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 (∼ 100 μg/cm2) 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 ∼0.4 μg/cm2 to ∼23.6 μg/cm2 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 ∼0.4 μg/cm2 of silver in the polymeric films caused a reduction of 6 log10 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 ∼0.4 μg/cm2 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.

A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 ؎ 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.

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.

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.

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 (10 6 and 10 8 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. monocytogenescontaminated food products.

Infection with Listeria monocytogenes during pregnancy is associated with miscarriage, preterm birth, and neonatal complications, including sepsis and meningitis. While the risk of these conditions is thought to be greatest during the third trimester of pregnancy, the determinants of fetoplacental susceptibility to infection, the contribution of gestational age, and the in vivo progression of disease at the maternal-fetal interface are poorly understood. We developed a nonhuman primate model of listeriosis to better understand antecedents of adverse pregnancy outcomes in early pregnancy. Four pregnant cynomolgus macaques (Macaca fascicularis) received a single intragastric inoculation between days 36 and 46 of gestation with 107 CFU of an L. monocytogenes strain isolated from a previous cluster of human listeriosis cases that resulted in adverse pregnancy outcomes. Fecal shedding, maternal bacteremia, and fetal demise were consistently noted within 7 to 13 days. Biopsy specimens of maternal liver, spleen, and lymph node displayed variable inflammation and relatively low bacterial burden. In comparison, we observed greater bacterial burden in the decidua and placenta and the highest burden in fetal tissues. Histopathology indicated vasculitis, fibrinoid necrosis, and thrombosis of the decidual spiral arteries, acute chorioamnionitis and villitis in the placenta, and hematogenous infection of the fetus. Vascular pathology suggests early impact of L. monocytogenes infection on spiral arteries in the decidua, which we hypothesize precipitates subsequent placentitis and fetal demise. These results demonstrate that L. monocytogenes tropism for the maternal reproductive tract results in infection of the decidua, placenta, and the fetus itself during the first trimester of pregnancy.

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.

Mechanisms by which the intestinal epithelium resists invasion by food-borne pathogens such as Listeria monocytogenes are an evolving area of research. Intestinal P glycoprotein is well known to limit the absorption of xenobiotics and is believed to act as a cytotoxic defense mechanism. The aim of this study was to determine if intestinal P glycoprotein is involved in host defense against L. monocytogenes. Caco-2 cells and a Pglycoprotein-overexpressing subclone (Caco-2/MDR) were employed in addition to mdr1a ؊/؊ mice and wildtype controls. In vitro invasion assays and in vivo experiments were employed to measure bacterial invasion and dissemination. In addition, L. monocytogenes proteins were labeled with [35 S]methionine, and the transepithelial transport across Caco-2 monolayers was characterized in both directions. Overexpression of P glycoprotein in Caco-2/MDR cells led to increased resistance to L. monocytogenes invasion, whereas P-glycoprotein inhibition led to increased invasion. Flux of [35 S]methionine-labeled L. monocytogenes proteins was significantly greater in the basolateral-to-apical direction than in the apical-to-basolateral direction, indicating dependence on an apically located efflux transporter. Moreover, inhibiting P glycoprotein reduced the basolateral-to-apical flux of the proteins. Early dissemination of L. monocytogenes from the gastrointestinal tract was significantly greater in the mdr1a ؊/؊ mice than in wild-type controls. Expression and function of intestinal P glycoprotein is an important determinant in resistance to early invasion of L. monocytogenes.P glycoprotein is the 170-kDa product of the human MDR1 gene and is arguably one of the most extensively studied members of the ATP-binding cassette superfamily of transport proteins (28). P glycoprotein is best known for its ability to transport drug substrates out of cells in a variety of tissues, including the intestine (1,12,27). Both the expression and the function of P glycoprotein have been linked to considerable variability in oral drug absorption; however, the precise physiological role of intestinal P glycoprotein is unknown. Because P glycoprotein is well conserved throughout evolution and has a broad substrate affinity, it is widely believed to act as a cytotoxic protection mechanism. Given its apical distribution on the enterocyte, P glycoprotein is exquisitely positioned to limit the absorption of substances that the cell perceives as harmful. Thus, it is conceivable that P glycoprotein restricts the absorption of other, nondrug substances in the intestine. Proteins facilitating invasion of pathogenic bacteria would be ideal candidates given the purported mechanism of action of P glycoprotein.Listeria monocytogenes is a food-borne pathogen responsible for considerable morbidity and mortality (11,24). Although multiple sites of invasion have been proposed, the vanguard of the body's interaction with L. monocytogenes is the intestinal epithelial barrier. Intestinal epithelial cells come in contact with not only the bacter...