Helicobacter pylori biofilm: a protective environment for bacterial recombination.

AIMS: The aim of this work was to investigate the interaction between two Helicobacter pylori strains in promoting genetic transfer, when grown in the biofilm mode. METHODS AND RESULTS: Biofilms produced by H. pylori 9/10 (A), H. pylori 15/4 (B) and their mixture (C) were studied for biomass production and cell viability. The genetic heterogeneity of 45 clones, coming from mature biofilm of co-cultured H. pylori strains was studied by both RAPD and cagA (EPIYA motifs)/vacA virulence genes analysis. Helicobacter pylori A, B and C developed a well-structured biofilm without significant differences in viability. No significant differences were recorded between A and B biomass measurement, whereas C biofilm expressed a significant (P < 0.001) higher adhesive capability when compared with A and B biofilms. C-clones DNA-fingerprintings showed an high genetic heterogeneity (mean similarity value = 0.528). The 60% of C-clones displayed vacA allelic combination s1i1m1m2 associated with cagA EPIYA motif pattern P1P2P3P3P3. CONCLUSIONS: Biofilms developed by multiple H. pylori strains are more complex than those associated with single strains. Such condition might promote the genetic exchange favouring the generation of more virulent strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The 'biofilm niche' represents a successful strategy and a suitable environment for promoting bacterial population persistence by recombination events.

Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour.

AIMS: This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development. METHODS AND RESULTS: Extracellular DNA was purified and characterized in a 2-day-old mature biofilm developed by the reference strain H. pylori ATCC 43629, the clinical isolate H. pylori SDB60 and the environmental strain H. pylori MDC1. Subsequently, the role of eDNA in the H. pylori biofilm was evaluated by adding DNase I during biofilm formation and on mature biofilms. Extracellular DNA was detected in the 2-day-old EPS biofilm matrix of all analysed H. pylori strains. The DNA fingerprintings, performed by RAPD analysis, on eDNA and intracellular DNA (iDNA), showed some remarkable differences. The data obtained by microtitre biofilm assay as well as colony forming unit count and CLSM (confocal laser scanning microscopy) qualitative analysis did not show any significant differences between the DNase I-treated biofilms and the corresponding not treated controls both in formation and on mature biofilms. CONCLUSIONS: In this study, we provide evidence that eDNA is a component of the EPS matrix of H. pylori biofilm. The different profiles of eDNA and iDNA indicate that lysed cells are not the primary source of eDNA release, suggesting that other active mechanisms might be involved in this process. Moreover, the biomass assay suggests that eDNA may not be the main component of biofilm matrix, suggesting that it could be primarily involved in other mechanisms such as recombination processes, via transformation, contributing to the wide genomic variability of this micro-organism defined as a 'quasi-species'. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of eDNA in H. pylori biofilm can contribute to the active dynamic exchange of information aimed to reach the best condition for the bacterial survival in the host and in the environment.

5-lipoxygenase and cyclooxygenase-2 in porcine parasitic bronchopneumonia: immunohistochemical and biochemical investigations.

Eicosanoids are products of arachidonic acid metabolism and have numerous biological roles. The present study aimed to investigate the role of 5-lipoxygenase (5-LOX)- and cyclooxygenase-2 (COX-2)- dependent enzymatic pathways in the pathogenesis of porcine parasitic bronchopneumonia caused by Metastrongylus spp. Pulmonary tissue samples from healthy control and parasitized pigs were processed for histopathological, immunohistochemical and biochemical investigations. In control animals, immunohistochemistry demonstrated that 5-LOX and COX-2 expression was almost exclusively limited to the bronchiolar epithelial cells. Parasitized pigs had greater 5-LOX- and COX-2- specific immunoreactivity, involving a wide range of cell types within foci of granulomatous and eosinophilic bronchopneumonia. Biochemical investigations demonstrated the presence of 5-LOX (and the related product Leukotriene B(4)) and COX-2 (and the related product prostaglandin E(2); PGE(2)) in all tissues under study. COX-2 activity and PGE(2) concentration were significantly higher in diseased lungs compared with normal healthy controls. These findings demonstrate that 5-LOX and COX-2 are differentially expressed in normal versus lungworm-infected lungs and therefore suggest that both biochemical pathways are likely to be involved in the pathogenesis of porcine parasitic bronchopneumonia.

Ileal tract and Peyer's patch innervation in scrapie-free versus scrapie-affected ovines.

Ileal Peyer's patches (PPs) are involved early during sheep scrapie infection. This study qualitatively and semi-quantitatively evaluated ileal tract and PP innervation in 29 Sarda ovines of different age, PrP genotype and scrapie status. A prominent network of fibres was detected within PPs, mainly located in interfollicular lymphoid and stromal components. Intrafollicular fibres were rarely observed, with no apparent differences between scrapie-free and scrapie-affected animals, or among ovines carrying different PrP genotypes. In adult sheep, independent of their scrapie status, nerve fibres could be detected infrequently, close to the follicle-associated epithelium. Fibres were also detected within newly formed follicles and intrafollicular microgranulomas.

Lipoprotein Lp(a) and cerebrovascular disease in the elderly: correlations with the severity of extracranial carotid atherosclerosis assessed by ultrasonography.

High levels of lipoprotein Lp(a) are related to cerebrovascular disease clinical manifestations, as well as to the severity of extracranial carotid atherosclerosis assessed by ultrasonography. In order to investigate the relationship of Lp(a) to the severity of carotid atherosclerosis in the elderly, 100 subjects, aged 78.5 +/- 0.6 yrs underwent an echo-color-doppler scanning of carotids; atherosclerosis severity, assessed as maximum percentage stenosis, presence of complicated plaque and Intima-Media Thickness (IMT), was related to Lp(a) levels, assayed by an immunoenzymatic procedure. A slight association between Lp(a) and CVD clinical manifestations was apparent only in subjects under 78 yrs and for Lp(a) values above 25 mg/dL. Lp(a) levels were not related either to the degree of stenosis, the presence of complicated plaque, or IMT. As for other selected risk factors, while no relationship was found for clinical CVD and IMT, the maximum percentage of stenosis and the presence of complicated plaques were positively related to LDL-cholesterol in subjects under 78 yrs. We can conclude that Lp(a), albeit unrelated to the severity of extracranial vessel atherosclerosis, maintains a role as cerebrovascular risk factor in the elderly, being slightly related to clinical manifestations; however its discriminant power is lower than in middle-aged people and further decreases throughout ageing.