Detection and isolation of low levels of E. coli O157:H7 in cilantro by real-time PCR, immunomagnetic separation, and cultural methods with and without an acid treatment.

UNLABELLED: Leafy greens such as cilantro, contaminated with Escherichia coli O157:H7, have been implicated in cases of human illnesses. High levels of microflora in fresh cilantro make recovery of low numbers of E. coli O157:H7 difficult. To improve upon current methods, immunomagnetic separation (IMS) techniques in combination with real-time PCR (RTiPCR) and selective enrichment protocols were examined. Rinsates were prepared from cilantro samples inoculated with low (~0.02 CFU/g) and slightly higher (~0.05 CFU/g) levels of E. coli O157:H7. Rinsate portions were enriched in modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C. After 5 h, selective agents were added to samples and further incubated at 42 °C overnight. Detection and recovery were attempted at 5 and 24 h with and without IMS. IMS beads were screened by RTiPCR for simultaneous detection of stx1, stx2, and uidA SNP. Additionally, broth cultures and IMS beads were streaked onto selective agar plates (Rainbow(®) agar, R&F(®) E. coli O157 Chromogenic medium, TC-SMAC and CHROMagar™ 0157) for isolation of E. coli O157:H7. Both broth cultures and IMS beads were also acid treated in Trypticase Soy Broth pH 2 prior to plating to selective media to improve upon cultural recovery. Although E. coli O157 strains were detected in most samples by PCR after 5 h enrichment, cultural recovery was poor. However, after 24 h enrichment, both PCR and cultural recovery were improved. Acidification of the broths and the IMS beads prior to plating greatly improved recovery from 24 h enrichment broths by suppressing the growth of competing microorganisms. PRACTICAL APPLICATION: Detection and recovery of Escherichia coli O157:H7 in fresh produce matrices (e.g., cilantro) can be complicated by high background microflora present in these foods. Rapid detection by molecular methods combined with effective enrichment and isolation procedures such as using immunomagnetic separation (IMS) techniques can quickly identify potential hazards to public health. Additional techniques such as acidification of enrichment broths can exploit acid resistance characteristics of pathogens such as E. coli O157:H7, facilitating their isolation in complex food matrices.

Efficacy of a post enrichment acid treatment for isolation of Escherichia coli O157:H7 from alfalfa sprouts.

The enrichment, detection and isolation procedure in the current US FDA BAM have been shown effective for Escherichia coli O157:H7 in a wide variety of foods. Recently reported modifications to the enrichment protocol, including post-enrichment immunomagnetic separation (IMS) procedures have improved sensitivity of the method for alfalfa sprouts. However, cultural isolation on selective agar plates still presents a challenge in this food matrix. The focus of this study was to reduce levels of competing microflora and enhance isolation of E. coli O157:H7 on selective agars. We optimized the use of a short acid treatment after enrichment and with post-enrichment IMS beads. The optimized acid treatments were then evaluated in alfalfa sprouts artificially contaminated with E. coli O157:H7. After 5h enrichment of alfalfa sprouts contaminated at 0.2cfu/g, there was significant improvement in isolation on the selective plates following acid treatment of two types of IMS beads. Following 24h enrichment of alfalfa sprouts contaminated at low levels, E. coli O157:H7 was only recovered from 8/25 samples when no IMS or acid treatments were used. The use of only the acid treatment improved recovery to 19/25 samples. Following IMS of the enrichment broths, acid treatment increased isolation to 23/25 for Pathatrix™ and 24/25 for BeadRetriever™ IMS. Acid treatment (pH 2) of the enrichment broth for 30min or IMS beads for 7min is a simple and rapid way to greatly improve isolation of E. coli O157 from alfalfa sprout enrichments by reducing the interfering microflora on the selective media.

Optimization and evaluation of a modified enrichment procedure combined with immunomagnetic separation for detection of E. coli O157:H7 from artificially contaminated alfalfa sprouts.

Escherichia coli O157:H7 has been linked to foodborne disease outbreaks with alfalfa sprouts. Detection of the organism in sprouts by standard cultural methods can be difficult due to the high background microflora. The objective of this study was to develop and optimize an enrichment protocol with and without post-enrichment immunomagnetic separation (IMS) for the rapid detection by real-time PCR (RTiPCR) and cultural recovery of E. coli O157:H7 from artificially contaminated alfalfa sprouts. Initially we found that the FDA BAM procedure, enriching samples in modified buffered peptone water with pyruvate and at 37°C for 5h, followed by the addition of acriflavin, cefsulodin and vancomycin (mBPWp+ACV) and static incubation at 42°C gave poor results for both PCR detection and isolation for alfalfa sprouts artificially contaminated at 0.2cfu/g. The addition of post-enrichment IMS improved detection but not isolation. This procedure was modified and optimized by changing to mBPWp with cefsulodin and vancomycin at 42°C and shaking for 24h with and without IMS prior to PCR detection and cultural isolation. Using the resulting protocol we were able to detect E. coli O157:H7 in 100% of samples of alfalfa sprouts contaminated at 0.2cfu/g. This was validated for five strains of E. coli O157:H7. Isolation was 84% without added post-enrichment IMS and 100% with IMS. The optimized procedure was effective for detection and isolation of E. coli O157:H7 from this difficult food matrix.

Detection of E. coli O157:H7 in raw ground beef by Pathatrix™ immunomagnetic-separation, real-time PCR and cultural methods.

Detection of Escherichia coli O157:H7 by conventional cultural methods can be difficult in food matrices with a high background flora such as raw ground beef. Raw ground beef samples, artificially contaminated separately with five strains of E. coli O157:H7 at low (~0.2 cfu/g) and high (~2 cfu/g) levels, were enriched by two enrichment protocols; buffered peptone water (BPW) at 37 °C for 5h and 24h and modified buffered peptone water with pyruvate (mBPWp) for 5h at 37 °C followed by adding selective agents and incubating at 42 °C to 24h. Detection of added E. coli O157:H7 by real-time PCR (RTiPCR) and recovery on isolation agars was performed before and after PATHATRIX™ immunomagnetic separation (IMS). RTiPCR detection and cultural recovery of inoculated E. coli O157:H7 after 5h enrichment were poor at 0.21-0.24 cfu/g. The addition of IMS after 5h enrichment did not improve RTiPCR detection but markedly improved recovery by culturing. By extending enrichment to 24h, RTiPCR detection improved to 76% for either enrichment protocol without IMS. When 24h enrichment was followed by IMS, RTiPCR detection was also further improved. Cultural recovery after 24h enrichment was 56% and 84% without IMS and 100% and 92% after IMS for BPW and mBPWp respectively. Extended enrichment to 24h followed by IMS was found to be sensitive and reliable for detection and cultural recovery from raw ground beef using either enrichment method.

Comparison of Petrifilm Staph Express Count System with the bacteriological analytical manual direct-plating method for enumeration of Staphylococcus aureus in artificially contaminated hard cheese.

The 3M Petrifilm Staph Express Count System was compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) direct-plate count method for the enumeration of Staphylococcus aureus in 6 types of artificially contaminated hard cheese (Asiago, Cheddar, Gruyère, Parmesan, Romano, and Swiss). Five different samples of each cheese type were inoculated with S. aureus (ATCC 25923) to achieve low, medium, and high inoculum levels. S. aureus was enumerated by the Petrifilm and BAM methods, and the results were compared. Multivariate analysis of variance revealed no significant differences (P<0.05) between the 2 methods. The Petrifilm method compared favorably with the BAM procedure. The rapid method was more convenient to use, considerably faster, and less expensive to perform than the BAM method.