Comparative characterisation of human and ovine non-aureus staphylococci isolated in Sardinia (Italy) for antimicrobial susceptibility profiles and resistance genes.

We present the comparative characterisation of 195 non-aureus staphylococci (NAS) isolates obtained from sheep (n = 125) and humans (n = 70) in Sardinia, Italy, identified at the species level by gap gene polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis with AluI. Isolates were tested phenotypically with a disc diffusion method and genotypically by PCR, for resistance to 11 antimicrobial agents including cationic antiseptic agents. Among the ovine isolates, Staphylococcus epidermidis (n = 57), S. chromogenes (n = 29), S. haemolyticus (n = 17), S. simulans (n = 8) and S. caprae (n = 6) were the most prevalent species, while among human isolates, S. haemolyticus (n = 28) and S. epidermidis (n = 26) were predominant, followed by S. lugdunensis and S. hominis (n = 4). Of the 125 ovine isolates, 79 (63.2%) did not carry any of the resistance genes tested, while the remainder carried resistance genes for at least one antibiotic. The highest resistance rates among ovine isolates were recorded against tetracycline (20.8%), and penicillin (15.2%); none was resistant to methicillin and two exhibited multidrug resistance (MDR); one of which was positive for the antiseptic resistance smr gene. By contrast, most human isolates (59/70, 84.3%) were resistant to ⩾1 antimicrobials, and 41 (58.6%) were MDR. All 52 (74.3%) penicillin-resistant isolates possessed the blaZ gene, and 33 of 70 (47.1%) harboured the mec gene; of these, seven were characterised by the Staphylococcal Chromosomal Cassette (SCCmec) type IV, 6 the type V, 5 of type III and one representative each of type I and type II. The majority (57.1%) was erythromycin-resistant and 17 isolates carried only the efflux msrA gene, 11 the methylase ermC gene and an equal number harboured both of the latter genes. Moreover, 23 (32.8%) were tetracycline-resistant and all but one possessed only the efflux tetK gene. qacA/B and smr genes were detected in 27 (38.6%) and 18 (25.7%) human NAS, respectively. These results underline a marked difference in species distribution and antimicrobial resistance between ovine and human-derived NAS.

Biofilm formation and virulence factor analysis of Staphylococcus aureus isolates collected from ovine mastitis.

AIMS: To perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines. METHODS AND RESULTS: The potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly-ß and hly-γ), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 71·3% of the Staph. aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (61·6%) possessed all lukD-E/lukM/lukPV83 genes, 123 (47·7%) harboured both hly-ß/hly-γ genes, whereas almost all (97·3%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (69·4%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins. CONCLUSIONS: In this search, several putative virulence determinants have been identified in ovine Staph. aureus isolates collected in Sardinia. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis.

Native pears of Sardinia affect Penicillium expansum pathogenesis.

Penicillium expansum causes blue mould rot, a serious post-harvest disease of pome fruits and is the main producer of the mycotoxin patulin. The occurrence of natural resistance against different hostpathogens, has been evidenced in some pear accessions of the Sardinian germoplasm. The aim of this research was to correlate P. expansum growth and patulin production on these indigenous pear accessions. In vitro and in vivo experiments were carried out with seven accessions ('Sarmentina', 'Vacchesa', 'De Puleu', 'De su Duca', 'Natalina', 'Oliena', 'Laconi 5') belonging to the CNR-ISPA ex situ collection and one national control cultivar ('Abate'). A wild type P. expansum from our collection was isolated from blue mould-decayed Sardinian pear fruit and selected for its aggressiveness and patulin production. The in vivo assay was carried out using 5 x 2 cm (Ø x thickness) sterilized fruit discs wounded and inoculated by a 10(5)UFC/mL concentration of P. expansum. Fruit discs were incubated at 23 degrees C for 7 days before analysis. The in vitro experiments, aimed at monitoring over time P. expansum mycelial growth and patulin accumulation, were performed with a standard medium (PDA) and a pear puree Agar Medium (PAM). Petri dishes with PDA and PAM were inoculated centrally with P. expansum conidia (10(5)UFC/ml) and then incubated at 23 degrees C for 7 days. Mycelial growth on Sardinian PAMs was inhibited in comparison to 'Abate' PAM and PDA. In particular, the accessions 'Sarmentina' and 'Vacchesa' showed the maximum inhibitory activity both in vitro and in vivo. Patulin production was detected by high-pressure liquid chromatography-mass spectrometry. The mycotoxin concentration in Sardinian PAMs was lower than that detected in PDA medium, pointing out a positive correlation between fungal growth inhibition and patulin production. The lowest concentration of patulin was found in 'Sarmentina' PAM. Based on these findings, some of Sardinian pear accessions seems to affect P. expansum pathogenesis and inhibit patulin production. Further researches are necessary to assess the mechanism of this biocontrol activity.