Comparison of Enrichment Conditions for Rapid Detection of Low Numbers of Sublethally Injured Escherichia coli O157in Food

2009 ◽  
Vol 72 (9) ◽  
pp. 1862-1868 ◽  
Author(s):  
VICKY JASSON ◽  
ANDREJA RAJKOVIC ◽  
LEEN BAERT ◽  
JOHAN DEBEVERE ◽  
MIEKE UYTTENDAELE
Keyword(s):  
Escherichia Coli ◽  
Bile Salts ◽  
False Negative ◽  
Raw Milk ◽  
Detection Methods ◽  
Fermented Sausage ◽  
E Coli ◽  
Negative Results ◽  
Peptone Water ◽  
Sprouted Seeds

A comparative study of lag phases and growth rates of healthy, stressed, and sublethally injured Escherichia coli O157 cells in 10 enrichment broths was performed. The evaluation of enrichment protocols was validated by different end point detection methods (two PCR and two combined capture-plate methods). Tryptic soy broth b [TSB (b)] provided the fastest growth (μmax = 1.00 ± 0.06 h−1) but failed to recover oxidative-stressed E. coli O157. TSB (a), TSB–yeast extract medium, TSB supplemented with 8 mg/liter novobiocin plus 16 mg/liter vancomycin (TSB+), buffered peptone water (BPW), and BPW supplemented with 8 mg/liter vancomycin (BPW+V) enabled resuscitation of E. coli O157 cells independent from precultural conditions. Modified TSB plus 10 mg/liter novobiocin (mTSB+N), EC medium, EC reduced bile salts medium (ECred), TSB (b), and TSB supplemented with 8 mg/liter novobiocin plus 16 mg/liter vancomycin plus 2 mg/liter rifampin plus 1 mg/liter K-Telluriet plus 1.5 g/liter bile salts no. 3 (TSB++) all failed to recover E. coli O157 cells for at least one type of stress. The use of TSB (a), TSB+, BPW, and BPW+V was compared with that of mTSB+N (International Organization for Standardization reference broth) for reliable detection of low numbers of healthy, stressed, and sublethally injured E. coli O157 (approximately 10 CFU/10 g) from foods (sprouted seeds, fermented sausage, raw milk, and raw ground beef). When low numbers of healthy cells were inoculated, BPW, BPW+V, TSB, TSB+, and mTSB+N enabled growth until detectable numbers within 6 h of enrichment at 41.5°C. Results showed that mTSB+N failed to recover to detectable numbers E. coli O157 cells sublethally injured by freeze and food stresses, in contrast to what was obtained with BPW and BPW+V. This study highlights that using mTSB+N for recovery of E. coli O157 from foods may yield false-negative results.

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.

scholarly journals Comparison of Colilert-18 with miniaturised most probable number method for monitoring of Escherichia coli in bathing water

2015 ◽  
Vol 14 (1) ◽  
pp. 121-131 ◽  
Author(s):  
Ananda Tiwari ◽  
Seppo I. Niemelä ◽  
Asko Vepsäläinen ◽  
Jarkko Rapala ◽  
Seija Kalso ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
False Negative ◽  
Reference Method ◽  
Water Quality Monitoring ◽  
Coliform Bacteria ◽  
Most Probable Number ◽  
Probable Number ◽  
E Coli ◽  
Negative Results ◽  
Bathing Water

The purpose of this equivalence study was to compare an alternative method, Colilert-18 Quanti-Tray (ISO 9308-2) with the European bathing water directive (2006/7/EC) reference method, the miniaturised most probable number (MMPN) method (ISO 9308-3), for the analysis of Escherichia coli. Six laboratories analysed a total of 263 bathing water samples in Finland. The comparison was carried out according to ISO 17994:2004. The recovery of E. coli using the Colilert-18 method was 7.0% and 8.6% lower than that of the MMPN method after 48 hours and 72 hours of incubation, respectively. The confirmation rate of presumptive E. coli-positive wells in the Colilert-18 and MMPN methods was high (97.8% and 98.0%, respectively). However, the testing of presumptive E. coli-negative but coliform bacteria-positive (yellow but not fluorescent) Colilert-18 wells revealed 7.3% false negative results. There were more false negatives in the naturally contaminated waters than in the samples spiked with waste water. The difference between the recovery of Colilert-18 and the MMPN method was considered not significant, and subsequently the methods are considered as equivalent for bathing water quality monitoring in Finland. Future bathing water method equivalence verification studies may use the data reported herein. The laboratories should make sure that any wells showing even minor fluorescence will be determined as positive for E. coli.

Evaluation of Culture- and PCR-Based Detection Methods for Escherichia coli O157:H7 in Inoculated Ground Beef†

2005 ◽  
Vol 68 (8) ◽  
pp. 1566-1574 ◽  
Author(s):  
TERRANCE M. ARTHUR ◽  
JOSEPH M. BOSILEVAC ◽  
XIANGWU NOU ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
False Positive ◽  
False Negative ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Perishable Product ◽  
Specificity And Sensitivity

Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P > 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results.

scholarly journals Single tube confirmatory tests forEscherichia coli

1980 ◽  
Vol 85 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Keyword(s):  
Escherichia Coli ◽  
False Positive ◽  
False Negative ◽  
Gas Production ◽  
Single Tube ◽  
E Coli ◽  
Peptone Water ◽  
Confirmatory Tests ◽  
Laboratory Trial ◽  
The Difference

SummaryIn a multi-laboratory trial, lauryl tryptose mannitol broth (LTMB) and minerals-modified glutamate medium with added tryptophan (MMGM + T) were compared as single tube tests for the confirmation ofEscherichia coliin water; the confirmed results were also compared with the production of gas from minerals-modified glutamate medium without added tryptophan (MMGM). LTMB and MMGM + T gave similar gas and indole results with about 90% of the water samples in most of the laboratories. When compared with the ‘correct’ results as judged by acid and gas production from lactose peptone water and indole from tryptone water, the difference in the rate of false positive reactions between LTMB and MMGM + T was insignificant; but LTMB gave a significantly lower rate of false negative reactions than MMGM + T. Gas production from MMGM without added tryptophan gave significantly higher rates of both false positive and false negative reactions. Lauryl sulphate is therefore a suitable inhibitory surfactant for use in single tubemedia for the confirmation ofE. coli, which can be recommended.

Evaluation of a rapid fluorogenic method for the detection ofEscherichia coliin dairy products

1991 ◽  
Vol 58 (4) ◽  
pp. 477-483 ◽  
Author(s):  
Hassan R. Sarhan ◽  
Leslie R. Williams ◽  
Howard A. Foster
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Dairy Products ◽  
Membrane Filtration ◽  
False Negative ◽  
Raw Milk ◽  
False Positive Reaction ◽  
Negative Results ◽  
False Negative Results ◽  
Soft Cheese

SummaryA rapid fluorogenic medium was evaluated for the detection ofEscherichia coliin dairy products. The medium was capable of detectingEsch. coliafter 7·5 h incubation at 41·5 °C. Samples of pasteurized milk (136), raw milk (63), soft cheese (60) and pasteurized cream (39) were examined with media based on 4-methylumbelliferyl-β-D-glucuronide (MUG-7) and Violet red bile agar and there were no significant differences between the numbers ofEsch. colidetected on the two media. MUG-7 medium had a specificity of 98·6% and the small number of organisms giving a false positive reaction were identified asKlebsiella pneumoniae. The incidence of false negative results was ∼2%. MUG-7 medium was suitable for pour plate, spread plate and membrane filtration methods. Possible applications of the method are discussed.

scholarly journals Evaluation of Escherichia coli O157:H7 Growth Media for Use in Test-and-Hold Procedures for Ground Beef Processing†

2006 ◽  
Vol 69 (5) ◽  
pp. 1007-1011 ◽  
Author(s):  
MICHAEL N. GUERINI ◽  
TERRANCE M. ARTHUR ◽  
STEVEN D. SHACKELFORD ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
Ground Beef ◽  
Threshold Level ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Growth Media ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Peptone Water

Since the mid-1990s, the beef industry has used a process called test and hold, wherein beef trim and ground beef are tested to keep products contaminated with Escherichia coli O157:H7 out of commerce. Current O157:H7 detection methods rely on a threshold level of bacterial growth for detection, which is dependent on the growth medium used. Twelve media were examined for growth and doubling time: buffered peptone water (BPW), SOC (which contains tryptone, yeast extract, KCl, MgCl2, and glucose), buffered peptone water plus SOC (BPW-SOC), Bacto-NZYM, RapidChek E. coli O157:H7 medium, BioControl EHEC8 culture medium, Neogen Reveal for E. coli O157:H7—Eight Hour medium (Neogen Reveal 8), BAX System medium for E. coli O157:H7 (BAX), BAX System medium for E. coli O157:H7 MP (BAX-MP), modified E. coli broth, nutrient medium, and tryptic soy broth (TSB). All media were tested at 37 or 42°C under static or shaking conditions. The eight media with the highest total CFU per milliliter and most rapid doubling times were BPW-SOC, NZYM, RapidChek, EHEC8, Neogen Reveal 8, BAX, BAX-MP, and TSB. The ability of these eight media to enrich E. coli O157:H7 in ground beef was further evaluated through time-course experiments using immunomagnetic separation. Of these media, TSB was the easiest to prepare, had a wide application base, and was the least expensive. In the test-and-hold process, the normal ratio of medium to product is 1:10. In this study, a 1:3 ratio worked as well as a 1:10 ratio. Processors using test-and-hold procedures could use 1 liter of TSB to enrich for E. coli O157:H7 in a 375-g sample instead of the usual 3.375 liters, thus saving reagents, time, and labor while maintaining accuracy.

scholarly journals Prevalence and molecular characterisation of shiga-toxin-producing Escherichia coli in raw milk cheeses from Lazio region, Italy

2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Selene Marozzi ◽  
Paola De Santis ◽  
Sarah Lovari ◽  
Roberto Condoleo ◽  
Stefano Bilei ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Raw Milk ◽  
Foodborne Diseases ◽  
Control Plan ◽  
Lazio Region ◽  
Standard Methods ◽  
E Coli ◽  
Regional Control ◽  
Unpasteurized Milk

In recent years, the incidence of foodborne diseases caused by shiga toxin-producing <em>Escherichia coli</em> (STEC) has increased globally. For this reason, within the specific regional control plan for the detection of STEC in food products in Italy, the presence of STEC in unpasteurized milk cheeses was investigated. In total 203 samples obtained from March 2011 to December 2013 were analysed, with two standard methods (ISO 16654:2001 and ISO 13136:2012). Two strains of <em>E. coli</em> O157 were isolated (2/161, 1.2%) but did not carry any virulence-associated genes and 22 <em>stx</em>-positive samples (22/146, 15.1%) were detected in enrichment cultures, mostly from ovine cheeses. Only two strains isolated from different ovine cheeses carried <em>stx</em> gene and none of these was <em>eae</em>-positive. This study confirms the presence of <em>stx</em>-positive <em>E. coli</em> and suggests that this type of food cannot be excluded as a potential vehicle of STEC.

scholarly journals Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil

2007 ◽  
Vol 59 (2) ◽  
pp. 508-512 ◽  
Author(s):  
B.R. Paneto ◽  
R.P. Schocken-Iturrino ◽  
C. Macedo ◽  
E. Santo ◽  
J.M. Marin
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Antimicrobial Agents ◽  
Raw Milk ◽  
Diffusion Method ◽  
Chain Reaction ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Polymerase Chain ◽  
Raw Milk Cheese

The occurrence of toxigenic Escherichia coli in raw milk cheese was surveyed in Middle Western Brazil. Fifty samples of cheese from different supermarkets were analyzed for E.coli. The isolates were serotyped and screened for the presence of verotoxigenic E. coli (VTEC) and enterotoxigenic E. coli (ETEC) by Polymerase Chain Reaction (PCR). The susceptibility to thirteen antimicrobial agents was evaluated by the disk diffusion method. E.coli were recovered from 48 (96.0%) of the samples. The serogroups identified were O125 (6.0%), O111 (4.0%), O55 (2.0%) and O119 (2.0%). Three (6.0%) and 1(2.0%) of the E.coli isolates were VTEC and ETEC, respectively. Most frequent resistance was observed to the following antimicrobials: cephalothin (60.0%), nalidixic acid (40.0%), doxycyclin (33.0%), tetracycline (31.0%) and ampicillin (29.0%).

Contamination of Intact Apples after Immersion in an Aqueous Environment Containing Escherichia coli O157:H7†

1999 ◽  
Vol 62 (5) ◽  
pp. 444-450 ◽  
Author(s):  
R. L. BUCHANAN ◽  
S. G. EDELSON ◽  
R. L. MILLER ◽  
G. M. SAPERS
Keyword(s):  
Escherichia Coli ◽  
Outer Core ◽  
Core Region ◽  
Effect Of Temperature ◽  
Aqueous Environment ◽  
Escherichia Coli O157 ◽  
Dye Uptake ◽  
E Coli ◽  
Golden Delicious ◽  
Peptone Water

The extent and location of Escherichia coli O157:H7 contamination after intact apples were immersed in cold (2°C) 1% peptone water containing approximately 3 × 107 CFU/ml was assessed using four apple varieties, Golden Delicious, McIntosh, Red Delicious, and Braeburn. Room temperature and refrigerated apples were used to determine the effect of temperature differential on E. coli infiltration. The highest levels of E. coli were associated with the outer core region of the apple, followed by the skin. Apples were subsequently treated by immersing them for 1 min in 2,000 mg/liter sodium hypochlorite, followed by a 1-min tapwater rinse. This treatment reduced pathogen levels by 1- to 3-log cycles but did not eliminate the microorganism, particularly from the outer core region. While E. coli was not detected in the inner core of most apples, warm fruit immersed in cold peptone water occasionally internalized the pathogen. The frequency and extent of internalization of the pathogen was less when cold apples were immersed in cold peptone water. Subsequent dye uptake studies with Golden Delicious apples indicated that approximately 6% of warm apples immersed into a cold dye solution accumulated dye via open channels leading from the blossom end into the core region. However, dye uptake did not occur when the dye solution was warmer than the apple.

scholarly journals Detection of virulence genes and antimicrobial resistance profiles of Escherichia coli isolates from raw milk and artisanal cheese in Southern Brazil

2019 ◽  
Vol 40 (1) ◽  
pp. 163 ◽  
Author(s):  
Leandro Parussolo ◽  
Ricardo Antônio Pilegi Sfaciotte ◽  
Karine Andrezza Dalmina ◽  
Fernanda Danielle Melo ◽  
Ubirajara Maciel Costa ◽  
...  
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Antimicrobial Susceptibility ◽  
Virulence Genes ◽  
Antimicrobial Agents ◽  
Raw Milk ◽  
Diffusion Method ◽  
Public Health Issue ◽  
Southern Brazil ◽  
E Coli

The serrano artisanal cheese is a typical product from South region of Brazil, which is produced by skilled cheesemakers using raw milk. The contamination of this food by Escherichia coli has a great impact on public health, since it could threat the consumers’ health. The study evaluated the presence of virulence genes, antimicrobial susceptibility profiles and bofilm-production ability of Escherichia coli isolates obtained from raw milk and artisanal cheese produced in Southern Brazil. A total of 117 isolates of E. coli were characterized by multiplex PCR to detect the following virulence genes: eae for enteropatogenic E. coli (EPEC), lt and st for enterotoxigenic E. coli (ETEC), stx for shiga toxin-producing E. coli (STEC), stx and eae for enterohemorrhagic E. coli (EHEC), ipaH for enteroinvasive E. coli (EIEC) and aggR for enteroaggregative E. coli (EAEC). In addition, antimicrobial susceptibility profile to 22 antimicrobial agents was also performed by disk diffusion method, and we searched for extended-spectrum beta-lactamases (ESBL) and/or carbapenemase- producing isolates. Isolates that were positive for ESBL and carbapenemase were further investigated for the presence of the genes: blaTEM, blaSHV, blaOXA, blaCTX-M, for ESBL and blaOXA-48 for carbapenemase. Further, isolates had their ability to form biofilms investigated by the red Congo agar method. Virulence genes of E. coli were identified in 21.37% of the tested isolates, which were classified as EPEC (the most prevalent pathotype) and ETEC or EAEC. Ten (8.55%) of the total studied E. coli isolates revealed a multidrug-resistant profile, since they were resistant to three or more antimicrobial classes; whereas four isolates (3.42%) were classified as ESBL-producers and showed the presence of blaTEM gene. None of the isolates exhibited carbapenemase activity nor did they carry carbapenemase genes. From the total of E. coli isolates, 79 (67.52%) were considered potential biofilm producers. These results address a serious public health issue, since artisanal cheeses pose a risk to consumers’ health, since may be sources of dissemination of diarrheogenic E. coli, that can cause from subclinical to severe and fatal infections in children and adults, and also emphasize the need to improve adaptations/adjustments in the manufacturing processes of these products.

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