Penetration of Escherichia coli O157:H7 into iceberg lettuce tissues and the effect of chlorine treatment on cell viability were evaluated. Attachment of different inoculum levels (10(9), 10(8), and 10(7) CFU/ml) was examined by determining the number of cells at the surface and the cut edge of lettuce leaves (2 by 2 cm). E. coli O157:H7 attached preferentially to cut edges at all inoculum levels, with greater attachment per cm2 of lettuce at higher inoculum levels. A longer attachment time allowed more cells to attach at both sites. Immunostaining with a fluorescein isothiocyanate-labeled antibody revealed that cells penetrated into lettuce leaves from cut edges. Cells showed greater penetration when lettuce was held at 4 degrees C compared with 7, 25, or 37 degrees C and were detected at an average of 73.5 +/- 16.0 microm below the surfaces of cut tissues. Penetrating cells were mostly found at the junction of lettuce cells. The viability of attached cells after treatment with 200 mg/liter (200 ppm) of free chlorine for 5 min was examined by plating on tryptic soy agar and by a nalidixic acid elongation method. Although chlorine treatment caused significant reduction in attachment (0.7- and 1.0-log reduction at surfaces and cut edges, respectively), cells remained attached at high numbers (7.9 and 8.1 log CFU/cm2 at surfaces and cut edges, respectively). Elongated cells were observed in stomata and within the tissues of the lettuce, indicating they were protected from contact with chlorine.

Attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens on iceberg lettuce was evaluated by plate count and confocal scanning laser microscopy (CSLM). Attachment of each microorganism (approximately 10(8) CFU/ml) on the surface and the cut edge of lettuce leaves was determined. E. coli O157:H7 and L. monocytogenes attached preferentially to cut edges, while P. fluorescens attached preferentially to the intact surfaces. Differences in attachment at the two sites were greatest with L. monocytogenes. Salmonella Typhimurium attached equally to the two sites. At the surface, P. fluorescens attached in greatest number, followed by E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium. Attached microorganisms on lettuce were stained with fluorescein isothiocyanate and visualized by CSLM. Images at the surface and the cut edge of lettuce confirmed the plate count data. In addition, microcolony formation by P. fluorescens was observed on the lettuce surface. Some cells of each microorganism at the cut edge were located within the lettuce tissues, indicating that penetration occurred from the cut edge surface. The results of this study indicate that different species of microorganisms attach differently to lettuce structures, and CSLM can be successfully used to detect these differences.

A dose-response model using rhesus monkeys as a surrogate for pregnant women indicates that oral exposure to 10 7 CFU of Listeria monocytogenes results in about 50% stillbirths. Ten of 33 pregnant rhesus monkeys exposed orally to a single dose of 10 2 to 10 10 CFU of L. monocytogenes had stillbirths. A log-logistic model predicts a dose affecting 50% of animals at 10 7 CFU, comparable to an estimated 10 6 CFU based on an outbreak among pregnant women but much less than the extrapolated estimate (10 13 CFU) from the FDA-U.S. Department of Agriculture-CDC risk assessment using an exponential curve based on mouse data. Exposure and etiology of the disease are the same in humans and primates but not in mice. This information will aid in risk assessment, assist policy makers, and provide a model for mechanistic studies of L. monocytogenes-induced stillbirths.

We examined (i) the persistence of Escherichia coli O157:H7 as a postpasteurization contaminant in fermented dairy products; (ii) the ability of E. coli O157:H7 strains with and without the general stress regulatory protein, RpoS, to compete with commercial starter cultures in fermentation systems; and (iii) the survival of E. coli O157:H7 in the yogurt production process. In commercial products inoculated with 10(3) CFU/ml, E. coli O157:H7 was recovered for up to 12 days in yogurt (pH 4.0), 28 days in sour cream (pH 4.3), and at levels > 10(2) CFU/ml at 35 days in buttermilk (pH 4.1). For the starter culture competition trials, the relative inhibition of E. coli O157:H7 in the experimental fermentation systems was, in decreasing order, thermophilic culture mixture, Lactobacillus delbrueckii subsp. bulgaricus R110 alone, Lactococcus lactis subsp. lactis D280 alone, Lactococcus lactis subsp. cremoris D62 alone, and Streptococcus thermophilus C90 alone showing the least inhibition. Recovery of the rpoS mutant was lower than recovery of its wild-type parent by 72 h or earlier in the presence of individual starter cultures. No E. coli O157:H7 were recovered after the curd formation step in yogurt manufactured with milk inoculated with 10(5) CFU/ml. Our results show that (i) postprocessing entry of E. coli O157:H7 into fermented dairy products represents a potential health hazard; (ii) commercial starter cultures differ in their ability to reduce E. coli O157:H7 CFU numbers in fermentation systems; and (iii) the RpoS protein appears to most effectively contribute to bacterial survival in the presence of conditions that are moderately lethal to the cell.

Listeria monocytogenes, isolated from outbreaks in either human or nonhuman primate populations, was administered orally at doses ranging from 10 6 to 10 10 CFU. Four of 10 treated animals delivered stillborn infants. L. monocytogenes was isolated from fetal tissue, and the pathology was consistent with L. monocytogenes infection as the cause of pregnancy loss. For all pregnancies resulting in stillbirths, L. monocytogenes was isolated from maternal feces, indicating that L. monocytogenes had survived and had probably colonized the gastrointestinal tract. Antibodies and antigen-specific lymphocyte proliferation against Listeria increased in animals that had stillbirths.Listeriosis resulting from exposure to food containing the bacterium L. monocytogenes causes serious disease, with case fatality rates between 20 and 40% (33). Listeriosis is especially serious in susceptible populations such as immunocompromised persons and pregnant women (11,14,16,20,24,26,29,32,35). For healthy nonpregnant adults, listeriosis has a relatively low incidence, presumably due to its low infectivity in immunocompetent individuals.Pregnancy-related listeriosis primarily affects the fetus or neonate. The maternal reaction to the presence of Listeria infection is generally an influenza-like episode with fever, backache, and perhaps diarrhea (7,11,13,24,29). The effect of fetal Listeria infection is dependent on the point in gestation time when infection occurs. First-trimester infection leads to spontaneous abortion, whereas second-and third-trimester infections lead to preterm birth followed by neonatal illness or fetal death with preterm delivery of a stillborn (7,11,13).The rhesus monkey (Macaca mulatta), with a reproductive cycle and placenta comparable to those of humans (31), is widely used as an experimental model for human reproduction and development. As with humans, exposure to L. monocytogenes in pregnant nonhuman primates may result in abortions, stillbirths, or neonatal deaths (4, 27; J. Paul-Murphy, J. E. Markovits, I. Wesley, and J. A. Roberts, Lab. Anim. Sci. 40:547 [abstr.], 1990). For humans and nonhuman primates, the pathogenesis and morphological findings associated with stillbirths due to L. monocytogenes are essentially the same (1,4,28,37).Despite several epidemiological studies confirming the relationship between L. monocytogenes and specific foods (soft cheeses, undercooked chicken, paté, etc.) (2, 30), an infectious dose has not been established for healthy or susceptible human populations due to the delay between exposure and the onset of symptoms. The severe ramifications of the disease in highrisk human populations such as pregnant women precludes the use of humans in volunteer feeding studies. Recently, a draft risk assessment of L. monocytogenes in ready-to-eat foods (36) reviewed human epidemiological and animal study data. The risk assessment concluded that mouse studies provide the only acceptable data for developing dose-response information at this time and acknowledged the difficulty with the use of...