Comparative growth kinetics of Listeria monocytogenes and Salmonella enterica on dehydrated enoki and wood ear mushrooms during rehydration and storage.

Specialty mushrooms have been implicated in foodborne illness outbreaks in the U.S. in recent years. These mushrooms are available to consumers in both their fresh and dried states. Dehydrating mushrooms is a convenient way to increase shelf life. The dehydration process results in a lowered water activity (aw) of the commodity, creating an environment where both spoilage and pathogenic bacteria cannot proliferate. Prior to food preparation and consumption, these mushrooms are typically rehydrated and possibly stored for later use which could lead to increased levels of pathogens. This study examined the survival and growth of Listeria monocytogenes and Salmonella enterica on dehydrated enoki and wood ear mushrooms during rehydration and subsequent storage. Mushrooms were heat dehydrated, inoculated at 3 log CFU/g, and rehydrated at either 5 or 25°C for 2 h. Rehydrated mushrooms were stored at 5, 10, or 25°C for up to 14 d. L. monocytogenes and S. enterica survived on enoki and wood ear mushroom types during rehydration at 5 and 25°C, with populations often <2.39 log CFU/g. During subsequent storage, no growth was observed on wood ear mushrooms, regardless of the rehydration or storage temperature, with populations remaining <2.39 log CFU/g for both pathogens. When stored at 5°C, no growth was observed for either pathogen on enoki mushrooms. During storage at 10 and 25°C, pathogen growth rates and populations after 14 d were generally significantly higher on the enoki mushrooms rehydrated at 25°C; the highest growth rate (3.56 ± 0.75 log CFU/g/d) and population (9.48 ± 0.62 log CFU/g) after 14 d for either pathogen was observed by S. enterica at 25°C storage temperature. Results indicate a marked difference in pathogen survival and proliferation on the two specialty mushrooms examined in this study and highlight the need for individual product assessments. Data can be used to assist in informing guidelines for time and temperature control for the safety of rehydrated mushrooms.

Method of Inoculation Influences the Survival of Salmonella enterica on Retail and Orchard Peaches.

Challenge studies associated with fruits and vegetables generally utilize wet bacterial inoculation methods. However, a recent salmonellosis outbreak in the U.S. was linked to peaches plausibly contaminated via fugitive dust from a nearby animal operation. This outbreak has highlighted the need for a suitable inert carrier which can be used for the dry transfer of Salmonella enterica to produce. The purpose of this study was 1) to examine the population stability of S. enterica and its surrogate, Enterococcus faecium, in different dry matrices during extended storage to identify suitable carriers and 2) to evaluate the survival of S. enterica on peaches based on the mode of contamination (i.e., wet vs. dry). S. enterica and E. faecium were cultivated on tryptic soy agar (TSA) and inoculated into corn-cob small animal litter, sand, or silica at 10-11 log CFU/g. Matrices were mixed by hand and stored at 25°C and 33% relative humidity for up to 120 d. S. enterica remained relatively stable in the silica and litter, with no significant decrease in population after 14 and 28 d, respectively. E. faecium significantly reduced in all matrices, with the greatest reduction observed in silica (2.86 log CFU/g after 120 d). Additional carriers would need to be assessed for E. faecium which could maintain its population stability. Silica was ultimately selected for the dry carrier of S. enterica. Peaches available at retail or from orchards were inoculated with S. enterica using the silica carrier or by spot or dip inoculation methods at 5 log CFU/peach and stored at 5°C and 80% relative humidity for up to 28 d. The population of S. enterica significantly reduced on all peaches except for the dry inoculated orchard peaches, where the population remained stable (4.62 ± 0.35 log CFU/peach after 28 d). Results from this study determined that the mode of contamination influences the survival of S. enterica on peaches and that dry inoculation methods should be considered for produce in some instances.

Survival of Listeria monocytogenes on Frozen Vegetables during Long-term Storage at -18 and -10°C.

Two recent listeriosis outbreaks have occurred in the United States and Europe due to contaminated individually quick-frozen (IQF) vegetables. While one of the outbreaks was due to frozen vegetables considered ready-to-eat (RTE), the other was linked to frozen corn whose packaging contained cooking instructions and was considered not-ready-to-eat (NRTE). However, consumers may thaw certain frozen vegetables and consume without cooking. Since no data is available on the survivability of L. monocytogenes on IQF vegetables during frozen storage, this study examined the population of six different strains (comprising lineages 1/2a, 1/2b, and 4b) on IQF vegetables during long-term storage. Individual strains were inoculated onto an IQF vegetable mix at 4 log CFU/g and stored at -18 or -10°C for 360 days. Although fluctuations in populations of all strains were observed on the vegetables during storage, no significant differences based on strain, lineages, or temperature were observed. Overall, L. monocytogenes populations were only reduced by up to 0.47 and 0.59 log CFU/g after 360 days at -18 and -10°C, respectively. Results from this study suggest that L. monocytogenes is able to persist on IQF vegetables for extended time periods with no significant reduction in population. Future studies could evaluate the survival and growth of L. monocytogenes on IQF vegetables during thawing and storage.

Long-Term Survival of Listeria monocytogenes in Nut, Seed, and Legume Butters.

Nut, seed, and legume butters have become increasingly popular with consumers. Listeria monocytogenes contamination of a variety of butters has resulted in several recalls, although no known outbreaks have been identified. L. monocytogenes has been shown to survive on a variety of seeds for up to 6 months, legumes and nuts for over 12 months, and in peanut butter and peanut-chocolate spreads for 21 to 60 weeks depending on formulation; however, long-term survival in other butters has not yet been characterized. In this study, the survival of L. monocytogenes in various nut, seed, legume, and chocolate-containing butters (n = 10) based on inoculation level, storage temperature, and the pH, aw, and nutrient contents of the butters was examined. First, butters were inoculated with L. monocytogenes at 4 log CFU/g and stored at either 5 or 25°C with enumeration and/or enrichment at intervals over 12 months. L. monocytogenes survived in all butters examined with no significant change in population after storage at 5°C, whereas the population was reduced to <1.70 log CFU/g in as little as 3 months at 25°C; the only exception was for sunflower butter, where L. monocytogenes decreased approximately 1 log CFU/g. Subsequently, all butters were inoculated at 1 log CFU/g and stored at 25°C for 6 months with enrichment during storage. L. monocytogenes was detected in all butters, except pecan butter, after 6-month storage. Butters containing chocolate did not inhibit L. monocytogenes survival, regardless of the inoculation level. Results indicate there may be an association between high-fat and carbohydrate level and survivability of L. monocytogenes in various types of butters. This work highlights the need to mitigate the potential for cross-contamination of L. monocytogenes into nut, seed, and legume butters due to the potential for long-term survival.

Effect of Time, Temperature, and Transport Media on the Recovery of Listeria monocytogenes from Environmental Swabs.

ABSTRACT: Environmental monitoring for Listeria monocytogenes in food processing environments is key for ensuring the safety of ready-to-eat foods. For sampling, swabs are often hydrated with a wetting or transport medium that may contain neutralizers and other ingredients. After swabbing the environment, the swabs may then be transported or shipped cold to an off-site laboratory for testing, ideally within 48 h. Extended shipping times may subject the pathogen to increased temperatures in the presence of the wetting medium, organics, and other chemicals from the processing facility that could confound detection. This study evaluated growth and detection of L. monocytogenes on stainless steel exposed to either buffer or sodium hypochlorite before drying. Swabs were rehydrated with Butterfield's phosphate buffer, neutralizing buffer, Letheen broth, or Dey-Engley neutralizing broth before swabbing. Swabs were stored in the presence of no added food, cheese whey, or ice cream under both optimal (4°C) and suboptimal (15°C) temperatures for up to 72 h. Overall, there was no growth of L. monocytogenes at 4°C through 72 h of storage, although enrichment from these swabs was dependent on the presence and type of food matrix. Pathogen growth during storage at 15°C was more variable and depended on both the food matrix and transport media used, with Dey-Engley and Letheen broths allowing for the highest population increases. Overall, more enrichments resulting in L. monocytogenes detections were observed when using Letheen broth and neutralizing buffer than Dey-Engley broth, which resulted in fewer detections at 15°C. Logistic regression and Cochran-Mantel-Haenszel analyses determined that storage temperature, transport media, and food matrix all significantly affected detection of L. monocytogenes, whereas storage time did not have a clear effect on recovery from swabs.

Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization.

Survival of hepatitis A virus in spinach during low temperature storage.

Spinach leaves are frequently consumed raw and have been involved with past foodborne outbreaks. In this study, we examined the survival of hepatitis A virus (HAV) on fresh spinach leaves in moisture- and gas-permeable packages that were stored at 5.4 +/- 1.2 degrees C for up to 42 days. Different eluents including phosphate-buffered saline (PBS), pH 7.5 (with and without 2% serum), and 3% beef extract (pH 7.5 and 8) were compared for how efficiently they recovered viruses from spinach by using a simple elution procedure (<1 h). The recoveries were compared and determined by a plaque assay with FRhK-4 cells. Culture grade PBS containing 2% serum was found to be appropriate for HAV elution from spinach leaves, with an average recovery of 45% +/- 10%. Over 4 weeks of storage at 5.4 +/- 1.2 degrees C, HAV in spinach decreased slightly more than 1 log, with 6.75% of the original titer remaining. HAV survived under refrigerated temperatures on spinach leaves with a D-value of 28.6 days (equivalent to an inactivation rate of -0.035 log of HAV per day, r(2) = 0.88). In comparison, HAV in PBS containing 2% serum under the same storage conditions remained constant throughout 7 weeks. The inactivation rate of -0.035 log each day for HAV on spinach leaves was possibly due to the interaction of the virus and the leaf.

Comparison of assurance gold salmonella EIA, BAX for screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays for detection of Salmonella in alfalfa sprouts and sprout irrigation water.

The Assurance Gold Salmonella EIA, BAX for Screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays were compared with official cultural methods described in the Bacteriological Analytical Manual (BAM) for analysis of alfalfa sprouts and sprout irrigation water for the presence of Salmonella. The lower limits of detection of 4 serovars of Salmonella cells (S. tennessee, S. muenchen, S. mbandanka, and S. cubana) in pure culture were determined as approximately log10 2, 5, and 6 for the BAX, GENE-TRAK, and Gold EIA, respectively. Despite its low detection limit, the BAX did not perform as well as the other assays in analyzing contaminated sprouts and sprout irrigation water. For 4 different lots of sprouts and sprout irrigation water samples inoculated with the 4 serovars at low [1-2 colony forming units (CFU/g)] and high (68-180 CFU/g) levels, the BAX detected Salmonella in 58/64 (90.6%) of the samples, compared with 64/64 (100%) by the GENE-TRAK, Gold EIA, and BAM methods. Assay performance was also compared for analysis of naturally contaminated sprouts and sprout irrigation water with 3 lots of alfalfa sprouted seeds associated with different salmonellosis outbreaks. Positive assay results for the naturally contaminated samples were Gold EIA 41, GENE-TRAK 36, BAM 33, and BAX 13.