Formulations of ready-to-eat (RTE) foods with antimicrobial compounds constitute an important safety measure against foodborne pathogens such as Listeria monocytogenes. While the efficacy of many commercially available antimicrobial compounds has been demonstrated in a variety of foods, the current understanding of the resistance mechanisms employed by L. monocytogenes to counteract these stresses is limited. In this study, we screened in-frame deletion mutants of two-component system response regulators associated with the cell envelope stress response for increased sensitivity to commercially available antimicrobial compounds (nisin, lauric arginate, -polylysine, and chitosan). A virR deletion mutant showed increased sensitivity to all antimicrobials and significantly greater loss of membrane integrity when exposed to nisin, lauric arginate, or -polylysine (P < 0.05). The VirR-regulated operon, dltABCD, was shown to be the key contributor to resistance against these antimicrobial compounds, whereas another VirR-regulated gene, mprF, displayed an antimicrobial-specific contribution to resistance. An experiment with a ␤-glucuronidase (GUS) reporter fusion with the dlt promoter indicated that nisin does not specifically induce VirRdependent upregulation of dltABCD. Lastly, prior exposure of L. monocytogenes parent strain H7858 and the ⌬virR mutant to 2% potassium lactate enhanced subsequent resistance against nisin and -polylysine (P < 0.05). These data demonstrate that VirRS-mediated regulation of dltABCD is the major resistance mechanism used by L. monocytogenes against cell envelope-damaging food antimicrobials. Further, the potential for cross-protection induced by other food-related stresses (e.g., organic acids) needs to be considered when applying these novel food antimicrobials as a hurdle strategy for RTE foods. Control of Listeria monocytogenes in ready-to-eat (RTE) foods is an important food safety goal due to the high mortality rate associated with listeriosis, particularly in susceptible populations, such as pregnant women, the elderly, and those with a compromised immune system (1). L. monocytogenes is of particular concern for those RTE foods that support growth of this pathogen to high levels during refrigerated storage, which can potentially cause a life-threatening disease. L. monocytogenes harbors a variety of stress coping mechanisms that allow it to survive under suboptimal environmental conditions associated with foods (e.g., acidic, osmotic, and/or temperature stress) (2). The ability of L. monocytogenes to tolerate and grow under such a wide range of adverse conditions elevates the likelihood of foodborne transmission to a human host. Thus, a multipronged approach (e.g., prevention of postprocessing contamination and reformulation of RTE foods with antimicrobials) to limit L. monocytogenes in foods along the farm-to-fork continuum is critical to reduce the potential for foodborne illnesses involving this organism (3).Natural antimicrobials are commonly applied to RTE foods to control foodb...

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.Escherichia coli O157:H7 remains a significant cause of food recalls and human illness despite the implementation of government regulations and process interventions to reduce transmission by contaminated foods. Ground beef remains a primary vehicle of food-borne dissemination, but a variety of foods have been involved in outbreaks (21). Cattle are considered a primary source in outbreaks involving nonbeef foods which become contaminated by environmental or waterborne E. coli O157:H7. The low infectious dose of this pathogen (12, 28) requires new or additional intervention strategies to further reduce its prevalence and numbers entering the food chain.Practices to prevent or reduce E. coli O157:H7 contamination of beef carcasses are primarily applied at the processing level, with the most common treatments being hide washes, trimming of contaminated carcass parts, steam vacuuming, hot water and acid washes, and steam treatment (1, 2, 4, 5). Despite the implementation of these practices, there continue to be significant numbers of recalls and beef-linked illness caused by this pathogen. The prevalence of cattle shedding E. coli O157:H7 prior to processing ranges from 2% to 42%, which correlates with the frequency of carcass contamination (7). One of the challenges to the development of preharvest interventions is the transmission of E. coli O157:H7 between animals and by environmental sources, like contaminated water (8, 27). Competitive exclusion and vaccination have been evaluated as preharvest interventions, and both have been reported to reduce but not eliminate E. coli O157:H7 from cattle (3,9,16,19).Chitosan has been used to make microparticles (i.e., chitosan micropa...

A most-probable-number (MPN) method was used to enumerate Listeria monocytogenes in 2,320 commercial ice cream scoops manufactured on a production line that was implicated in a 2015 listeriosis outbreak in the United States. The analyzed samples were collected from seven lots produced in November 2014, December 2014, January 2015, and March 2015. L. monocytogenes was detected in 99% (2,307 of 2,320) of the tested samples (lower limit of detection, 0.03 MPN/g), 92% of which were contaminated at <20 MPN/g. The levels of L. monocytogenes in these samples had a geometric mean per lot of 0.15 to 7.1 MPN/g. The prevalence and enumeration data from an unprecedented large number of naturally contaminated ice cream products linked to a listeriosis outbreak provided a unique data set for further understanding the risk associated with L. monocytogenes contamination for highly susceptible populations.

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The presence of the foodborne pathogen Listeria monocytogenes on cold-smoked salmon is a major concern for the seafood industry. Understanding processing and postprocessing handling factors that affect the ability of this pathogen to grow on cold-smoked salmon is critical for developing effective control strategies. In this study, we investigated the effect of curing method and freeze-thawing of cold-smoked salmon on (i) physicochemical properties and (ii) subsequent growth of genetically diverse strains of L. monocytogenes (inoculated after freeze-thawing) and endogenous lactic acid bacteria. The majority of the measured physicochemical properties were unaffected by freezing and thawing. Overall, wet-cured cold-smoked salmon had higher pH, water activity, and moisture, as well as lower fat, water-phase salt, and phenolic content compared with dry-cured cold-smoked salmon. The curing method and freeze-thawing did not affect growth of endogenous lactic acid bacteria. Freeze-thawing cold-smoked salmon prior to inoculation led to pronounced growth of L. monocytogenes at 7°C. The increase in cell density between days 0 and 30 was significantly (P = 0.0078) greater for cold-smoked salmon that was frozen and thawed prior to inoculation compared with nonfrozen cold-smoked salmon. On dry-cured, freeze-thawed cold-smoked salmon, L. monocytogenes had a lag phase ranging from 3.7 ± 0.1 to 11.2 ± 1.4 days compared with salmon that was wet cured and freeze-thawed, on which L. monocytogenes began to grow within 24 h. Variation in growth among L. monocytogenes strains was also observed, indicating the significance of assessing multiple strains. Further efforts to understand the impact of processing and postprocessing handling steps of cold-smoked salmon on the growth of genetically diverse L. monocytogenes will contribute to improved challenge study designs and data. This, in turn, will likely lead to more reliable and unbiased risk assessments and control measures.

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A precise and accurate method for enumeration of low level of Listeria monocytogenes in foods is critical to a variety of studies. In this study, paired comparison of most probable number (MPN) and direct plating enumeration of L. monocytogenes was conducted on a total of 1730 outbreak-associated ice cream samples that were naturally contaminated with low level of L. monocytogenes. MPN was performed on all 1730 samples. Direct plating was performed on all samples using the RAPID'L.mono (RLM) agar (1600 samples) and agar Listeria Ottaviani and Agosti (ALOA; 130 samples). Probabilistic analysis with Bayesian inference model was used to compare paired direct plating and MPN estimates of L. monocytogenes in ice cream samples because assumptions implicit in ordinary least squares (OLS) linear regression analyses were not met for such a comparison. The probabilistic analysis revealed good agreement between the MPN and direct plating estimates, and this agreement showed that the MPN schemes and direct plating schemes using ALOA or RLM evaluated in the present study were suitable for enumerating low levels of L. monocytogenes in these ice cream samples. The statistical analysis further revealed that OLS linear regression analyses of direct plating and MPN data did introduce bias that incorrectly characterized systematic differences between estimates from the two methods.

The objective of this study was to compare the survival of non-O157 Shiga toxin–producing Escherichia coli (STEC) with E. coli O157:H7 during pepperoni production. Pepperoni batter was inoculated with 7 log CFU/g of a seven-strain STEC mixture, including strains of serotypes O26, O45, O103, O111, O121, O145, and O157. Sausages were fermented to pH ≤4.8, heated at 53.3°C for 1 h, and dried for up to 20 days. STEC strains were enumerated at designated intervals on sorbitol MacConkey (SMAC) and Rainbow (RA) agars; enrichments were completed in modified EC (mEC) broth and nonselective tryptic soy broth (TSB). When plated on SMAC, total E. coli populations decreased 2.6 to 3.5 log after the 1-h heating step at 53.3°C, and a 4.9- to 5-log reduction was observed after 7 days of drying. RA was more sensitive in recovering survivors; log reductions on it were 1.9 to 2.6, 3.8 to 4.2, and 4.6 to 5.3 at the end of cook, and at day 7 and day 14 of drying, respectively. When numbers were less than the limit of detection by direct plating on days 14 and 20 of drying (representing a 5-log kill), no more than one of three samples in each experiment was positive by enrichment with mEC broth; however, STEC strains were recovered in TSB enrichment. Freezing the 7-day dried sausage for 2 to 3 weeks generated an additional 1- to 1.5-log kill. Confirmation by PCR revealed that O103 and O157 had the greatest survival during pepperoni productions, but all serotypes except O111 and O121 were occasionally recovered during drying. This study suggests that non-O157 STEC strains have comparable or less ability than E. coli O157 to survive the processing steps involved in the manufacture of pepperoni. Processes suitable for control of E. coli O157 will similarly inactivate the other STEC strains tested in this study.