Penetration and replication of Listeria monocytogenes within intestinal epithelial cells were studied by infecting the human enterocyte-like cell line Caco-2. Entry was due to directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D on bacterial entry and by electron microscopy showing bacteria inside membrane-limiting vacuoles at the early stage of infection. Only bacteria from pathogenic species (L. monocytogenes and Listeria ivanovii) were able to induce their own phagocytosis by Caco-2 cells, as opposed to Listeria seeligeri, Listeria welshimeri, and Listeria innocua. L. monocytogenes multiplied readily within Caco-2 cells, with an apparent generation time of about 90 min. Listeriolysin 0 was found to be a major factor promoting intracellular growth of L. monocytogenes. After being internalized at the same rate as that of its hemolytic revertant strain, a nonhemolytic mutant from L. monocytogenes failed to replicate significantly within Caco-2 cells. Electron microscopic study demonstrated that bacteria from the nonhemolytic mutant remained inside phagosomes during cellular infection, whereas hemolytic bacteria from L. monocytogenes were released free within the cytoplasm. This indicates that disruption of vacuole membranes by listeriolysin 0-producing strains of L. monocytogenes might be a key mechanism allowing bacteria to escape from phagosomes and to multiply unrestricted within cell cytoplasm.

Food-borne Listeria monocytogenes is a serious threat to human health, and new strategies to combat this opportunistic pathogen in foods are needed. Bacteriophages are natural enemies of bacteria and are suitable candidates for the environmentally friendly biocontrol of these pathogens. In a comprehensive set of experiments, we have evaluated the virulent, broad-host-range phages A511 and P100 for control of L. monocytogenes strains Scott A (serovar 4b) and WSLC 1001 (serovar 1/2a) in different ready-to-eat (RTE) foods known to frequently carry the pathogen. Food samples were spiked with bacteria (1 ؋ 10 3 CFU/g), phage added thereafter (3 ؋ 10 6 to 3 ؋ 10 8 PFU/g), and samples stored at 6°C for 6 days. In liquid foods, such as chocolate milk and mozzarella cheese brine, bacterial counts rapidly dropped below the level of direct detection. On solid foods (hot dogs, sliced turkey meat, smoked salmon, seafood, sliced cabbage, and lettuce leaves), phages could reduce bacterial counts by up to 5 log units. Variation of the experimental conditions (extended storage over 13 days or storage at 20°C) yielded similar results. In general, the application of more phage particles (3 ؋ 10 8 PFU/g) was more effective than lower doses. The added phages retained most of their infectivity during storage in foods of animal origin, whereas plant material caused inactivation by more than 1 log 10 . In conclusion, our data demonstrate that virulent broad-host-range phages, such as A511 and P100, can be very effective for specific biocontrol of L. monocytogenes in contamination-sensitive RTE foods.Listeria monocytogenes is an opportunistic human pathogen, widely distributed in the environment and transmitted to humans and animals via contaminated foods (50). The organism is well adapted to very different environmental conditions encountered in foods; it tolerates high levels of salt content (10 to 20%), can grow at pH values below 6, with low oxygen, and at temperatures down to 1°C (48). L. monocytogenes causes listeriosis, a severe disease which may result in septicemia, meningitis, encephalitis, or loss of the fetus during pregnancy (52). Although listeriosis is comparatively rare compared to other food-borne infections, the high mortality rate of 15 to 40% is of great concern (49, 52). Cases of sporadic and epidemic listeriosis are increasing (4, 6, 11), and it has been estimated that about 2,000 hospitalizations and 500 deaths occur annually in the United States as a result of the consumption of Listeria in foods (39). Although many foods can serve as vehicles for this pathogen, Listeria was often isolated from ready-to-eat (RTE) foods, such as milk and cheeses, cold-cut meats, smoked fish, seafood, and vegetables (45). RTE foods have been implicated in most of the major listeriosis outbreaks in the last 30 years (13,18,20,21,42,(45)(46)(47). Of particular concern is the fact that they are consumed directly, without a final bactericidal processing step. Since the preservation methods applicable to minimally processed RTE foods of...

We developed a rapid in vitro test for determining the association of Campylobacter jejuni and C. coli with HeLa cells. Association was expressed as a weighted mean of the number of bacteria associated with one cell in an association index (AI). The reproducibility of the AI was checked by repeating the test six times, using four strains chosen at random. Means and standard deviations of the means were 7.3 +/- 1.2, 6.8 +/- 0.9, 1.8 +/- 1.2, and 0.1 +/- 0.2. The experimental conditions for which the results are reliable have been standardized. Among 42 strains from human feces, two groups appeared: for 22 nonassociative strains (52%), AI values ranged from 0.0 to 2.1 (mean +/- SD, 0.5 +/- 0.6); for 20 associative strains (48%), AI values ranged from 3.5 to 8.3 (mean +/- SD, 6.2 +/- 1.4). Of these 42 strains, 17 were clinically documented. Diarrhea occurred more frequently in patients infected with associative strains than in those infected with noninvasive strains (7/7 versus 3/10, P = 0.01). Fever also occurred more frequently in patients infected with associative strains (6/7 versus 2/10, P = 0.03). Transmission electron microscopy and viable counts made after killing of extracellular bacteria by gentamicin support the fact that associated Campylobacter spp. are adherent to the cell membrane and are internalized into cytoplasmic vacuoles. The described test seems to be a convenient and rapid method for estimating the pathogenicity of a given strain.

No abstract

Listeria monocytogenes is an invasive bacterial pathogen capable of multiplying inside many host cells, including macrophages, enterocytes and hepatocytes. There is evidence to believe that secretion of listeriolysin O, an SH-activated exotoxin, is crucial for bacterial growth in host tissues. This exotoxin is stimulated in iron-deprived medium and mostly active at low pH (5.5). Electron microscopic studies showed that intracellular bacteria rapidly disrupt the vacuole membrane of phagosomes and freely multiply inside the cytosol of infected cells, thus escaping at an early stage of infection from the cellular microbicidal mechanisms. Vacuole disruption does not occur with a nonhemolytic mutant obtained by insertion of a single copy of transposon Tn1545 in the structural gene of listeriolysin O. These results strongly suggest that listeriolysin O is a major factor promoting intracellular growth of L. monocytogenes and that intracellular growth of virulent bacteria is initiated after escaping from the phagosomal compartment.