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.     

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.     

SUITABILITY OF SELECTIVE MEDIA FOR RECOVERY AND ENUMERATION OF SUBLETHALLY HEAT- AND ACID-INJURED LISTERIA MONOCYTOGENES

The suitability of PALCAM and modified Oxford (MOX) agars for recovering sublethally heat- and lactic acid-injured Listeria monocytogenes was investigated. L. monocytogenes LM101M, LM103M (meat isolates), and Scott A were suspended in tryptose phosphate broth (TPB), heated for up to 40 min at 54C, and surface plated onto tryptose phosphate agar (TPA), TPA + 4% NaCl (TPAS), PALCAM, and MOX. TPA and TPAS were used to determine total viable and sublethally injured populations, respectively. Heat-injured LM103M was recovered in the highest numbers on all media, followed by Scott A and LM101M (P<0.01). TPA allowed best recovery of all test strains, followed by PALCAMand MOX which were not different, and TPAS (P<0.01). For acid-injury studies, uninjured and heat-injured (54C for 20 min) test strains were suspended in phosphate-buffered TPB + 0.85% lactic acid (bTPBLA) at 25C for up to 24 h and plated as described above. Uninjured and heat-injured L. monocytogenes were recovered better from bTPBLA on MOX than on PALCAM (P<0.05). Heat injured L. monocytogenes LM103M was recovered better than LM101M but similar to Scott A on MOX and PALCAM (P<0.05), whereas Scott A was recovered similarly to LM101M and LM103M on MOX and PALCAM (P>0.05). Acid-injury of L. monocytogenes LM103M was enhanced by prior heat stress.     

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.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

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.     

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.     

MEMBRANE LIFTING TECHNIQUE FOR RAPID DETECTION OF ESCHERICHIA COLI 0157:H7 IN INOCULATED GROUND MEATS

A membrane lifting technique was developed for direct rapid detection of Escherichia coli 0157:H7 in inoculated ground meats. Duplicate groups of 2 meatballs were inoculated with volumes of 0.1-ml of a serial dilution (1:10) of E. coli 0157:H7 or a mixed culture containing one strain of E. coli 0157:H7 and a non-0157:H7E. coli serotype (E. coli ATCC 25922). Each meatball was sampled by sandwiching with 2 pieces of nitrocellulose membranes and pressing against each other to the center of the meatball. The membranes were in contact with the meats for 10 min to lift the bacteria. The membranes were removed and incubated on MacConkey-sorbitol agar plates with the meat contact side up. After 18 h incubation at 37C, an immunostain was performed directly on the membranes for detection of the presence or absence of E. coli 0157:H7. This method was found to be sensitive enough to detect as few as 1 to 2 cells of E. coli 0157:H7 inoculated on surfaces of 18-g meatballs. This method might be used as a rapid presumptive test for E. coli 0157:H7.     

DEVELOPMENT AND EVALUATION OF A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD FOR DETECTING ESCHERICHIA COLI O157 IN RAW MILK

In the present study, we report the performance of a loop-mediated isothermal amplification (LAMP) assay detecting foodborne pathogen Escherichia coli O157. Three pairs of primers were specially designed for recognizing eight distinct sequences of rfbE gene. Time and temperature conditions for amplification of E. coli O157 were optimized to be 40 min at 65C. The LAMP assay gave artificially contaminated raw milk sample detection limit level of 410 cfu/mL which corresponds to three to five cells per reaction tube, while the detection level of conventional polymerase chain reaction was 4.1  ×  10⁴ cfu/mL. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with the ISO 16654: 2001 reference method.

PRACTICAL APPLICATIONS

The loop-mediated isothermal amplification method reported here provides a powerful tool for the detection of Escherichia coli O157 in raw milk samples because of its specificity, sensitivity and rapidity.     

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.