a vertically integrated feedlot slaughter plant in Mexico. A total of 1,695 samples were collected from feedlots, holding pens, colon contents, hides, and carcasses. E. coli O157:H7 detection and confirmation was carried out using conventional microbiology techniques, immunomagnetic separation, latex agglutination, and the BAX system. A total of 97 E. coli O157 strains were recovered and screened for key virulence and metabolic genes using multiplex and conventional PCR. Eighty-eight (91.72%) of the strains carried stx2, eae, and ehxA genes. Ten isolates (8.25%) were atypical sorbitol-fermenting strains, and nine were negative for the flicH7 gene and lacked eae, stx1, stx2, and ehxA. One sorbitol-positive strain carried stx2, eae, tir, toxB, and iha genes but was negative for stx1 and ehxA. Pulsed-field gel electrophoresis (PFGE) analysis yielded 49 different PFGE subtypes, showing a high genetic diversity; however, the majority of the typical isolates were closely related (80 to 90% cutoff). Atypical O157 isolates were not closely related within them or to typical E. coli O157:H7 isolates. Identical PFGE subtypes were found in samples obtained from colon contents, feedlots, holding pens, and carcasses. Isolation of a sorbitol-fermenting E. coli O157 positive for a number of virulence genes is a novel finding in Mexico. These data showed that genetically similar strains of E. coli O157:H7 can be found at various stages of beef production and highlights the importance of preventing cross-contamination at the pre- and postharvest stages of processing.

scholarly journals Escherichia coli O157[ratio ]H7 diarrhoea associated with well water and infected cattle on an Ontario farm

1998 ◽  
Vol 120 (1) ◽  
pp. 17-20 ◽  
Author(s):  
S. G. JACKSON ◽  
R. B. GOODBRAND ◽  
R. P. JOHNSON ◽  
V. G. ODORICO ◽  
D. ALVES ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phage Type ◽  
Dairy Farm ◽  
Female Child ◽  
Well Water ◽  
Escherichia Coli O157 ◽  
Cattle Farm ◽  
E Coli ◽  
Unpasteurized Milk ◽  
Hydrogeological Investigation

A 16-month old female child living on an Ontario dairy farm was taken to hospital suffering from bloody diarrhoea. Escherichia coli O157[ratio ]H7 was isolated from her stool. Initial tests of well water samples were negative for E. coli by standard methods but culture of selected coliform colonies on sorbitol-MacConkey agar led to isolation of E. coli O157[ratio ]H7. E. coli O157[ratio ]H7 was also isolated from 63% of cattle on the farm. The E. coli O157[ratio ]H7 isolates from the child, the water and the cattle were phage type 14, produced verotoxins 1 and 2, and were highly related on analysis by pulsed field gel electrophoresis. The child did not have known direct contact with the cattle and did not consume unpasteurized milk. Hydrogeological investigation revealed the design and location of the well would allow manure-contaminated surface water to flow into the well. This investigation demonstrates that cattle farm well water is a potential source of E. coli O157[ratio ]H7 which may not be identified by standard screening for E. coli in water.

scholarly journals Escherichia Coli O157:H7: Distribution, molecular characterization, antimicrobial resistance patterns and source of contamination of sheep and goat carcasses at an export Abattoir, Mojdo, Ethiopia

2019 ◽  
Author(s):  
Solomon Abreham ◽  
Akafete Teklu ◽  
Eric Cox ◽  
Tesfaye Sisay Tessema
Keyword(s):  
Escherichia Coli ◽  
Molecular Characterization ◽  
Virulence Genes ◽  
Antibiotic Sensitivity ◽  
Latex Agglutination ◽  
Human Infection ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Source Of Infection ◽  
Resistance Patterns

Abstract Background : Cattle have been identified as a major reservoir of E. coli O157:H7 for human infection; the ecology of the organism in sheep and goats is less understood. This study was carried out to determine prevalence, source of infection, antibiotic resistance and molecular characterization of Escherichia coli O157: H7 isolated from sheep and goat. Methods : Systematic random sampling was carried out at Modjo export abattoir, Ethiopia, from November 2012 to April 2013 to collect 408 samples from 72 sheep and 32 goats. Samples collected were skin swabs, fecal samples, intestinal mucosal swabs and the inside and outside part of carcasses as well as carcass in contacts such as workers hands, knife, hook and carcass washing water. Then, samples were processed following standard bacteriological procedures. Non-Sorbitol fermenting colonies were tested on latex agglutination test and the positives are subjected to PCR for detection of attaching and effacing genes ( eaeA) and shiga toxin producing genes ( stx1 and stx2 ). All E. coli O157:H7 isolates were checked for their susceptibility pattern towards 15 selected antibiotics. Results : E. coli O157:H7 were detected in only 20/408 samples (4.9%). Among these 20 positive samples, 70% (14/20), 25% (5/20) and 5% (1/20) were from sheep, goats and knife samples, respectively. No significant associations were found between carcasses and the assumed sources of contaminations. Of all the 20 isolates virulence genes were found in 10 (50%) of them; 3 (15%) with only the eaeA gene and 7(35%) expressing eaeA and stx2 genes. All the isolates were susceptible to Norfloxacin (NOR) (100%). Conclusions : The presence of virulence genes shows E. coli O157:H7 is a potential source of human infection in Ethiopia. Key words : Abattoir, antibiotic sensitivity, CT-SMAC, E. coli O157:H7, IMS, Latex agglutination, multiplex PCR.

scholarly journals Prevalence and antibiotic resistance profile of Shiga-toxigenic Escherichia coli O157 (STEC) from retailed miscellaneous meat and fish types in Abuja, Nigeria

2021 ◽  
Vol 2 (2) ◽  
pp. 37-43
Author(s):  
Adaeze Joy Alu ◽  
Gabriel K. Omeiza ◽  
James A. Ameh ◽  
Enem S.I
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Enrichment Culture ◽  
Intestinal Bacteria ◽  
Latex Agglutination ◽  
Multi Drug Resistance ◽  
Escherichia Coli O157 ◽  
Resistance Profile ◽  
E Coli ◽  
Antibiotic Resistance Profile

Most Escherichia coli strains are harmless intestinal bacteria of animals, but some are implicated in food infection/poisoning especially Shiga toxin (or Vero toxin) producing E. coli (STEC) due to consumption of meat. This study was conducted to determine the prevalence and antibiotic resistance profile of Shigatoxigenic Escherichia coli O157 (STEC) from retailed miscellaneous fish and meat types in Abuja, Federal Capital Territory, Nigeria. A total of 256 meat and fish consisting of cow muscles, intestines, rumen-sacs, livers and tails, cat-fish, frozen fish (mackerel and herrings) were examined. Escherichia coli were isolated by enrichment culture cefixime-tellurite sorbitol MacConkey agar (CT-SMAC), morphological, biochemical, serotype latex agglutination and disk diffusion methods. Of the 256 samples, 138 (53.9%) were contaminated with E. coli and 28 (21.7%) E. coli strains were positive for Shigatoxigenic Escherichia coli O157 (STEC). Meat muscles had the highest prevalence of STEC (7.41%) among meat samples, followed by rumen-sacs (6.0%), intestines (5.77%), tails (4.0%), and the prevalence of STEC in Fish includes Cat-fish intestine (26.7%), skin (21.4%), Mackerel intestine (26.7%), skin (14.3%), and Herrings skin (15.4%), gill (7.1%). All the STEC assessed indicated multi-drug resistance, with the isolates showing 100% resistant to ampicilin, and erythromycin, nitrofurantoin (95.7%), amoxicilin clavulanic acid (84.3%), sulphamethaxazole/trimethoprim (75%), streptomycin (75%), tetracycline (66.17%), and gentamycin (53.6%). The isolates were susceptible to ciprofloxacin (66.7%), Cefoxitin (66.7%), amikacin (39.3%), and chloramphenicol (35.7%). The implication of STEC in this study suggests that contaminated meat types are sold to consumers and can result to serious foodborne hazards. Prescription of ciprofloxacin and cefoxicilin are recommended against this organism. Application of good hygienic procedures in meat and fish handling processes and proper boiling before consumption can mitigate the risk of infection due to resistance STEC strains.

Antibacterial Activities of Semipurified Fractions of Quercus infectoria against Enterohemorrhagic Escherichia coli O157:H7 and Its Verocytotoxin Production

2008 ◽  
Vol 71 (6) ◽  
pp. 1223-1227 ◽  
Author(s):  
SUPAYANG PIYAWAN VORAVUTHIKUNCHAI ◽  
SAKOL SUWALAK
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Latex Agglutination ◽  
Enterohemorrhagic Escherichia Coli ◽  
Natural Food ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Broth Microdilution Method ◽  
Quercus Infectoria

Escherichia coli O157:H7 is one of the most important foodborne pathogens, causing nonbloody and bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Use of antibiotics has been demonstrated to result in increased levels of verocytotoxin (VT) production as well as antibiotic resistance. Quercus infectoria was investigated for its antibacterial activity against E. coli O157:H7 and other VT-producing enterohemorrhagic E. coli (VTEC). The MIC was determined by a broth microdilution method, and the MBC was assessed by subculturing the bacteria from the wells that showed no apparent growth onto Mueller-Hinton agar. The fractions Qi2, Qi3, and Qi4 of Q. infectoria were demonstrated to possess good antibacterial activity, with MICs and MBCs ranging from 250 to 500 μg/ml. The effect of the effective fraction, Qi4, on the production of VT was determined using a reversed passive latex agglutination. The results indicate that at 20 h, fraction Qi4 markedly inhibits the release of VT1 and VT2 from VTEC cells at both inhibitory and subinhibitory concentrations. Furthermore, verotoxicity assay demonstrated that bacterial cultures treated with fraction Qi4 exerted less toxic effect on Vero cells. These in vitro results clearly indicate that the fraction Qi4 might constitute a promising natural food additive for the control of food poisoning by E. coli O157:H7 as well as other VTEC strains.

Frequency of Escherichia coli O157:H7 in Turkish Cattle

2002 ◽  
Vol 65 (10) ◽  
pp. 1637-1640 ◽  
Author(s):  
AYSUN YILMAZ ◽  
HUSEYIN GUN ◽  
HUSEYIN YILMAZ
Keyword(s):  
Escherichia Coli ◽  
Urease Activity ◽  
Sampling Method ◽  
Immunomagnetic Separation ◽  
Systematic Sampling ◽  
Escherichia Coli O157 ◽  
Macconkey Agar ◽  
E Coli ◽  
Systematic Sampling Method ◽  
Age And Sex

In this study, five abattoirs in Istanbul were visited between January 2000 and April 2001. During these visits, 330 cattle were selected by a systematic sampling method. Cattle were examined clinically and breed, age, and sex were recorded. Rectal swabs were taken immediately after slaughter. Immunomagnetic separation was performed, and sorbitol-negative colonies were selected on sorbitol MacConkey agar with cefixime and tellurite (CT-SMAC agar). These colonies were checked for 4-methylenebelliferyl-β-d-glucuronide, indol, rhamnose, and urease activity and motility. Serotypes of bacteria were determined by using antisera specific for Escherichia coli O157 and H7. All cattle selected were clinically healthy. Of 88 sorbitol-negative colonies selected on CT-SMAC agar, isolates from only 14 (4.2%) cattle reacted with anti-O157, and 13 of these isolates also reacted with anti-H7. E. coli O157:H7 was isolated from all breeds, but the numbers of isolates were largest for Holstein and Swiss Brown cows. E. coli O157:H7 was most frequently isolated from 2-year-old cattle. Similarly, it was most frequently isolated from male cattle. E. coli O157:H7 was isolated from cattle slaughtered in four of the five abattoirs studied.

Oral and Rectal Administration of Bacteriophages for Control of Escherichia coli O157:H7 in Feedlot Cattle

2009 ◽  
Vol 72 (2) ◽  
pp. 241-250 ◽  
Author(s):  
ERICA A. ROZEMA ◽  
TYLER P. STEPHENS ◽  
SUSAN J. BACH ◽  
ERASMUS K. OKINE ◽  
ROGER P. JOHNSON ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nalidixic Acid ◽  
Plaque Assay ◽  
Rectal Administration ◽  
Host Bacterium ◽  
Escherichia Coli O157 ◽  
Bacteriophage Therapy ◽  
Potassium Tellurite ◽  
Fecal Shedding ◽  
E Coli

This study compared oral and rectal administration of O157-specific bacteriophages for mitigating the fecal shedding of Escherichia coli O157 by experimentally inoculated steers. Fecal shedding of nalidixic acid–resistant (NalR) E. coli O157:H7 was monitored over 83 days after oral (ORL; 3.3 × 1011 PFU), rectal (REC; 1.5 × 1011 PFU), both oral and rectal (O+R; 4.8 × 1011 PFU), or no (CON; control) treatment with a four-strain O157-specific bacteriophage cocktail in multiple doses. Bacteriophages were enumerated by plaque assay, and NalR E. coli O157:H7 by direct plating on sorbitol MacConkey agar supplemented with cefixime, potassium tellurite, and nalidixic acid. Orally treated steers produced the fewest NalR E. coli O157:H7 culture-positive samples (P < 0.06) compared with REC and O+R steers, but this number was only nominally lower (P = 0.26) than that for the CON steers. The overall mean shedding level (log CFU per gram of feces) was higher for REC steers (P < 0.10) than for steers of the other treatment groups. Despite the shedding of higher mean bacteriophage levels (log PFU per gram of feces) by ORL and O+R than by CON and REC steers, there was no difference (P > 0.05) in the number of E. coli O157–positive samples among treatments. Bacteriophage was isolated from CON steers, indicating that these steers acquired the bacteriophage from the environment and shed the phage at a level similar to that of REC steers (P = 0.39). Continuous bacteriophage therapy may be an efficacious method for mitigating shedding of E. coli O157:H7 in cattle, providing that the host bacterium does not develop resistance. This therapy may be especially advantageous if nontreated cattle can acquire this biocontrol agent from the feedlot environment.

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