Aims: A rapid detection system specific for Listeria monocytogenes and based on the polymerase chain reaction (PCR) was developed. Methods and Results: Primers annealing to the coding region of the actA gene, critically involved in virulence and capable of discrimination between two different alleles naturally occurring in L. monocytogenes, have been utilized. The procedure was applied to recover L. monocytogenes cells in artificially contaminated fresh Italian soft cheeses (mozzarella, crescenza and ricotta). Low levels of L. monocytogenes were detected in mozzarella and crescenza homogenates (0·04–0·4 and 4 CFU g−1, respectively) whereas in ricotta the detection limit was higher (40 CFU g−1). Conclusions: This PCR-based assay is highly specific as primers used recognize the DNA from different L. monocytogenes strains of clinical and food origin, while no amplification products result with any other Listeria spp. strains. Significance and Impact of the Study: This study highlighted a low-cost and rapid procedure that can be appropriated for the detection in real time of low L. monocytogenes levels in soft cheese.
In Listeria monocytogenes the acid tolerance response (ATR) takes place through a programmed molecular response which ensures cell survival under unfavorable conditions. Much evidence links ATR with virulence, but the molecular determinants involved in the reactivity to low pHs and the behavior of acid-exposed bacteria within host cells are still poorly understood. We have investigated the effect of acid adaptation on the fate of L. monocytogenes in human macrophages. Expression of genes encoding determinants for cell invasion and intracellular survival was tested for acid-exposed bacteria, and invasive behavior in the human myelomonocytic cell line THP-1 activated with gamma interferon was assessed. Functional approaches demonstrated that preexposure to an acidic pH enhances the survival of L. monocytogenes in activated human macrophages and that this effect is associated with an altered pattern of expression of genes involved in acid resistance and cell invasion. Significantly decreased transcription of the plcA gene, encoding a phospholipase C involved in vacuolar escape and cell-to-cell spread, was observed in acid-adapted bacteria. This effect was due to a reduction in the quantity of the bicistronic plcA-prfA transcript, concomitant with an increase in the level(s) of the monocistronic prfA mRNA(s). The transcriptional shift from distal to proximal prfA promoters resulted in equal levels of the prfA transcript (and, as a consequence, of the inlA, hly, and actA transcripts) under neutral and acidic conditions. In contrast, the sodC and gad genes, encoding a cytoplasmic superoxide dismutase and the glutamate-based acid resistance system, respectively, were positively regulated at a low pH. Morphological approaches confirmed the increased intracellular survival and growth of acid-adapted L. monocytogenes cells both in vacuoles and in the cytoplasm of interferon gamma-activated THP-1 macrophages. Our data indicate that preexposure to a low pH has a positive impact on subsequent challenge of L. monocytogenes with macrophagic cells.
The effects of 12 fatty acids, naturally occurring in milk from several mammalian species, on the survival and invasion ability of Listeria monocytogenes, a food‐borne pathogen, were determined. The survival was tested in the presence of 200 μg ml−1 fatty acids suspended in brain hearth infusion broth or in storage conditioning solution (NaCl 1%) of Mozzarella cheese, an Italian soft unripened cheese, at pH 7·0 or 5·0. Lauric (C12:0), linoleic (C18:2) and linolenic (C18:3) acids exerted the strongest bactericidal activity. The invasive efficiency of L. monocytogenes, determined in the Caco‐2 enterocyte‐like cell line, was strongly decreased in the presence of the fatty acids tested (from about 20 to 500‐fold). This research suggests that naturally occurring fatty acids of milk, supplemented in milk derivatives, could affect both bacterial growth and invasiveness and consequently, could serve as barriers towards L. monocytogenes infection.
We developed a method of identification of Listeria monocytogenes based on colony hybridization with nonradioactively labeled DNA probes, represented by the hly and inlA virulence-associated genes. The procedure described in this paper results simple, rapid, specific and reproducible. Since it can be performed in a short time, the above technique can be applied to detect L. monocytogenes from different source and constitutes a noteworthy and alternative tool to identify this gram-positive pathogenic bacterium.
Invasion of gingival and junctional epithelial cells has been recently proposed as a potentially relevant mechanism in the pathogenesis and recurrence of periodontal disease. The gram negative anaerobe Prevotella nigrescens was shown to be involved in the development of periodontal lesions in man, suggesting a possible involvement of invasivity as a mean to circumvent the host immune surveillance and other hostile factors. Appropriately designed invasion assays demonstrated that P. nigrescens efficiently invades human epithelial cells, through a mechanism whose efficiency is influenced by the phase of growth, by the multiplicity of infection, and by the cell line used, and that requires microfilament integrity, but is not affected by an impairment of microtubule organization. Intracellular replication assays suggested that P. nigrescens probably multiplies within Kb epithelial cells, causing extensive cell alterations. Invasion of gingival epithelial cells could consequently be a basic step in the virulence mechanism of the species.
Incorporation of labelled precursors into neutral lipids and phospholipids of hypertrophied guinea-pig cardiac ventricles during normobaric hypoxia (breathing gas mixture of N2: O2, 9: 91%) was studied. The lipids of the heart were isotopically labelled in vivo with [1-14C] palmitate and [2-3H] glycerol, and, after a period of 2 hours, the incorporation of the two injected precursors into mitochondrial and microsomal fractions was determined. Eighty hours after the onset of the hypoxic treatment, the specific activities of isolated lipids showed a more elevated incorporation into lipid classes of hypoxic right and left ventricles with respect to that of the control. For both precursors, the percent increase of incorporation into microsomal and mitochondrial neutral lipids was higher than into phospholipids. In the microsomal fraction the increase of 14C-radioactivity incorporation was more pronounced than 3H-incorporation. 3H/14C incorporation ratio appeared decreased in hypoxic ventricles. The data in this experiment suggest that the net increase of 3H-glycerol and 14C-palmitate incorporation into myocardial lipid classes was a compensatory response of the heart to the hypoxic stimulus.
This study analysed the invasiveness of Listeria monocytogenes into enterocyte-like Caco-2 cells in which iron depletion was achieved by picolinic acid treatment. Both entry and intracellular multiplication varied depending on the endogenous iron content of bacterial and eukaryotic cells. The behaviour within enterocytes was correlated with a 10-fold increased transcription of the actA gene observed in bacterial cells grown under conditions of iron stress.
