MOLECULAR CHARACTERIZATION OF STAPHYLOCOCCUS PSEUDINTERMEDIUS: RESEARCH ON VIRULENCE FACTORS, ANTIBIOTIC-RESISTANCE AND QUORUM-SENSING

Staphylococcus pseudintermedius (S. pseudintermedius) is one of the most important pathogens in small animal veterinary medicine. In the dog, it is a commensal bacterium able to cause severe diseases (mainly dermatological), which rapidly evolve to persistent and chronic infections. This pathogen is also considered a public health problem due to the rapid evolution and spread of antibiotic-resistant clones, in particular, the worldwide dissemination of Multi-Drug Resistant (MDR) strains and methicillin-resistant S. pseudintermedius (MRSP). Nowadays, its zoonotic potential is under constant investigations. The first chapter of the thesis centers the attention on the typing techniques used to study a population of S. pseudintermedius isolated from the skin of dogs and raw milk of Holstein-Fresian dairy cattle. The aims of this study were the investigation of the genetic relatedness of different S. pseudintermedius strains using three molecular typing techniques; the Ribosomal Spacers Amplification PCR (RSA-PCR), the Random Amplification of Polymorphic DNA PCR (RAPD-PCR) and the Restriction Fragment Length Polymorphism PCR (RAPD-PCR). Moreover, the dissemination of species-specific genes coding for exfoliative toxins (siet, expA, and expB), enterotoxin (seCcanine) and a bicomponent leukocidin (lukS-F) was studied to understand the virulence of the strains. The study was carried out on a final population of 40 S. pseudintermedius strains isolated from 42 clinical cases of deep canine pyoderma and from 5 samples of raw milk from Holstein-Fresian dairy cattle with intramammary infection. After the microbiological examination and phenotypic typing, 47 S. pseudintermedius strains were collected and genetically analyzed. RSA-PCR revealed the presence of a unique biotype. The dendrogram obtained from RAPD-PCR disclosed two important results: i) the staphylococcal strains isolated from dogs and cows are genetically similar underlying the ability of this pathogen to colonize also dairy cattle; ii) the presence of a single cluster grouping all the S. pseudintermedius strains. Based on the presence of a specific restriction site in the phosphotransacetylase (pta) gene, RFLP-PCR confirmed the presence of 40 S. pseudintermedius and 7 non-S. pseudintermedius strains. The sequencing unveiled the effective presence of one strain of S. aureus and one of E. faecalis, 5 strains of S. schleiferi and 40 of S. pseudintermedius. All the S. pseudintermedius isolates were positive for siet gene, while expA and expB were amplified by, respectively, 10% and 17,5%. Specific enterotoxin seCcanine and leukotoxin luk-S-F were found with a prevalence of 35% and 92,5%, respectively. Therefore, the combined use of molecular typing techniques and virulence factors screening allows to better characterize S. pseudintermedius and to understand its zoonotic potential. The second chapter points out the attention to the problem of antibiotic resistance. Frequently, in Veterinary and Human medicine too, wide-spectrum antibiotics are prescribed to face with bacteria-based diseases without the microbiological examination and relative antibiotic-resistance screening. This could lead to the selection and spread of antibiotic-resistant strains. The aim of this work was to study the antibiotic-resistance profile of a large population of S. pseudintermedius strains collected in two Italian veterinary teaching hospitals, the first in Milan (Università degli Studi di Milano) and the second in Naples (Università degli Studi di Napoli “Federico II”) One-hundred sixteen and one-hundred twenty-six strains were collected in Milan and Naples, respectively. A panel of 10 different antibiotic molecules was tested with the Kirby-Bauer assay, qualitative PCR was used to describe the dissemination of mecA and tetM/K genes. The results showed a high rate of resistance for amoxicillin+clavulanate, clindamycin, tobramycin and tetracycline also confirmed by statistical significance. The prevalence of methicillin-resistant strains (MRSP) was 30% in Milan and18% in Naples also confirmed by the amplification of mecA gene. The cohort of methicillin-sensible strains in Naples (N= 103) was significantly more resistant compared to that from Milan (N= 81). In relation to the MDR population, all the MRSP in Milan and 91% in Naples are considered resistant to three or more pharmaceutical categories. These results totally agree with the recent report about the “Surveillance of antimicrobial resistance in Europe, 2017” and could be a starting point to set out a clearer use and prescription of antibiotics. The third chapter describes quorum-sensing and biofilm. It is known that bacteria are able to communicate via small peptides (Gram-positive) or lipids (Gram-negative) using a system called quorum-sensing. This complex behavior acts in response to external stimuli (e.g. cell density) and controls gene expression. Biofilm is one of the products of this communication, defined as a complex ecosystem of bacteria (sometimes also fungi and algae) enclosed in a self-produced matrix and adherent to a biotic or abiotic surface. This structure is frequently resistant to commonly used antibiotics and causes severe and more complex re-infections. The aim of the work was to describe the biofilm-forming ability of MDR and non-MDR S. pseudintermedius strains investigating the dissemination of virulence factors and antibiotic-resistance genes. A panel of 22 antibiotics was used to screen the antibiotic-resistance profiles of 73 S. pseudintermedius strains isolated from deep canine pyoderma. The biofilm-forming ability was investigated using a microtiter assay. The results clearly highlight a correlation between antibiotic-resistance and the ability to produce biofilm. All the MDR strains (N=42) are biofilm producers while 27 out of 31 non-MDR strains were able to produce biofilm. The last chapter of the thesis focused on an alternative strategy for the treatment of bacterial infections caused by Gram-positive and Gram-negative bacteria. For centuries, silver was used for its antimicrobial properties. Recently, the nanobiotechnology research area aimed to synthesize small particles (less than 100 nm) of silver metal to use as an alternative to traditional antibiotic-based therapies. The aim of this work was to synthesize silver nanoparticles using i) a Curcuma longa infusion and ii) the supernatant of E. coli. Furthermore, the antibacterial properties of synthesized nanoparticles were determined against 10 S. pseudintermedius strains and 10 Pseudomonas aeruginosa isolates by Kirby-Bauer disk diffusion assay and via Minimum Inhibitory Concentration (MIC). The anti-biofilm capacities were also investigated using an in vitro model of biofilm growth. Results showed that silver nanoparticles exert strong antibacterial activity compared to antibiotic alone and these properties are magnified when antibiotic was adjuvated with silver. Nanoparticles are also able to disrupt mature biofilm by directly linking to the cell wall of bacteria causing bacterial lysis. The use of silver nanoparticles obtained with green technology (e.g plant or organism derived molecules) is an eco-friendly alternative to classical antibiotic-based therapies and can be used to fight against both Gram-positive and Gram-negative infections.