Vertebrate secreted RNases (ribonucleases) are small proteins that play important roles in RNA metabolism, angiogenesis or host defence. In the present study we describe the antimicrobial properties of the N-terminal domain of the hcRNases (human canonical RNases) and show that their antimicrobial activity is well conserved among their lineage. Furthermore, all domains display a similar antimicrobial mechanism, characterized by bacteria agglutination followed by membrane permeabilization. The results of the present study show that, for all antimicrobial hcRNases, (i) activity is retained at the N-terminus and (ii) the antimicrobial mechanism is conserved. Moreover, using computational analysis we show that antimicrobial propensity may be conserved at the N-terminus for all vertebrate RNases, thereby suggesting that a defence mechanism could be a primary function in vertebrate RNases and that the N-terminus was selected to ensure this property. In a broader context, from the overall comparison of the peptides' physicochemical and biological properties, general correlation rules could be drawn to assist in the structure-based development of antimicrobial agents.

A study was made of the staphylococcal population on the skin and on the nasal mucosa and in the milk of 133 healthy goats. Of a total of 346 strains isolated and characterised as belonging to the genus Staphylococcus, 74 (21.4 %) were coagulase-positive (68 S. aureus and 6 S. hyicus), and 272 (78.6 %) coagulase-negative. The novobiocin-sensitive species S. huemolyticus (23.5 YO), S. wurneri (16.5 '%), S. epidermidis (1 1.8 YO), S. chromogenes (8.5 Yo), S. caprue (6.6 Yo) and S. hyicus (2.6 YO), and the novobiocin-resistant species S. xylosus (8.5 %), S. sczurz (7.4 %), S. saprophyticus (4.8 %), S. cohnii (2.2 "YO) , S. lentus (1.1 I % ) , S. equorum (1.1 Yo) and S. kloosii (1.1 "/") were identified. Twelve (4.4 %) of coagulase-negative strains remained unidentified. Strains isolated in the skin of the udder and teats of the 133 goats were mainly novobiocin-sensitive coagulase-negative staphylococci, the most prevalent species being S. huemolyticus, S. wurneri and S. epidermidis. Staphylococci indicative of subclinical infection were determined in the milk of 47 (35.3 %) of the 133 goats sampled. U.S.Samples were taken from 133 female Verata goats from 11 different extensive flocks in the province of Ciceres (Spain). All animals were in lactation, and were clinically healthy. SamplesSamples were collected from the nasal mucosa and the skin of the axillary fold, the udder surface and the teat, on sterile moist swabs. Milk (50 mi) was collected in sterilized containers, after washing and disinfecting the teats. All samples were rapidly transferred to the laboratory in an isothermic container at 4 "C. Mucosa and skin samples were streaked from the swabs onto sheep blood agar (5 %) (Oxoid). Milk samples (loo& were spread onto sheep blood agar (5%) (Oxoid) and Schleifer and Krimer's staphylococci selective medium (SCHLEIFER and KRAMER, 1980). Plates were incubated in aerobic conditions at 37°C for 24-72 hours. Colonies whose cell morphology resembled that of Gram-positive, catalase-positive cocci, were selected. O n e or two selected colonies were used to obtain purified cultures on Brain Heart Infusion agar (Difco). MicroorganismsA total of 359 strains of gram-positive, catalase-positive cocci were tested. Differentiation of staphylococci and minococci

The ability of 342 staphylococcal isolates from different anatomical sites in healthy goats to produce staphylococcal enterotoxins (SE) was investigated. SE were produced by 74.3% of the 70 coagulase-positive strains and by 22% of the coagulase-negative strains studied. Most enterotoxigenic strains were isolated from the skin of udders and teats and from milk. SEC was the SE type most frequently produced, either alone (67.9%) or in combination with others. Five coagulase-negative species not previously reported as SE producers were identified (Staphylococcus chromogenes, S. warneri, S. sciuri, S. saprophyticus, and S. lentus). SEA, SEB, and SEC were detected in the milk of 17 of the 133 healthy goats studied. These results suggest that the goat is an important reservoir of enterotoxigenic staphylococci, most of which produce SEC.

Objectives To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus biofilms. Methods Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths. Results Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified. Conclusions Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.