COMPARISON OF RAPID TECHNIQUES FOR THE DETECTION OF ESCHERICHIA COLI O157:H7

Four different commercial kits (EHEC-TEK of Organon Teknika, Durham, NC; HEC O157 of 3M, St. Paul, MN; and Coline dipstick and Coline one-step of AMP-COR Inc., Camden, NJ) were evaluated for the detection and recovery of E. coli O157:H7from 75 fresh meat samples and 23 artificially inoculated beef and pork samples. Of the total 75 samples tested, 21 (28%) were presumptive positive by HEC O157 and Coline dipstick, 18 (24%) by Coline one-step, and 12 (16%) by EHEC-TEK. None of the presumptive positive samples by any of the methods was confirmed as E. coli O157:H7 (false positive). Of the 23 positive spiked samples tested, 23 were recovered by Coline dipstick and one-step (100%), 22 (95.6%) by HEC O157, and 20 (86.9) were recovered by EHEC-TEK. In the confirmation step 17 of the 23 spiked samples produced characteristic colonies on MacConkey sorbitol agar (Difco) with 5-bromo-4-chloro-3-indoxy-β-D-glucuronide (Biosynth International) (MSA-BCIG) and were confirmed as E. coli O157:H7. No characteristic colonies on MSA-BCIG were detected for 6 of the spiked samples with initial inoculum levels of between 2 to 70 cells/g and, therefore, were not confirmed as E. coli O157:H7. A better enrichment medium, as well as improved selective plating and confirmation techniques, are needed to enhance the selective growth of E. coli O157:H7 and provide lower detection levels.     

DIRECT DETECTION OF ESCHERICHIA COLI 0157:H7 IN UNPASTEURIZED APPLE JUICE WITH AN EVANESCENT WAVE BIOSENSOR

An evanescent wave biosensor was used to detect Escherichia coli O157:H7 in unpasteurized apple juice. Light is launched from a 635 nm laser diode into silica or polystyrene optical waveguides, generating an evanescent field which extends from the waveguide surface. Fluorescent molecules within the evanescent field are excited resulting in an emission signal that the biosensor then detects and quantifies. A sandwich immunoassay was performed on the waveguides using cyanine 5 dye-labeled anti-E. coli O157:H7 antibodies for generation of the specific fluorescent signal. The lower limit of detection was between 6.0 × 10² and 6.0 × 10⁴ CFU/mL with silica waveguides and between 3.2 × 10⁴ and 3.2 × 10⁴ CFU/mL using polystyrene waveguides. One-hundred percent correct identification of true positive samples occurred at 6.0 × 10⁴ and 3.2 × 10⁴ CFU/mL for silica and polystyrene waveguides, respectively. Signals from a variety of non-E. coli O157 bacteria, including closely related enterotoxigenic strains of E. coli at concentrations of ˜ 10⁶ CFU/mL, were below the limits of detection. Assays were conducted in near real-time with results obtained within 15 min of sample processing.     

IMPROVEMENT OF SELECTIVITY FOR ISOLATION OF ESCHERICHIA COLI O157:H7 FROM GENERIC ESCHERICHIA COLI

A modified selective medium was developed to increase selectivity for isolation of Escherichia coli O157 from generic E. coli based on the knowledge that E. coli O157:H7 has more resistance against HCl condition than E. coli. As a preliminary experiment, four strains of E. coli O157:H7 (ATCC 35150, 43889, 43890, and 43894) were tested to determine the maximum concentration of 6N HCl (from 0 to 250 μL) added to 50 mL of MacConkey agar medium (MAC). The maximum level was 125 μL/50 mL (6N HCl/MAC), which E. coli O157:H7 strains could tolerate against the HCl concentration. After determination, comparative growth of 15 isolates of E. coli O157 and generic E. coli were evaluated on modified selective medium (HCl-MAC; with the addition of 125 μL/50 mL) and conventional MAC, respectively. All tested strains of E. coli O157 were grown on both media, whereas 9 out of 15 generic E. coli (60% of tested strains) were strongly inhibited on HCl-MAC. For selective isolation of E. coli O157 from generic E. coli, HCl-MAC has an effective potential for an implemental use. This information can extend as a baseline for use of HCl to conventional medium for successful isolation E. coli O157 from generic E. coli.     

ASSESSMENT OF IMPEDANCE MICROBIOLOGICAL METHOD FOR THE DETECTION OF ESCHERICHIA COLI IN FOODS

This study was conducted to determine the sensitivity and specificity of the impedance-based microbiological method for the detection of Escherichia coli in foods within 24 h of testing. A Malthus Microbiological Analyzer system (Malthus System V, Malthus Instruments Ltd., Bury, United Kingdom), and a modified Malthus Coliform Broth Medium (MCBM), and an incubation temperature of 44C were used. The sensitivity of the impedance method was determined by testing E. coli-negative food samples spiked with different concentrations of E. coli. The specificity of the method was determined by testing E. coli -negative food samples spiked with Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The test results were compared with those obtained by the Most Probable Number (MPN) method. Milk, milk products, raw and ready-to-eat meats, and vegetables were tested for the presence of E. coli by both methods. The sensitivity of the impedance method and the MPN method for the detection of foods containing 10¹ CFU/g was 100% and 84.4%, respectively. Both methods had a specificity of 100% for food samples spiked with 10¹ CFU/g E. coli. The specificity of the impedance and the MPN methods for the detection of E. coli in naturally contaminated milk and meat samples was 100% and 95.7% respectively. E. coli was detected in foods by the impedance method within 4–24 h of testing at a detection limit of 1 CFU/mL. These results demonstrate that the impedance method can be used as a rapid and sensitive method for the detection of E. coli in foods.     

A SIMPLE AND RAPID DETECTION BY PCR OF ENTEROPATHOGENIC ESCHERICHIA COLI IN NATURALLY CONTAMINATED VEGETABLES

Contaminated vegetables have been identified as one of the principal sources of foodborne illnesses. Escherichia coli is one of the bacteria that can contaminate vegetables and cause serious foodborne disease. The development of simple and rapid assays for detection of E. coli would enable official agencies and food industries to identify contaminated foodstuffs in a timelier manner. In this work, we detected E. coli contamination in four types of vegetables using a 24 h procedure. This method is very specific, rapid and simple to use in the laboratory. Indeed, the enrichment, DNA isolation and polymerase chain reaction (PCR) amplification procedures described here can also be used for detection of E. coli in other foods.

PRACTICAL APPLICATIONS

Foodborne disease remains an important public health threat worldwide; one of the most important food safety hazards is associated with raw vegetables. Several studies have found that products can support the growth of enteric bacterial pathogens such as Salmonella , Shigella and Escherichia coli . Culture techniques are universally recognized as the standard method for detecting pathogenic bacteria in foods. Bacteria are detected and subsequently identified by growth on solid selective culture media and by analysis of metabolic properties or serotyping. This process is lengthy and may last 5–10 days or more. In our investigation, the polymerase chain reaction (PCR) detection of E. coli in vegetables was realized within 24 h. The pre-enrichment step used in this study did not have any inhibiting effect on the PCR. Therefore, it became possible to rapidly and directly detect E. coli in raw vegetables by the PCR technique just by heating samples.     

一种快速提取禽源性大肠埃希氏菌外胰蛋白的方法

介绍一种快速提取禽源性大肠埃希氏菌外膜蛋白的方法。该法是对Ｋａｐｕｒ等发表的方法的改进。全过程只需超速离心一次,比Ｋａｐｕｒ等的方法缩短了４ｈ。所得样品可直接用于禽源性大肠埃希氏菌外膜蛋白模式的测定。     

SUITABILITY OF SELECTIVE MEDIA FOR RECOVERY OF HEAT-STRESSED ESCHERICHIA COLI O157:H7

Tryptone soya agar (TSA) and three selective media, BCM^1M O157:H7(+) agar (BCM), modified eosin methylene blue agar (MEMB), and sorbitol MacConkey agar (SMAC) were evaluated for recovery of two strains of E. coli O157:H7 (salami and cider isolates) heated at 56, 58, and 60C for up to 60 min in tryptone soya broth (TSB). TSA and MEMB were equally effective at recovery of heat-stressed (56, 58, and 60C) E . coli O 157:H7 and superior to SMAC and BCM (P 0.05). When heated at 56 and 58C, recovery of E. coli O157:H7 on MEMB and TSA was not significantly different (P > 0.05); recovery was poorer on SMAC, followed by BCM (P 0.05). There was no significant difference in recovery of E. coli O157:H7 on BCM and SMAC when strains were heated at 60C (P > 0.05).     

INFLUENCE OF GLUCOSE OXIDASE ON THE GROWTH OF ESCHERICHIA COLI 0157:H7, LISTERIA MONOCYTOGENES, AND SALMONELLA TYPHIMURIUM IN UNIVERSAL PREENRICHMENT BROTH

Glucose oxidase (GO), a food-grade enzyme, was compared with Oxyrase^TM oxygen reducing membrane fraction in Universal Preenrichment Broth (UPB) for enhancement of the growth of the facultatively anaerobic pathogens Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes Scott A. Oxidation-reduction potential (ORP) and pH changes in UPB following the addition of GO (4 units/ml) or Oxyrase^TM (0.5 units/ml) were measured. Microbial growth was evaluated at 0, 3, 6, 18, and 24 h of incubation using spiral plating. Nonenzyme supplemented UPB served as the control. Oxyrase^TM provided a higher oxygen scavenging action in terms of ORP decrease during the initial 6 h of incubation. However, no difference occurred in Eh between Oxyrase^TM and GO by 18 h, with both enzyme systems effectively reducing the Eh compared to that of the control. A 1.0 pH unit reduction was observed in GO-supplemented UPB after 18 h, indicating production of gluconic acid. The pH decrease in Oxyrase^TM - supplemented media was 0.2 units. By 6 h, the E. coli O157:H7 population was enhanced by 0.6 and 1.4 log CFU/ml in Oxyrase^TM -supplemented media, compared to the control and GO-supplemented media, respectively. By 18 h, 0.4 and 0.9 log CFU/ml growth enhancements of the E. coli O157:H7 populations were seen in GO- and Oxyrase^TM -supplemented media, respectively, compared to the control. By the end of 18 h, counts of S. typhimurium and L. monocytogenes increased by 0.6 and 0.2 log units, respectively, in GO-supplemented media compared to the control.     

RAPID TEST FOR DIAGNOSIS OF ENTEROHEMORRHAGIC E. COLI O157:H7 IN HUMAN STOOL SAMPLES

A new rapid test platform for the direct detection of E. coli O157:H7 in stool samples of infected patients was compared with the current standard methods. The new test, DIAPRO FAST-Q®, used biochip ICEflo ®technology that provided results within 20 min. Twenty-one stool samples from patients infected with E. coli O157:H7 or of unknown status were studied. Using the DIAPRO FAST-Q® method, within 20 min, we confirmed positive results for the seven known E. coli O157:H7 samples. While the standard culture method gave rise to only four positives in the remaining 14 unknown samples, the DIAPRO FAST-Q® detected E. coli O157:H7 in eight of these samples, which was confirmed subsequently by broth enrichment culture method.     

EVALUATION OF 5'NUCLEASE BASED DETECTION ASSAYS TO DETECT ESCHERICHIA COLI O157:H7 FROM FOOD PRODUCTS

Two 5'nuclease-based PCR methods (PCR-LS-50B and PCR-7200) were evaluated to determine their sensitivity for detecting Escherichia coli O157:H7 from pure cultures and in food samples enriched in different media and after different incubation periods. The PCR-7200 method was able to detect E. coli O157:H7 at ± 10² CFU/mL in pure culture in both mECB and EEB. In spiked meat samples, the PCR-7200 procedure was capable of detecting the eaeA gene at lower concentrations than the PCR-LS-50B procedure, regardless of the meat type or enrichment medium. Escherichia coli O157:H7 spiked at 0.3 CFU/mL was detectable after 9 h in EEB, but it was not detected in mECB within 24 h. An enrichment time of 4 h in mECB was needed to detect E. coli O157:H7 when spiked at higher levels (41 CFU/mL). The detection levels reported in this study are similar with other reported PCR-based detection techniques for E. coli O157:H7, however, the 5'nuclease-based assays are less labor intensive and capable of higher sample throughput because of their automated detection and analysis steps.     

利用大肠杆菌质粒检测与分离痘苗病毒的启动子

本文发现,痘苗病毒DNA一些巳知的启动子序列和一些功能尚不清楚的DNA片段,可以在大肠杆菌中起始氯霉素乙酰基转移酶(Chloramphenicol Acetyltrsnsferase,简称CAT)基因的转录和表达,使转化细菌呈现氯霉素抗性表型,这一结果证明,痘苗病毒的启动子可以被大肠杆菌的RNA多聚酶所识别并有效工作。同时发现不同启动子具有不同的强度,利用大肠杆菌质粒分离和检测痘苗病毒的启动子序列,不仅可以研究痘苗病毒基因组的表达调节特点,而且也为组建痘苗病毒表达载体提供了一个快速、简便可靠的方法。     

APPLICATIONS OF TIME-RESOLVED FLUOROIMMUNOASSAY TO DETECT MAGNETIC BEAD CAPTURED ESCHERICHIA COLI O157:H7

A time-resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, streptavidin coated magnetic beads conjugated with biotin-labeled anti E. coli O157:H7 were used to capture the bacteria. The bacteria were, at the same time, also labeled by a nonfluorescent, europium (Eu)-tagged anti-E. coli O157:H7 antibody. The sandwiched bacterial complexes were then concentrated using a magnetic particle concentrator and washed to remove other solution components. Upon addition of an enhancement buffer, the Eu-labels were then released from the antibodies and chelated to nitrilo-triacetic acid (NTA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu-(2-NTA)₃(TOPO)_2–3 micellar complexes. Delayed fluorescence associated with these complexes was measured and its intensity was used to estimate the original bacterial concentration spiked in hamburger. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method is able to detect 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the approach exhibited no or limited cross reactivity to Salmonella typhimurium, E. coli K-12 or Shigella dysenteriae spiked in hamburgers. Thus, the developed approach may be used as a rapid screening procedure for E. coli O157 bacteria in foods.     

DETECTION OF ESCHERICHIA COLI O157 IN RAW BEEF USING A CCD BASED LIGHT SCATTERING INSTRUMENT

A sensitive and easy-to-perform instrumentational method for the detection of Escherichia coli O157 in raw minced beef is described. The detection is based on a light scattering immunoassay and a charge-coupled device (CCD) direct readout spectrometer measuring the scattered light spectral signals at an optimized angle of 20° to the axis of transmitted light. Using latex particles coated with antibodies for E. coli O157, the method sensitivity has significantly improved comparing with the visual immunoassay assessment method when detecting the presence of this bacterium in spiked beef samples. The method is capable of detecting E. coli O157 at the level of 10³ cfu mL^-1 after 6 h of incubation of the spiked samples. This study has demonstrated a faster technique (within 8 h) for the detection of E. coli O157 in raw beef and a possible new application for the CCD based light scattering instrument.