The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control.

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot's matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food.

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.

Antibiotic resistances of starter and probiotic strains of lactic acid bacteria.

The antibiotic resistances of 45 lactic acid bacteria strains belonging to the genera Lactobacillus, Streptococcus, Lactococcus, Pediococcus, and Leuconostoc were investigated. The objective was to determine antibiotic resistances and to verify these at the genetic level, as is currently suggested by the European "qualified presumption of safety" safety evaluation system for industrial starter strains. In addition, we sought to pinpoint possible problems in resistance determinations. Primers were used to PCR amplify genes involved in beta-lactam antibiotic, chloramphenicol, tetracycline, and erythromycin resistance. The presence of ribosomal protection protein genes and the ermB gene was also determined by using a gene probe. Generally, the incidences of erythromycin, chloramphenicol, tetracycline, or beta-lactam resistances in this study were low (<7%). In contrast, aminoglycoside (gentamicin and streptomycin) and ciprofloxacin resistances were higher than 70%, indicating that these may constitute intrinsic resistances. The genetic basis for ciprofloxacin resistance could not be verified, since no mutations typical of quinolone resistances were detected in the quinolone determining regions of the parC and gyrA genes. Some starter strains showed low-level ampicillin, penicillin, chloramphenicol, and tetracycline resistances, but no known resistance genes could be detected. Although some strains possessed the cat gene, none of these were phenotypically resistant to chloramphenicol. Using reverse transcription-PCR, these cat genes were shown to be silent under both inducing and noninducing conditions. Only Lactobacillus salivarius BFE 7441 possessed an ermB gene, which was encoded on the chromosome and which could not be transferred in filter-mating experiments. This study clearly demonstrates problems encountered with resistance testing, in that the breakpoint values are often inadequately identified, resistance genes may be present but silent, and the genetic basis and associated resistance mechanisms toward some antibiotics are still unknown.