Phenotypic and genotypic study on antibiotic resistance and pathogenic factors of Staphylococcus aureus isolates from small ruminant mastitis milk in South of Italy (Sicily).

Staphyloccoccus aureus is the major cause of mastitis in small ruminants in the Mediterranean farms causing severe losses to dairy industry. Antibiotic treatment has been the most common approach to control these infections. Aim of this study was to investigate antimicrobial resistance (AMR), virulence factors and biofilm-related genes of 84 Sicilian strains of S. aureus isolated from sheep and goats milk during two different periods δT1 (2006-2009) and δT2 (2013-2015). Kirby Bauer method and Polymerase Chain Reaction (PCR) were utilized to monitor AMR and related genes (mecA, tetK, tetM, ermA, ermC). Moreover, toxin genes (tsst-1, sea-see, seg-sej, and sep) and biofilm genes (bap, ica, sasC) were studied. Twenty-six isolates (30.9%) showed multidrug resistance. The two groups showed similar results with exception for higher values of resistance for tilmicosin and lower for sulfamethoxazole and vancomycin of the second group. MecA gene was detected in one isolate. Tetracycline resistance was higher than 20%, with an increase in δT2 group. Toxin genes were found in 5 isolates (5.9%), belonging of δT2 group, while 57 of isolates (67.8%) showed biofilm related genes. The high presence of multi-resistant isolates suggests the need of more responsible use of antibiotic therapy for the control of these infections.

Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants.

Contagious agalactia (CA), an infectious disease of small ruminants, caused by Mycoplasma agalactiae, is responsible for severe losses to dairy sheep production with substantial socioeconomic impacts on small-scale farmers. The diagnosis of CA is still problematic, time-consuming and requires well-equipped labs for confirmation of outbreaks. Therefore, rapid, accurate and cost-effective diagnostic tests are urgently needed. This work aims to validate a novel Loop-Mediated Isothermal Amplification (LAMP) test, based on the p40 target gene, for the detection of M. agalactiae in dairy sheep in order to confirm its potential practical use as a rapid and cheap field test. The LAMP system proposed in this study consists of a portable device composed of real-time fluorometer with the automatic interpretation of results displayed in a tablet. A total of 110 milk samples (90 positives and 20 negatives) were analysed to optimise the analysis procedure and to investigate the efficacy and robustness of the LAMP method. All samples were analysed using LAMP and conventional real-time PCR to compare the diagnostic sensitivity of the methods. The sensitivity of the LAMP was 10-fold higher than that of real-time PCR, with a detection limit up to 103 CFU/ml. The LAMP assay was able to detect M. agalactiae in 81 of 90 (90%, 95%CI 0.84-0.96) positive milk samples compared to 69 (77%, 95%CI 0.59-0.95) positive samples detected by real-time PCR; no positive signal occurred for any of the negative milk samples in either test. Therefore, the LAMP assay was found to be more sensitive than real-time PCR, low-cost, easy to perform, fast and not affected by contamination, indicating its potential as an effective diagnostic tool in the field level for the diagnosis of CA.

A New Approach to Predict the Fish Fillet Shelf-Life in Presence of Natural Preservative Agents.

Three data sets concerning the behaviour of spoilage flora of fillets treated with natural preservative substances (NPS) were used to construct a new kind of mathematical predictive model. This model, unlike other ones, allows expressing the antibacterial effect of the NPS separately from the prediction of the growth rate. This approach, based on the introduction of a parameter into the predictive primary model, produced a good fitting of observed data and allowed characterising quantitatively the increase of shelf-life of fillets.

Evaluation of the Antibacterial Activity of Bergamot Essential Oils on Different Listeria Monocytogenes Strains.

Essential oils are aromatic and volatile substances extracted from plants and characterized by antimicrobial activity. The aim of the present study was to evaluate the antibacterial activity (agar disc-diffusion method) of seven different bergamot essential oils (BEOs) on eight Listeria monocytogenes strains. Minimal inhibitory concentration (MIC) of most efficient BEOs was estimated. Extremely variable results for agar disc-diffusion method for L. monocytogenes strains were reported. One of the tested microorganisms resulted insensible to all the BEOs; 3 strains showed an inhibition from weak to null and the remaining 4 a variable susceptibility. Among the BEOs tested, one showed a strong activity against four pathogenic strains. Four BEOs revealed weak, moderate or null activity in all the 7 sensitive strains, while for two oils only a weak or no activity was reported. MIC values were 0.625 µL/mL for the most efficient BEO, 2.5 and 5 µL/mL for the other samples that showed moderate inhibition. Experiment results are significantly related to the strains tested (P<0.01), rather than the BEO employed (P>0.01). In conclusion, we can consider BEO as a natural technological hurdle for Listeria monocytogenes in combination with other preservation strategies. Finally, this study underlines the necessity to evaluate the antimicrobial activity of EOs on a significant strains number of the same bacteria.