MOLECULAR CHARACTERIZATION OF STAPHYLOCOCCUS PSEUDINTERMEDIUS: RESEARCH ON VIRULENCE FACTORS, ANTIBIOTIC-RESISTANCE AND QUORUM-SENSING
Tesi di Dottorato
Data di Pubblicazione:
2020
Citazione:
MOLECULAR CHARACTERIZATION OF STAPHYLOCOCCUS PSEUDINTERMEDIUS: RESEARCH ON VIRULENCE FACTORS, ANTIBIOTIC-RESISTANCE AND QUORUM-SENSING / G. Meroni ; tutor: P. A. Martino ; coordinatore: V. Grieco. DIPARTIMENTO DI MEDICINA VETERINARIA, 2020 Feb 05. 32. ciclo, Anno Accademico 2019. [10.13130/meroni-gabriele_phd2020-02-05].
Abstract:
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 diss
Tipologia IRIS:
Tesi di dottorato
Keywords:
Antibiotic resistance; canine pyoderma; quorum-sensing; silver nanoparticles; Staphylococcus pseudintermedius; virulence genes
Elenco autori:
G. Meroni
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