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Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration–validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method. Inhibited detection of C. cayetanensis in cilantro samples can occur. Five commercial DNA cleanup kits were evaluated in inhibited cilantro samples. The kits reduced the inhibition levels of the internal amplification control. Results were conclusive at low and high levels of seeded oocysts. DNA cleanup minimized inhibition in cilantro with soil. [ABSTRACT FROM AUTHOR]

Cilantro was recently identified as a vehicle for protozoan illness. Current postharvest practices are not sufficient to inactivate protozoa on cilantro. Cold plasma is an emerging nonthermal waterless technology with potential applications in food processing that are currently being investigated to enhance the safety of herbs. The purpose of this study was to determine the impact of cold atmospheric plasma (CP) on the viability of Cryptosporidium parvum oocysts on cilantro. C. parvum oocysts were inoculated onto cilantro and treated with a CP jet for 0, 30, 90, and 180 s at a working distance of 10 cm with a flow of 1.42 × 10−3 m3/s. Oocyst viability was determined using HCT-8 cell culture infectivity assays. Overall, each treatment significantly reduced oocyst infectivity compared with the 0-s treatment control (P ≤ 0.02). Log inactivations of oocysts observed on cilantro were 0.84, 1.23, and 2.03 for the 30-, 90-, and 180-s treatment times, respectively. Drying and darkening of cilantro leaves was observed with treatments longer than 30 s. CP can reduce C. parvum infectivity on cilantro. With further research and optimization, this treatment technology has potential applications in postharvest processing of cilantro. Cold atmospheric plasma has the ability to inactivate C. parvum oocysts. Three minutes of treatment resulted in a 2.03-log inactivation of oocysts. Treatment times longer than 30 s impacted the raw-like characteristics of cilantro. Cold plasma can be optimized for commercial use on various produce commodities. [ABSTRACT FROM AUTHOR]

Leafy greens have been associated with foodborne disease outbreaks in different countries. To decrease microbial contamination of leafy greens, chemical agents are commonly used; however, a number of studies have shown these agents to have limited antimicrobial effect against pathogenic bacteria on vegetables. The objective of this study was to compare the antibacterial effect of Hibiscus sabdariffa calyx extracts (water, methanol, acetone, and ethyl acetate), sodium hypochlorite, acetic acid, and colloidal silver against foodborne bacteria on leafy greens. Thirteen foodborne bacteria were used in the study: Listeria monocytogenes, Shigella flexneri, Salmonella serotypes Typhimurium Typhi, and Montevideo, Staphylococcus aureus, Escherichia coli O157:H7, five E. coli pathotypes (Shiga toxin-producing, enteropathogenic, enterotoxigenic, enteroinvasive, and enteroaggregative), and Vibrio cholerae O1. Each foodborne bacterium was separately inoculated on romaine lettuce, spinach, and coriander leaves. Separately, contaminated leafy greens were immersed in four hibiscus extracts and in sanitizers for 5 min. Next, green leaves were washed with sterile tap water. Separately, each green leaf was placed in a bag that contained 0.1% sterile peptone water and was rubbed for 2 min. Counts were done by plate count using appropriate dilutions (in sterile peptone water) of the bacterial suspensions spread on Trypticase soy agar plates and incubated at 35 ± 2°C for 48 h. Statistically significant differences ( P < 0.05) were calculated with an analysis of variance and Duncan's test. All 13 foodborne bacteria attached to leafy greens. Roselle calyx extracts caused a significantly greater reduction ( P < 0.05) in concentration of all foodborne bacteria on contaminated romaine lettuce, spinach, and coriander than did the sodium hypochlorite, colloidal silver, and acetic acid. Dry roselle calyx extracts may potentially be a useful addition to disinfection procedures for romaine lettuce, spinach, and coriander. [ABSTRACT FROM AUTHOR]

The U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 4a describes a Luminex microbead-based suspension array used to screen colonies for 11 clinically relevant Shiga toxin-producing Escherichia coli (STEC) serogroups: 026, 045, 091, 0103, 0104, 0111, 0113, 0121, 0128, 0145, and 0157. We evaluated the usefulness of this method to identify STEC-positive enrichment samples before agar plating. Twelve E. coli strains were added to three types of fresh produce (bagged baby spinach, alfalfa sprouts, and cilantro) at levels near the detection limit of the test. A subset of these strains (six O serogroups) was similarly evaluated in raw milk. For comparison, portions of each of the 168 enrichment cultures were analyzed for serogroup by a real-time PCR assay and a Bio-Plex 200 assay with the bead-based suspensions. No falsepositive results were obtained. Of the 112 samples with a reported cycle threshold (CT) value, 101 undiluted, diluted, or extracted enrichment cultures also produced ratios above 5.0 in the Bio-Plex assay. When PCR CT values approached or were greater than 35, Bio-Plex detection became less reliable. Using undiluted or extracted enrichment cultures resulted in a significantly larger number of positive results. With the same enrichment material prepared for real-time PCR analysis as described in the BAM Chapter 4a, the STEC tnicrobead-based suspension array can accurately screen food enrichment cultures. [ABSTRACT FROM AUTHOR]

Analysis of fresh produce showed that enterotoxigenic Escherichia coli (ETEC) strains are most often found in cilantro and parsley, with prevalence rates of approximately 0.3%. Some ETEC strains also carried Shiga toxigenic E. coli (STEC) genes but had no STEC adherence factors, which are essential to cause severe human illness. Most ETEC strains in produce carried stable toxin and/or labile toxin genes but belonged to unremarkable serotypes that have not been reported to have caused human illnesses. [ABSTRACT FROM AUTHOR]

Shigella boydii CDPH (Chicago Department of Public Health) serotype 18 was implicated in an outbreak of foodborne illness in 1998. The suspected food vehicles were parsley and cilantro imported from Mexico used to prepare bean salad. Previous studies revealed that S. boydii CDPH serotype 18 can survive in bean salad, which contains organic acids and whose pH decreases over time. Acid challenge assays in acidified tryptic soy broth at pH 4.5, acidified Luria-Bertani broth at pH 4.5, and acidified M9 minimal salts medium at pH 2.5 containing amino acids, arginine, or glutamic acid were performed using S. boydii CDPH, S. boydii ATCC 35966, S. flexneri 3136, Escherichia coli O157:H7 dd8872, and E. coli O157:H7 dd642 to compare differences in acid tolerance. Differences in survival of exponential-phase cells were detected in acidified tryptic soy broth and Luria-Bertani broth at pH 4.5. In acidified minimal medium containing arginine, S. boydii strains were able to survive at pH 2.5. The arginine decarboxylase gene (adiA) present in S. boydii is involved in survival at extremely low pH. The discovery of adiA expression in S. boydii serotype 18 by use of an acidified minimal medium challenge and arginine decarboxylase biochemical assay is significant because arginine decarboxylase activity was thought to be unique to E. coli. Sequencing of the rpoS gene from the S. boydii outbreak strain indicates that it is 99% conserved compared with the E. coli K-12 rpoS gene and plays a vital role in survival under acidic conditions. [ABSTRACT FROM AUTHOR]

Cilantro (Coriandrum sativum) inoculated with Escherichia coli 0157:H7 at levels approximating 107 CFU/g was dipped in 200 ppm chlorine solution followed by low-dose gamma irradiation. Samples were plated on tryptic soy agar containing 50 μg/ml nalidixic acid (TSAN) as well as TSAN plates with two 7-ml layers of basal yeast extract agar (TSAN-TAL). Levels of E. coli 0157:H7 recovered from both types of media were determined over 11 days. Chlorination alone reduced counts by just over 1.0 log cycle, whereas irradiation at 1.05 kGy resulted in a 6.7-log reduction, and a combination of irradiation and chlorination reduced counts more than 7 log cycles. Trained panels performed analytical sensory tests at time intervals for 14 days to detect changes in yellowing, tip burn, browning, black rot, sliminess, off-aroma, and off-flavor. Sensory tests found no significant differences among attributes over time or dose in samples irradiated at 1.08 to 3.85 kGy. This study showed that combination treatments of chlorination and low-dose irradiation can significantly reduce levels of E. coli 0157:H7 in fresh cilantro while maintaining product quality. [ABSTRACT FROM AUTHOR]

A protocol has been developed to process cilantro and parsley samples for male-specific coliphages. Coliphage recovery depended on the duration of peptone rinsing, and whether the products were intact or cut. After 60 min of rinsing with 0.1% peptone, 78% of spiked coliphages were recovered from intact cilantro samples, and 60% of the spiked coliphages were recovered from cut cilantro samples. The protocol was field tested on a limited scale using cilantro and parsley samples from six retail outlets using enrichment-based and quantitative coliphage assays. Of the18 retail cilantro and parsley samples that were analyzed, 50% (9 of 18) of the cilantro samples were positive for male-specific coliphages using the enrichment-based assay compared to 39% (7 of 18) of the parsley samples. Using the quantitative coliphage assay, only 28% (5 of 18) of the cilantro samples were positive, and none of the parsley samples were positive. The number of male-specific coliphages ranged between 1 and 11 plaque-forming units per 10 g of cilantro samples. None of the samples was positive for Escherichia coli. The results suggest that simplified male-specific coliphage screening of herb samples is possible and that male-specific coliphages be used along with conventional bacteriological indicators to screen produce for presence of fecal contamination. [ABSTRACT FROM AUTHOR]