The effect of crisping, misting, and storage temperature on the survival or growth of Listeria monocytogenes and natural psychrotrophic bacteria on romaine lettuce.

L. monocytogenes has been linked to fresh produce and detected in the retail environment. This study simulated the retail practices (crisping, misting, and storage) of unbagged whole heads of romaine lettuce to determine the growth of L. monocytogenes and natural psychrotrophic microflora. Three nalidixic acid-resistant strains of L. monocytogenes strains were inoculated to each head of lettuce (≈5 log10 CFU/g). For crisping, 24 heads of romaine lettuce were immersed in tap water or electrolyzed water (EW; free chlorine: 55 ppm) for 5 min, followed by holding at 5 °C for 2 h. The water-crisped (WC), EW crisped (EWC), or non-crisped (NC) lettuces were placed in a commercial refrigerated cabinet for misting at 5 °C. After 24-h misting, heads of lettuce were placed in perforated drain boxes with cover at 5 °C or 15 °C. The tap water and EW crisping achieved 1.3 and 2.9 log10 CFU/g reduction of L. monocytogenes, respectively. Approximately 1 log additional reduction of L. monocytogenes in the non-crisped lettuce was shown after misting (p < 0.05), but no significant effect of misting on the population of L. monocytogenes was observed on WC or EWC lettuces (p > 0.05). Regardless of the storage temperature or misting, L. monocytogenes populations remained significantly (p < 0.05) lower on EWC lettuce than NC and WC lettuce. On days 4 and 7 of storage, the natural psychrotrophic bacteria on lettuce stored at 5 °C was significantly lower than stored at 15 °C, and its population was not affected by crisping and misting. These provide insight into the influence of retail lettuce handling practices on the risk of L. monocytogenes.

Recovery Rate of Cells of the Seven Regulated Serogroups of Shiga Toxin-Producing Escherichia coli from Raw Veal Cutlets, Ground Veal, and Ground Beef from Retail Stores in the Mid-Atlantic Region of the United States.

ABSTRACT: A total of 482 veal cutlet, 555 ground veal, and 540 ground beef samples were purchased from retail establishments in the mid-Atlantic region of the United States over a noncontiguous 2-year period between 2014 and 2017. Samples (325 g each) were individually enriched and screened via real-time PCR for all seven regulated serogroups of Shiga toxin-producing Escherichia coli (STEC). Presumptive STEC-positive samples were subjected to serogroup-specific immunomagnetic separation and plated onto selective media. Up to five isolates typical for STEC from each sample were analyzed via multiplex PCR for both the virulence genes (i.e., eae, stx1 and/or stx2, and ehxA) and serogroup-specific gene(s) for the seven regulated STEC serogroups. The recovery rates of non-O157 STEC from veal cutlets (3.94%, 19 of 482 samples) and ground veal (7.03%, 39 of 555 samples) were significantly higher (P < 0.05) than that from ground beef (0.93%, 5 of 540 samples). In contrast, only a single isolate of STEC O157:H7 was recovered; this isolate originated from 1 (0.18%) of 555 samples of ground veal. Recovery rates for STEC were not associated with state, season, packaging type, or store type (P > 0.05) but were associated with brand and fat content (P < 0.05). Pulsed-field subtyping of the 270 viable and confirmed STEC isolates from the 64 total samples testing positive revealed 78 pulsotypes (50 to 80% similarity) belonging to 39 pulsogroups, with ≥90% similarity among pulsotypes within pulsogroups. Multiple isolates from 43 (67.7%) of 64 samples testing positive had an indistinguishable pulsotype. STEC serotypes O26 and O103 were the most prevalent serogroups in beef and veal, respectively. These findings support related findings from regulatory sampling studies over the past decade and confirm that recovery rates for the regulated STEC serogroups are higher for raw veal than for raw beef samples, as was observed in the present study of meat purchased at food retailers in the mid-Atlantic region of the United States.

Inactivation of Shiga Toxin-Producing Escherichia coli in Refrigerated and Frozen Meatballs Using High Pressure Processing.

High pressure processing (HPP) was evaluated to inactivate Shiga toxin-producing Escherichia coli (STEC) in raw meatballs. Ground meat (>90% lean) was inoculated (ca. 7.0 log CFU/g) with a rifampicin-resistant cocktail of eight STEC strains (O26:H11, O45:H2, O103:H2, O104:H4, O111:H-, O121:H19, O145:NM, and O157:H7). Inoculated ground beef, ground veal, or a mixture of ground beef, pork, and veal were separately mixed with liquid whole eggs and seasonings, shaped by hand into meatballs (40 g each), and stored at -20 or at 4 °C for at least 18 h. Samples were then exposed to 400 or 600 MPa for 0 to 18 min. There were no differences (p > 0.05) in pathogen reduction related to the species of meat used or for meatballs that were refrigerated (0.9 to 2.9 log CFU/g) compared to otherwise similar meatballs that were stored frozen (1.0 to 3.0 log CFU/g) prior to HPP treatment. However, less time was needed to achieve a ≥ 2.0 log CFU/g reduction at 600 MPa (1 to 3 min) compared to 400 MPa (at least 9 min). This work provides new and practically useful information on the use of HPP to inactivate STEC in raw meatballs.

Viability of Shiga Toxin-Producing Escherichia coli, Salmonella, and Listeria monocytogenes within Plant versus Beef Burgers during Cold Storage and following Pan Frying.

ABSTRACT: The viability of Shiga toxin-producing Escherichia coli (STEC), Salmonella, and Listeria monocytogenes within plant- and beef-based burgers was monitored during storage and cooking. When inoculated (ca. 3.5 log CFU/g) into 15-g portions of plant- or beef-based burgers, levels of STEC and Salmonella decreased slightly (≤0.5-log decrease) in both types of burgers when stored at 4°C, but increased ca. 2.4 and 0.8 log CFU/g, respectively, in plant-based burgers but not beef-based burgers (≤1.2-log decrease), after 21 days at 10°C. For L. monocytogenes, levels increased by ca. 1.3 and 2.6 log CFU/g in plant burgers after 21 days at 4 and 10°C, respectively, whereas pathogen levels decreased slightly (≤0.9-log decrease) in beef burgers during storage at 4 and 10°C. Regarding cooking, burgers (ca. 114 g each) were inoculated with ca. 7.0 log CFU/g STEC, Salmonella, or L. monocytogenes and cooked in a sauté pan. Cooking plant- or beef-based burgers to 62.8°C (145°F), 68.3°C (155°F), or 73.9°C (165°F) delivered reductions ranging from ca. 4.7 to 6.8 log CFU/g for STEC, ca. 4.4 to 7.0 log CFU/g for L. monocytogenes, and ca. 3.5 to 6.7 log CFU/g for Salmonella. In summary, the observation that levels of all three pathogens increased by ca. 1.0 to ca. 2.5 log CFU/g in plant-based burgers when stored at an abusive temperature (10°C) highlights the importance of proper storage (4°C) to lessen risk. However, because all three pathogens responded similarly to heat in plant-based as in beef-based burgers, well-established cooking parameters required to eliminate STEC, Salmonella, or L. monocytogenes from ground beef should be as effective for controlling cells of these same pathogens in a burger made with plant-sourced protein.

Inactivation of Shiga Toxin-Producing Escherichia coli and Listeria monocytogenes within Plant versus Beef Burgers in Response to High Pressure Processing.

ABSTRACT: We evaluated high pressure processing to lower levels of Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes inoculated into samples of plant or beef burgers. Multistrain cocktails of STEC and L. monocytogenes were separately inoculated (∼7.0 log CFU/g) into plant burgers or ground beef. Refrigerated (i.e., 4°C) or frozen (i.e., -20°C) samples (25 g each) were subsequently exposed to 350 MPa for up to 9 or 18 min or 600 MPa for up to 4.5 or 12 min. When refrigerated plant or beef burger samples were treated at 350 MPa for up to 9 min, levels of STEC were reduced by ca. 0.7 to 1.3 log CFU/g. However, when refrigerated plant or beef burger samples were treated at 350 MPa for up to 9 min, levels of L. monocytogenes remained relatively unchanged (ca. ≤0.3-log CFU/g decrease) in plant burger samples but were reduced by ca. 0.3 to 2.0 log CFU/g in ground beef. When refrigerated plant or beef burger samples were treated at 600 MPa for up to 4.5 min, levels of STEC and L. monocytogenes were reduced by ca. 0.7 to 4.1 and ca. 0.3 to 5.6 log CFU/g, respectively. Similarly, when frozen plant and beef burger samples were treated at 350 MPa up to 18 min, reductions of ca. 1.7 to 3.6 and ca. 0.6 to 3.6 log CFU/g in STEC and L. monocytogenes numbers, respectively, were observed. Exposure of frozen plant or beef burger samples to 600 MPa for up to 12 min resulted in reductions of ca. 2.4 to 4.4 and ca. 1.8 to 3.4 log CFU/g in levels of STEC and L. monocytogenes, respectively. Via empirical observation, pressurization did not adversely affect the color of plant burger samples, whereas appreciable changes in color were observed in pressurized ground beef. These data confirm that time and pressure levels already validated for control of STEC and L. monocytogenes in ground beef will likely be equally effective toward these same pathogens in plant burgers without causing untoward effects on product color.

Survey of Intact and Nonintact Raw Pork Collected at Retail Stores in the Mid-Atlantic Region of the United States for the Seven Regulated Serogroups of Shiga Toxin-Producing Escherichia coli.

A total of 514 raw pork samples (395 ground or nonintact and 119 intact samples) were purchased at retail stores in Pennsylvania, Delaware, and New Jersey between July and December 2017. All raw pork samples were screened for serogroup O26, O45, O103, O111, O121, O145, or O157:H7 cells of Shiga toxin-producing Escherichia coli (STEC-7) using standard microbiological and molecular methods. In short, 21 (5.3%) of the 395 ground or nonintact pork samples and 3 (3.4%) of the 119 intact pork samples tested positive via the BAX system real-time PCR assay for the stx and eae virulence genes and for the somatic O antigens for at least one of the STEC-7 serogroups. However, none of these 24 presumptive-positive pork samples subsequently yielded a viable isolate of STEC displaying a STEC-7 serogroup-specific surface antigen in combination with the stx and eae genes. These data suggest that cells of STEC serogroups O26, O45, O103, O111, O121, O145, or O157:H7 are not common in retail raw pork samples in the mid-Atlantic region of the United States.

Meat Bars: A Survey To Assess Consumer Familiarity and Preparation Parameters and a Challenge Study To Quantify Viability of Shiga Toxin-Producing Escherichia coli Cells during Processing and Storage.

Meat bars are dried snacks containing a mixture of meat, berries, and nuts. To explore consumer awareness of meat bars, we conducted two online, nationally representative surveys and established that 70.8% (743 of 1,050) of U.S. citizens were unfamiliar with this product. When asked to check all answers that applied, most of the 545 respondents (who were recruited based on their familiarity with meat bars) preferred beef (n = 385) as the protein source, followed by chicken (n = 293), pork (n = 183), and turkey (n = 179). Most meat bars were purchased from grocery stores (n = 447), followed by online orders (n = 130) and outdoor stores (n = 120). When asked specifically whether they made their own meat bars, 17.8% of respondents (97 of 545) replied "yes," the majority (52 of 97, 54%) of which obtained recipes online. Some 69.1% (67 of 97) measured the internal temperature of the meat during dehydration, but only 10.3% (10 of 97) confirmed the internal temperature by using a thermometer. Given the paucity of information available on the fate of pathogenic or spoilage bacteria associated with meat bars, as another component of this study, batter was prepared with or without encapsulated citric acid (ECA; 0.74%) added to a formulation of ground beef (65%; 90% lean, 10% fat), chopped pecans (15%), golden flaxseed flour (9.7%), chopped cranberries (5.0%), chopped sunflower seeds (3.1%), sea salt (1.1%), black pepper (0.8%), and celery powder (0.35%). Batter was inoculated (ca. 6.5 log CFU/g) with Shiga toxin-producing Escherichia coli (STEC), portioned by hand (40 ± 0.1 g each), and then dried in a commercial dehydrator. Regardless of the drying treatment, inclusion of ECA in the batter resulted in a pH decrease from ca. 5.5 to ca. 4.7 to 5.0 in the finished product. Without ECA, when meat bars were dried at 62.8°C for 6 h, 71.1°C for 4 h, or 62.8°C for 2 h and then 71.1°C for 2 h, levels of STEC decreased by ca. 6.2, 6.3, or 5.2 log CFU/g, respectively. With ECA, STEC decreased by ca. 6.0, 6.6, or 6.0 log CFU/g in meat bars dried at 62.8°C for 6 h, 71.1°C for 4 h, or 62.8°C for 2 h and then 71.1°C for 2 h, respectively. Our results confirmed that a ≥5.0-log reduction in STEC could be achieved in meat bars formulated with or without ECA under all dehydration conditions tested.

Thermal Inactivation of Salmonella in Pâté Made from Chicken Liver.

HIGHLIGHTS: Cooking may reduce the potential risk of salmonellosis associated with liver pâté. A 5-log reduction was achieved when inoculated pâté was cooked to an internal temperature of ≥73.8°C. A 5-log reduction was achieved when pâté was made with inoculated liver fried for >8 min at 140°C. Findings of this study may be useful for establishing cooking guidelines for liver and pâté.

Prevalence, Levels, and Viability of Salmonella in and on Raw Chicken Livers.

We surveyed chicken livers from various sources for the presence and levels of Salmonella. The pathogen was recovered from 148 (59.4%) of 249 chicken livers purchased at retail stores in Delaware, New Jersey, and Pennsylvania over about a 9-month period. Positive samples harbored Salmonella at levels of 6.4 most probable number (MPN)/g to 2.4 log CFU/g. The percentage of Salmonella-positive livers purchased at retail outlets in New Jersey (72%, 59 of 82 livers) was significantly higher (P < 0.05) than the percentage for livers purchased in Delaware (48%, 36 of 75 livers); however, this percentage was not significantly different (P > 0.05) from that for livers purchased in Pennsylvania (57.6%, 53 of 92 livers). The pathogen was also recovered more often (P = 0.019) from livers that were packaged by retailers (81 of 121 livers, 66.9%) than from livers packaged directly by processors (67 of 128 livers, 52.3%). In related studies, 12 (5.8%) of 207 chicken livers harvested from birds on a research farm tested positive for Salmonella at levels of 0.4 to 2.2 MPN/g. The recovery rate of Salmonella was 4.4% (6 of 135 livers) from livers with the gall bladder attached and 8.3% (6 of 72 livers) from livers when the gall bladder was removed at harvest on the research farm. We also quantified the levels of a nine-strain cocktail (ca. 6.5 log CFU/g) of Salmonella strains inoculated externally onto or internally into livers both before and after extended cold storage. Storage for at least 2 days at 4°C or 15 days at -20°C resulted in a decrease of about 1.0 log CFU/g in pathogen levels. Given the relatively high recovery rate (ca. 6.0 to 60.0%) and high (possibly illness causing) levels (0.4 MPN/g to 2.4 CFU/g) of Salmonella associated with chicken livers in the present study, further interventions for processors are needed to lower the prevalence and levels of this pathogen on poultry liver.

Assessment of Microbiological Safety and Quality of Marinades Used To Treat Beef and That Were Collected over a 12-Month Period from Specialty Retailers Near Raleigh, North Carolina.

In total, 115 marinade samples (58 fresh marinades and 57 spent marinades) were collected over 12 months from specialty retailers (four individual stores) near Raleigh, NC. These marinades were screened for total mesophilic aerobic plate count (M-APC), total psychrotrophic aerobic plate count (P-APC), and Enterobacteriaceae. These marinades were also screened for the seven regulated serogroups of Shiga toxin-producing Escherichia coli. Stores A and B used immersion to marinade raw beef cuts, whereas stores C-1 and C-2 used vacuum tumbling. In general, marinade temperatures at the stores ranged from 1.8 to 6.6°C, and beef cuts were marinated from a few minutes to up to 3 days. Regardless of the process used to marinade meat, levels of M-APC and P-APC in fresh marinades ranged from 3.4 to 4.7 and 1.4 to 1.8 log CFU/mL, respectively, whereas Enterobacteriaceae were not detected in any fresh marinades, even after enrichment. However, levels of M-APC, P-APC, and Enterobacteriaceae in spent marinades collected from stores C-1 and C-2 (ca. 3.6 to 7.1 log CFU/mL) were significantly higher (P < 0.05) compared with levels of these same types of bacteria enumerated from spent marinades collected at stores A and B (ca. ≤0.7 to 4.9 log CFU/mL). None of the 115 marinade samples tested positive for Shiga toxin-producing E. coli by using a BAX system real-time PCR assay. No significant (P > 0.05) association was observed between microbial levels (i.e., M-APC, P-APC, and Enterobacteriaceae) and the temperature or duration of the marination process. Levels of M-APC, P-APC, and Enterobacteriaceae in spent marinades were significantly affected by the marination method (P < 0.05), with levels, in general, being higher in marinades used for tumbling. Thus, retailers must continue to keep marinade solutions and meat at a safe temperature (i.e., ≤4°C) and to properly and frequently sanitize the equipment and environment in both the processing area and deli case.

Sanitizer efficacy in preventing cross-contamination of heads of lettuce during retail crisping.

This study was conducted to provide information regarding mitigation of cross-contamination through the use of sanitizer during crisping at retail outlets. Seven non-inoculated heads and one inoculated head (≈5 log CFU/g) of lettuce were placed into commercial sink filled with 76 L of tap water (TW), electrolyzed water (EW, free chlorine: 43 ± 6 ppm), lactic acid and phosphoric acid-based sanitizer (LPA, pH 2.89), or citric acid-based sanitizer (CA, pH 2.78) and soaked for 5 min. Two subsequent batches (eight non-inoculated heads per batch) were soaked in the same solution. Soaking with EW significantly reduced the population of S. enterica (2.8 ± 1.5 log CFU/g), E. coli O157:H7 (3.4 ± 1.1 log CFU/g), and L. monocytogenes (2.6 ± 0.7 log CFU/g) inoculated on Romaine lettuce compared to TW, LPA, and CA (p < 0.05). On Red leaf lettuce, EW significantly reduced populations of S. enterica and E. coli O157:H7, but not L. monocytogenes compared to other treatments. No significant difference was noted between TW, LPA, and CA in reducing foodborne pathogens (p > 0.05) or preventing cross-contamination. Soaking with EW prevented cross-contamination among lettuce heads and controlled bacterial populations in crisping water for three consecutive batches. EW may be an effective option as a sanitizer to minimizing the cross-contamination of leafy greens during the retail crisping.

Potential transfer of extended spectrum ß-lactamase encoding gene, blashv18 gene, between Klebsiella pneumoniae in raw foods.

This study investigated the transfer frequency of the extended-spectrum ß-lactamase-encoding gene (blaSHV18) among Klebsiella pneumoniae in tryptic soy broth (TSB), pasteurized milk, unpasteurized milk, alfalfa sprouts and chopped lettuce at defined temperatures. All transconjugants were characterized phenotypically and genotypically. KP04(ΔKM) and KP08(ΔKM) isolated from seed sprouts and KP342 were used as recipients in mating experiments with K. pneumoniae ATCC 700603 serving as the donor. In mating experiments, no transconjugants were detected at 4 °C in liquid media or chopped lettuce, but detected in all media tested at 15 °C, 24 °C, and 37 °C. At 24 °C, the transfer of blaSHV18 gene occurred more frequently in alfalfa sprouts (5.15E-04 transconjugants per recipient) and chopped lettuce (3.85E-05) than liquid media (1.08E-05). On chopped lettuce, transconjugants were not detected at day 1 post-mating at 15 °C, but observed on day 2 (1.43E-05). Transconjugants carried the blaSHV18 gene transferred from the donor and the virulence gene harbored by recipient. More importantly, a class 1 integrase gene and resistance to tetracycline, trimethoprim/sulfamethoxazole were co-transferred during mating. These quantitative results suggest that fresh produce exposed to temperature abuse may serve as a competent vehicle for the spread of gene encoding for antibiotic resistance, having a potential negative impact on human health.

Effect of the food production chain from farm practices to vegetable processing on outbreak incidence.

The popularity in the consumption of fresh and fresh-cut vegetables continues to increase globally. Fresh vegetables are an integral part of a healthy diet, providing vitamins, minerals, antioxidants and other health-promoting compounds. The diversity of fresh vegetables and packaging formats (spring mix in clamshell container, bagged heads of lettuce) support increased consumption. Unfortunately, vegetable production and processing practices are not sufficient to ensure complete microbial safety. This review highlights a few specific areas that require greater attention and research. Selected outbreaks are presented to emphasize the need for science-based 'best practices'. Laboratory and field studies have focused on inactivation of pathogens associated with manure in liquid, slurry or solid forms. As production practices change, other forms and types of soil amendments are being used more prevalently. Information regarding the microbial safety of fish emulsion and pellet form of manure is limited. The topic of global climate change is controversial, but the potential effect on agriculture cannot be ignored. Changes in temperature, precipitation, humidity and wind can impact crops and the microorganisms that are associated with production environments. Climate change could potentially enhance the ability of pathogens to survive and persist in soil, water and crops, increasing human health risks. Limited research has focused on the prevalence and behaviour of viruses in pre and post-harvest environments and on vegetable commodities. Globally, viruses are a major cause of foodborne illnesses, but are seldom tested for in soil, soil amendments, manure and crops. Greater attention must also be given to the improvement in the microbial quality of seeds used in sprout production. Human pathogens associated with seeds can result in contamination of sprouts intended for human consumption, even when all appropriate 'best practices' are used by sprout growers.