Nancy G. Faith

Surfaces modified with nanometer-thick silver-impregnated polymeric films that kill bacteria but support growth of mammalian cells.

Silver is widely used as a biocidal agent in ointments and wound dressings. However, it has also been associated with tissue toxicity and impaired healing. In vitro characterization has also revealed that typical loadings of silver employed in ointments and dressings (approximately 100 microg/cm(2)) lead to cytotoxicity. In this paper, we report the results of an initial study that sought to determine if localization of carefully controlled loadings of silver nanoparticles within molecularly thin films immobilized on surfaces can lead to antimicrobial activity without inducing cytotoxicity. Polymeric thin films of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were prepared by layer-by-layer deposition and loaded with approximately 0.4 microg/cm(2) to approximately 23.6 microg/cm(2) of silver nanoparticles. Bacterial killing efficiencies of the silver-loaded films were investigated against Staphylococcus epidermidis, a gram-positive bacterium, and it was determined that as little as approximately 0.4 microg/cm(2) of silver in the polymeric films caused a reduction of 6log(10)CFU/mL (99.9999%) bacteria in suspensions incubated in contact with the films (water-borne assays). Significantly, whereas the antibacterial films containing high loadings of silver were found to be toxic to a murine fibroblast cell line (NIH-3T3), the polymeric films containing approximately 0.4 microg/cm(2) of silver were not toxic and allowed attachment, and growth of the mammalian cells. Thus, the results of this study go beyond prior reports by identifying silver-impregnated, polymeric thin films that are compatible with in vitro mammalian cell culture yet exhibit antibacterial activity. These results support the hypothesis that localization of carefully controlled loadings of silver nanoparticles within molecularly thin polymeric films can lead to antimicrobial activity without cytotoxicity. More broadly, this strategy of modifying surfaces with minimal loadings of bioactive molecules indicates the basis of approaches that may permit management of microbial burden in wound beds without impairment of wound healing.

Validation of pepperoni processes for control of Escherichia coli O157:H7

The outbreak of Escherichia coli O157:H7 linked with dry-cured salami in late 1994 prompted regulatory action that required manufacturers of fermented products to demonstrate a 5-log unit reduction in counts of this pathogen during processing. Therefore, pepperoni batter (75% pork:25% beef with a fat content of ca. 32%) was inoculated with a pediococcal starter culture and a five-strain mixture of E. coli O157:H7 (≥2 × 107 CFU/g) and stuffed into 55-mm diameter fibrous casings 47 cm in length. The viability of the pathogen was monitored before stuffing, after fermentation, after thermal processing, and/or after drying. Chubs were fermented at 96°F (36°C) and 85% relative humidity (RH) to pH ≤ 5.0 and then dried at 55°F (13°C) and 65% RH to a moisture/protein ratio of ≤1.6:1 (modified method 6 process). Counts of the pathogen decreased about 1.2 log units after fermentation and drying. In subsequent experiments, heating chubs after fermentation to internal temperatures of 145°F (63°C) instantaneous or 128°F (53°C) for 60 min resulted in a ≥5-log unit decrease in numbers of strain O157:H7 without visibly affecting the texture or appearance of the product. These data revealed that a traditional nonthermal, process for pepperoni was only sufficient to eliminate relatively low levels (ca. 2 log CFU/g) of E. coli O157:H7, whereas heating to internal temperatures of 145°F (63°C) instantaneous or 128°F (53°C) for 60 min delivered a 5 to 6 log unit reduction in counts of the pathogen in pepperoni.

Viability of Escherichia coli O157:H7 in Fermented Semidry Low-Temperature-Cooked Beef Summer Sausage

The population of inoculated Escherichia coli O157:H7 was monitored during the manufacture and storage of a semidry beef summer sausage processed by fermentation and cooking at a low temperature by heating to an internal temperature of 130°F (54°C). The all-beef batter (11% fat and nonmeat ingredients) was inoculated with the commercial starter culture Pediococcus acidilactici HP (≥8.6 log CFU/g of batter) and a five-strain mixture of E. coli O157:H7 (≥7 log CFU/g) and then hand stuffed into 2.5-inch (64-mm) diameter fibrous casings. The sausages were fermented at an initial temperature of 85°F (29°C) to a final temperature of 105°F (41°C) over ca. 13 h at 80% relative humidity (RH) to pH 4.6 or pH 5.0. After fermentation to pH 4.6, the internal temperature of the chubs was raised to 130°F (54°C) instantaneous over 3.6 h at 60% RH. After fermentation to pH 5.0, the internal temperature of the chubs was raised to 130°F (54°C) over 3.6 h at 60% RH and the chubs were maintained under these conditions for 0, 30, or 60 min. he chubs were cold water showered for 15 min and then chilled at 39°F (4°C) for 6 h before being vacuum packaged and stored at 39°F (4°C) or 77°F (25°C) for 7 days. Regardless of the target pH, fermentation alone resulted in only a 1.39-log CFU/g decrease in pathogen numbers. However, fermentation to pH 4.6 and heating to an internal temperature of 130°F (54°C) instantaneous reduced counts of E. coli O157:H7 by ≥7.0 log units to below detection levels (<10 CFU/g). Pathogen numbers remained below levels detectable by direct plating, but viable E. coli O157:H7 cells were recovered by enrichment of samples during sausage storage at either refrigeration or abuse temperatures. In contrast, fermentation to pH 5.0 and heating to an internal temperature of 130°F (54°C) instantaneous resulted in a 3.2-log-unit decrease in counts of E. coli O157:H7. No appreciable reductions in pathogen numbers were observed thereafter following storage at either 39°F (4°C) or 77°F (25°C) for 7 days. Fermentation to pH 5.0 and heating to an internal temperature of 130°F (54°C) instantaneous followed by holding for 30 or 60 min resulted in about a 5- or 7-log reduction, respectively, in pathogen numbers. For chubs held for 30 min at 130°F (54°C), pathogen numbers decreased to 2.02 and <1.0 log CFU/g at 39°F (4°C) and 77°F (25°C), respectively, after 7 days; viable cells were only observed by enrichment after storage at 77°F (25°C). For chubs held for 60 min at 130°F (54°C), pathogen numbers remained below levels detectable by direct plating, but viable cells were recoverable by enrichment after 7 days at both storage temperatures. These data will be useful guidelines to manufacturers for developing processing conditions to further ensure the safety of this category of fermented sausages relative to food-borne pathogens such as serotype O157:H7 strains of E. coli .

A/J Mice Are Susceptible and C57BL/6 Mice Are Resistant to Listeria monocytogenes Infection by Intragastric Inoculation

ABSTRACT Previous studies demonstrated that the innate resistance of mice to Listeria monocytogenes infection by intravenous or intraperitoneal inoculation is regulated principally by the Hc locus on mouse chromosome 2. The A/J and C57BL/6 mouse strains were identified as prototype L. monocytogenes-susceptible and -resistant strains, respectively. In the present study, we compared the relative susceptibilities of A/J and C57BL/6 mice to intragastric (i.g.) inoculation with L. monocytogenes. The results of our study indicate that A/J mice are significantly more susceptible than C57BL/6 mice to an i.g. challenge with L. monocytogenes. This was reflected in the estimated 50% lethal doses for the two strains (106 and 108 CFU for A/J and C57BL/6 mice, respectively) and a more rapid and severe dissemination of the infection to the spleen and liver in A/J mice than in C57BL/6 mice. Histopathological examination of tissues from the infected mice confirmed the greater severity of disease in A/J mice. Clearance of a primary infection enhanced the resistance of both A/J and C57BL/6 mice to reinfection with L. monocytogenes via the gastrointestinal tract. However, the relative difference in susceptibility between the two strains was evident even after immunization. The A/J mouse holds promise as a model for investigating the pathogenesis of gastrointestinal listeriosis because of its ability to develop systemic infection following challenge with numbers of organisms similar to those recovered from some L. monocytogenes-contaminated food products.

Viability of Escherichia coli O157:H7 in Salami Following Conditioning of Batter, Fermentation and Drying of Sticks, and Storage of Slices

The fate of Escherichia coli O157:H7 was monitored in salami during conditioning of batter, fermentation and drying of sticks, and storage of slices. The raw batter (75% pork: 25% beef, wt/wt, fat content about 20%) was inoculated with a pediococcal starter culture (about 10(8) CFU/g) and a five-strain cocktail of E. coli O157:H7 ( > or = 2 x 10(7) CFU/g) and stuffed into 104-mm diameter fibrous casings. After being refrigerated at 4 degrees C or being tempered at 13 degrees C, frozen at -20 degrees C, and thawed at 4 degrees C, or being frozen at -20 degrees C, and thawed at 4 degrees C, the inoculated batter was fermented at 24 degrees C and 90% relative humidity (RH) to pH < or = 4.8, dried at 13 degrees C and 65% RH to a moisture/protein ratio of < or = 1.9:1, and then stored at 4 or 21 degrees C under air or vacuum. For salami sticks sampled immediately after drying, appreciable differences were evident among the various batter-conditioning treatments; pathogen numbers were reduced from original levels by 2.1, 1.6, or 1.1 log10 units when batter was tempered, frozen, and thawed, frozen and thawed, or refrigerated, respectively. Similarly, regardless of storage temperature or atmosphere, within 7 days salami slices cut from sticks prepared from batter that was tempered, frozen, and thawed (2.7- to 4.9-log10-unit reduction) or frozen and thawed (2.3- to 4.8-log10-unit reduction) displayed a greater impact on pathogen numbers than slices cut from sticks prepared from batter that was refrigerated (1.6- to 3.1-log10-unit reduction). The effects of batter conditioning notwithstanding, a greater reduction in levels of E. coli O157:H7 was observed when slices were stored at 21 degrees C compared to otherwise similar slices stored at 4 degrees C. After storage for 60 days the pathogen was only detected by enrichment in slices stored at 21 degrees C, whereas pathogen levels ranged from 1.4 to 4.5 log10 CFU/g in slices stored at 4 degrees C. Differences related to storage atmosphere were first observed after slices were stored for 21 days. Such differences were more readily demonstrable after 60 and 90 days, with pathogen numbers for treatments that were statistically different ranging from 0.6- to 1.5-log10 units higher on slices stored under vacuum than in air. These data emphasize the need to implement multiple barriers to appreciably reduce numbers of E. coli O157:H7 in salami.

Viability of Escherichia coli O157:H7 in pepperoni during the manufacture of sticks and the subsequent storage of slices at 21, 4 and −20 ° C under air, vacuum and CO2

A raw, pepperoni batter (75% pork:25% beef with a fat content of about 32%) was inoculated with a pediococcal starter culture (about 10(8) cfu/g) and a five-strain cocktail of Escherichia coli O157:H7 (> or = 2 x 10(7) cfu/g), mixed with non-meat ingredients, and then hand-stuffed into 55 mm fibrous casings to form sticks. The numbers of the pathogen were determined before stuffing, after fermentation, after drying/slicing, and after periods of storage. For storage, slices were packaged under air, vacuum or CO2 and stored at -20, 4 and 21 degrees C. Sticks were fermented at 36 degrees C and 85% relative humidity (RH) to < or = pH 4.8 and then dried at 13 degrees C and 65% RH to a moisture/protein ratio (M/Pr) of < or = 1.6:1. Fermentation and drying resulted in the numbers of the pathogen decreasing by about 2 log10 units. During storage, the temperature rather than the atmosphere had the greater effect on pathogen numbers. The greatest reductions in numbers were observed during storage at 21 degrees C, when numbers decreased to about 2 and 3.8 log10 cfu/g within 14 days in product stored under air and vacuum, respectively, and a 5 log10 reduction was observed for both atmospheres within 28 days. Regardless of the storage atmosphere, numbers did not decrease below 3.6 or 3.7 log10 cfu/g after 90 days of storage at -20 or 4 degrees C, respectively. These data confirm that fermentation and drying are sufficient to eliminate only about 2 log10 cfu/g of E. coli O157:H7 from fermented sausage, and that additional strategies, such as storage for at least 2 weeks at ambient temperature in air, are required to achieve a 5 to 6 log10 reduction in the numbers of the pathogen in sliced pepperoni.

The Aryl Hydrocarbon Receptor Is Required for Optimal Resistance to Listeria monocytogenes Infection in Mice

The aryl hydrocarbon receptor (AhR) is part of a powerful signaling system that is triggered by xenobiotic agents such as polychlorinated hydrocarbons and polycyclic aromatic hydrocarbons. Although activation of the AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin or certain polycyclic aromatic hydrocarbons can lead to immunosuppression, there is also increasing evidence that the AhR regulates certain normal developmental processes. In this study, we asked whether the AhR plays a role in host resistance using murine listeriosis as an experimental system. Our data clearly demonstrate that AhR null C57BL/6J mice (AhR−/−) are more susceptible to listeriosis than AhR heterozygous (AhR+/−) littermates when inoculated i.v. with log-phase Listeria monocytogenes. AhR−/− mice exhibited greater numbers of CFU of L. monocytogenes in the spleen and liver, and greater histopathological changes in the liver than AhR+/− mice. Serum levels of IL-6, MCP-1, IFN-γ, and TNF-α were comparable between L. monocytogenes-infected AhR−/− and AhR+/− mice. Increased levels of IL-12 and IL-10 were observed in L. monocytogenes-infected AhR−/− mice. No significant difference was found between AhR+/− and AhR−/− macrophages ex vivo with regard to their ability to ingest and inhibit intracellular growth of L. monocytogenes. Intracellular cytokine staining of CD4+ and CD8+ splenocytes for IFN-γ and TNF-α revealed comparable T cell-mediated responses in AhR−/− and AhR+/− mice. Previously infected AhR−/− and AhR+/− mice both exhibited enhanced resistance to reinfection with L. monocytogenes. These data provide the first evidence that AhR is required for optimal resistance but is not essential for adaptive immune response to L. monocytogenes infection.

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

ABSTRACT Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.

Viability of Escherichia coli O157:H7 in ground and formed beef jerky prepared at levels of 5 and 20% fat and dried at 52, 57, 63, or 68°C in a home-style dehydrator

Beef jerky batter was prepared to fat contents of about 5 and 20% and inoculated with about 10(8) cfu g(-1) of a five-strain inoculum of Escherichia coli O157:H7. Pathogen numbers were determined in the raw batter and in the strips formed from it after drying at 52, 57, 63, and 68 degrees C for times that ranged from 2 to 20 h. For both the high and low fat products, pathogen numbers were reduced by about 5 log10 cfu g(-1) within 4 h drying at 68 degrees C and within 8 h drying at 63 degrees C. At 57 degrees C, a 5-log10-unit reduction was achieved within 10h drying for the 5% fat product and within 16 h drying for the 20% fat product. At 52 degrees C, a 5-log10-unit reduction was achieved within 10 h drying for the 5% fat product and within 20 h drying for the 20% fat product. In at least one of the three trials for all four drying temperatures tested, the pathogen was present following enrichment of the samples in synthetic media. The calculated D values decreased from 2.59, 2.48, 1.23, and 1.17 as the temperature increased from 52, 57, 63, and 68 degrees C and as the fat content decreased from 20 to 5%. However, there was no direct correlation between the moisture-to-protein ratio and either the doneness of the strips or the viability of the pathogen. These data indicate that the fat content and the time and temperature at which strips are dried directly impact the viability of E. coli O157:H7 in ground and formed beef jerky.

Survival of Escherichia coli O157:H7 in Full- and Reduced-Fat Pepperoni after Manufacture of Sticks, Storage of Slices at 4°C or 21°C under Air and Vacuum, and Baking of Slices on Frozen Pizza at 135, 191 and 246°C

Pepperoni batter was prepared with fat contents of about 15, 20, and 32% (wt/wt) and inoculated with a pediococcal starter culture and ≥2.0 X 10 7 CFU/g of a five-strain inoculum of Escherichia coli O157:H7. The batter was fermented at 96°F (ca. 36°C) and 85% relative humidity (RH) to pH 4.8 and then dried at 55°F (ca. 13°c) and 65% RH to a moisture/protein ratio of ≤1.6:1. For storage, slices were packaged under air or vacuum and stored at 39°F (ca. 4°C) and 70°F (ca. 21°C). For baking, frozen slices were placed on retail frozen cheese pizzas that were subsequently baked at 275°F (ca. 135°C), 375°F (ca. 191°C), or 475°F (ca. 246°C) for 0 to 20 min. Appreciable differences related to fat levels were observed after drying; pathogen numbers decreased by 1.04, 1.31 and 1.62 log 10 units in sticks prepared from batter at initial fat levels of 15, 20, and 32%, respectively. During storage, the temperature rather than the atmosphere had the greater effect on pathogen numbers, with similar viability observed among the three fat levels tested. At 70°F (ca. 21°C), compared to original levels, pathogen numbers decreased by ≥5.56 and ≥4.53 log,0 units within 14 days in slices stored under air and vacuum, respectively, whereas at 39°F (ca. 4°C) numbers decreased by ≤2.43 log 10 CFU/g after 60 days of storage under either atmosphere. Baking, as expected, resulted in greater reductions in pathogen numbers as the temperature and/or time of baking increased. However, it was still possible to recover the pathogen by enrichment after baking frozen slices on frozen pizza at 475°F (ca. 246°C) for 10 min or at 375°F (ca. 191°C) for 15 min. The calculated D values for all three temperatures tested increased as the fat content of the batter increased from 15 to 20 to 32%. The present study confirmed that fermentation and drying were sufficient to reduce levels of E. coli O157:H7 in pepperoni sticks by 5.5-log 10 -unit total reduction of the pathogen. Baking slices on frozen pizza for at least 15 min at 475°F (ca. 246°C) or 20 min at 375°F (ca. 191°C) was necessary to reduce pathogen numbers to below detection by both direct plating and enrichment.