Background: Clinical and experimental observations in animal models indicate that intestinal commensal bacteria are involved in the initiation and amplification of inflammatory bowel disease (IBD). No paediatric reports are available on intestinal endogenous microflora in IBD. Aims: To investigate and characterise the predominant composition of the mucosa-associated intestinal microflora in colonoscopic biopsy specimens of paediatric patients with newly diagnosed IBD. Methods: Mucosa-associated bacteria were quantified and isolated from biopsy specimens of the ileum, caecum and rectum obtained at colonoscopy in 12 patients with Crohn's disease, 7 with ulcerative colitis, 6 with indeterminate colitis, 10 with lymphonodular hyperplasia of the distal ileum and in 7 controls. Isolation and characterisation were carried out by conventional culture techniques for aerobic and facultative-anaerobic microorganisms, and molecular analysis (16S rRNA-based amplification and realtime polymerase chain reaction assays) for the detection of anaerobic bacterial groups or species. Results: A higher number of mucosa-associated aerobic and facultative-anaerobic bacteria were found in biopsy specimens of children with IBD than in controls. An overall decrease in some bacterial species or groups belonging to the normal anaerobic intestinal flora was suggested by molecular approaches; in particular, occurrence of Bacteroides vulgatus was low in Crohn's disease, ulcerative colitis and indeterminate colitis specimens. Conclusion: This is the first paediatric report investigating the intestinal mucosa-associated microflora in patients of the IBD spectrum. These results, although limited by the sample size, allow a better understanding of changes in mucosa-associated bacterial flora in these patients, showing either a predominance of some potentially harmful bacterial groups or a decrease in beneficial bacterial species. These data underline the central role of mucosa-adherent bacteria in IBD.

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.

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 (0AE04-0AE4 and 4 CFU g )1 , respectively) whereas in ricotta the detection limit was higher.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.

Bovine lactoferrin (BLf) and its derivative peptide lactoferricin B (LfcinB) are known for their antimicrobial activity towards several pathogens, including Listeria monocytogenes, a food-borne Gram-positive invasive bacterium that infects a wide variety of host cells, including professional phagocytes. To add further information on the antibacterial effects of these compounds, the influence of BLf, LfcinB and the antimicrobial centre of LfcinB, the hexapeptide LfcinB 4-9 , on the invasive behaviour of L. monocytogenes was analysed in IFN-ª-activated human macrophagic cells (THP-1). Significant inhibition of bacterial entry in THP-1 cells was observed at LfcinB concentrations that were unable to produce any bacteriostatic or bactericidal effect, compared with BLf and LfcinB 4-9 peptide. This inhibition occurred when LfcinB was incubated during the bacterial infection step and was not due only to competition for common glycosaminoglycan receptors. Assays performed through a temperature shift from 4 to 37 8C showed that inhibition of invasion took place at an early post-adsorption step, although an effect on a different step of intracellular infection could not be ruled out. INTRODUCTIONListeria monocytogenes is an invasive Gram-positive bacterium associated with severe food-borne diseases in humans that infects a wide variety of host cells, including nonprofessional and professional phagocytic cells (especially macrophages) (Vazquez-Boland et al., 2001). C3bi and C1q complement receptors are involved in uptake by phagocytic cells (Alvarez-Dominguez et al., 1993;Drevets et al., 1993), although non-opsonic receptor-ligand interactions also occur (Pierce et al., 1996). The bacterial surface protein ActA, essential for intracellular actin-based motility, may be a L. monocytogenes ligand that mediates recognition of heparan sulphate proteoglycan receptors present on the surface of both professional and non-professional phagocytes (Alvarez-Dominguez et al., 1997; Henry-Stanley et al., 2003).Naturally occurring antimicrobial substances that are resistant to gastric fluid and capable of preventing listerial infection at the level of enterocytes and/or macrophages may be useful in hindering the spread of this pathogen to host tissues. Among biologically active milk proteins, lactoferrin (Lf), a multifunctional iron-binding protein found in milk and colostrum, as well as in various other secretions (Vorland, 1999), and lactoferricin (Lfcin), a 25-residue, pepsin-generated Lf fragment (Tomita et al., 1994), have been shown to possess antibacterial activity towards Gramnegative and Gram-positive bacteria, including L. monocytogenes (Arnold et al., 1980;Payne et al., 1990;Tomita et al., 1994;Branen & Davidson, 2000). Decreased L. monocytogenes invasion of human intestinal cultured cells in the presence of Lf from bovine milk (BLf) (Antonini et al., 1997) has been correlated to an interaction with listerial surface proteins (Conte et al., 1999).Although different mechanisms of Lf antimicrobial defence have been identified, th...

Listeria monocytogenes, an intracellular facultative food-borne pathogen, was reported to induce apoptosis in vitro and in vivo in a variety of cell types with the exception of murine macrophages. These cells represent the predominant compartment of bacterial multiplication and die as a result of necrosis. In this study we showed that human non-activated and IFN-gamma-activated macrophagic-like (THP-1) cells infected with L. monocytogenes, mainly die by necrosis rather than by an apoptotic process. Two natural products derived from bovine milk, lactoferrin and its derivative peptide lactoferricin B, are capable of regulating the fate of infected human macrophages. Bovine lactoferrin treatment of macrophages protects them from L. monocytogenes-induced death whereas lactoferricin B, its derivative peptide, determines a shifting of the equilibrium from necrosis to apoptosis.