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 (Universit\ue0 degli Studi di Milano) and the second in Naples (Universit\ue0 degli Studi di Napoli \u201cFederico II\u201d) 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 \u201cSurveillance of antimicrobial resistance in Europe, 2017\u201d 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

Performance of a large limited streamer tube cell in drift mode

The performance of a large (3x3 $cm^2$) streamer tube cell in drift mode is shown. The detector space resolution has been studied using cosmic muons crossing an high precision silicon telescope. The experimental results are compared with a GARFIELD simulation.Comment: 18 pages, 7 figures. Accepted by Nucl. Instr. and Methods

Synthesis of 2-substituted-6-hydroxy and 6-methoxy benzothiazoles from 1,4-benzoquinone

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UR 3.13 - MAXIMUM OBSERVABLE SHAKING (MOS) MAPS OF ITALY

The main goal of UR 3.13 is to establish a work flow for a multi-layer map that includes the seismicity of Italy in terms of Maximum Observable Shaking (MOS), and the near-field/far-field boundaries (NF/FF) with respect to the major seismogenic faults mapped within the DISS database. Here we will discuss only the procedure to derive the MOS-map of Italy. Our approach merges updated knowledge on the Italian regional tectonic setting and on the Source Zone (SZ) definition and broadband scenario-like calculation of expected maximum shaking on a given area. For a given SZ, broadband ground shaking is computed for a rupture model derived from a Maximum Credible Earthquake (MCE) and its associated Typical Fault (TF). Amplitude spectra for deterministic Low Frequency and stochastic High Frequency waveforms are reconciled at intermediate frequency, where their domain of validity overlaps, to derive broadband synthetics and compute the associated shaking. As the MCE and TF float along the SZ, broadband ground motion is computed at each point surrounding the given fault and the maximum among observable shaking according to that scenario is plotted on the MOS map. So far the procedure was entirely successfully tested on the Macro Region MR4 (central-northern Apennine), while more detailed analysis is done on the MCE and TF suggested for the Colfiorito earthquake. Here our broadband ground motion scenario shows, besides a complex pattern of variation, a southwestern area of high PGA values, at about 20 km distance from the fault, likely associated to with the properties of the spatio-temporal complexity of the rupture process. For the purpose of the project a complete new map of SZ and MCE is under compilation, grouping seismogenic sources according to Mw and faulting mechanisms. This goal can be achieved most efficiently by targeted numerical simulations that cover the parameter range of interest (in terms of magnitude and distance etc) and consider a large suite earthquake rupture scenarios

Implementation of the Hierarchical Reference Theory for simple one-component fluids

Combining renormalization group theoretical ideas with the integral equation approach to fluid structure and thermodynamics, the Hierarchical Reference Theory is known to be successful even in the vicinity of the critical point and for sub-critical temperatures. We here present a software package independent of earlier programs for the application of this theory to simple fluids composed of particles interacting via spherically symmetrical pair potentials, restricting ourselves to hard sphere reference systems. Using the hard-core Yukawa potential with z=1.8/sigma for illustration, we discuss our implementation and the results it yields, paying special attention to the core condition and emphasizing the decoupling assumption's role.Comment: RevTeX, 16 pages, 2 figures. Minor changes, published versio

High sensitivity double beta decay study of 116-Cd and 100-Mo with the BOREXINO Counting Test Facility (CAMEO project)

The unique features (super-low background and large sensitive volume) of the CTF and BOREXINO set ups are used in the CAMEO project for a high sensitivity study of 100-Mo and 116-Cd neutrinoless double beta decay. Pilot measurements with 116-Cd and Monte Carlo simulations show that the sensitivity of the CAMEO experiment (in terms of the half-life limit for neutrinoless double beta decay) is (3-5) 10^24 yr with a 1 kg source of 100-Mo (116-Cd, 82-Se, and 150-Nd) and about 10^26 yr with 65 kg of enriched 116-CdWO_4 crystals placed in the liquid scintillator of the CTF. The last value corresponds to a limit on the neutrino mass of less than 0.06 eV. Similarly with 1000 kg of 116-CdWO_4 crystals located in the BOREXINO apparatus the neutrino mass limit can be pushed down to m_nu<0.02 eV.Comment: 29 pages, LaTex, 9 eps figure

A WebGIS tool for the dissemination of earthquake data

In 2004 a new seismic hazard map of Italy (MPS Working Group, 2004) has been released by a task force that produced an amount of new or updated data, such as a new version of the earthquake catalogue (CPTI04; CPTI Working Group, 2004) and an updated seismogenic zonation. A set of WebGIS tools has been designed for the data dissemination to the scientific community and the general public. The design of the WebGIS tools focused first on the consultation of the DBM04 macroseismic database (DBM Working Group, 2005), which contains the macroseismic intensity data-points (IDP) of the earthquakes listed in the CPTI04 catalogue. The WebGIS tool design and development process had to fulfill: 1) simplicity, 2) responsiveness and 3) readiness for future extensions. The specific requirements for the macroseismic database consultation interface were: - data access by place and by earthquake; - IDP maps with queryable points; - data download in both tabular and map format; - easily upgradable content; - quick and user friendly interface

Deliverable 1 # A3.13.1-2-3-4-5

We investigate wave motion through numerical simulations that take into account primarily the ground acceleration in response to a given earthquake rupture that radiates seismic waves. The shaking that potential sources might cause is plotted on maps that provide a general overview of the hazard over a large area, and that can be used as the starting point for further detailed investigations. Here, we establish a procedure to compute ground motion that spans the entire frequency range of engineering interest (i.e., broad-band), and we derive the maximum shaking that is caused by expected earthquakes throughout Italy (i.e. the maximum observable shaking; MOS). Our approaches merge updated knowledge of the Italian regional tectonic setting and of source-zone definitions (Valensise and Pantosti, 2001; Basili et al., 2008) and scenario-like calculations of the expected MOS in any given area

Biofilm-forming ability and virulence factors of methicillin-resistant Staphylococcus pseudintermedius from canine pyoderma

Staphylococcus pseudintermedius (SP) has been associated with high antibiotic-resistance rates (e.g. methicillin) in European countries [1,3]. This condition could be also related to the ability to produce biofilm [2]. The aim of this study was to investigate the presence of methicillin-resistant SP strains and determine their ability to produce biofilm and some crucial virulence factors. Forty-two SP strains, previously determined as multi drug resistant (MDR) by the disk diffusion method using a panel of 17 antimicrobial agents, were selected from our collection and tested phenotypically for the minimum inhibitory concentration (MIC) of methicillin and genotypically for the presence of mecA and blaZ genes. The ability to produce biofilm was assessed phenotypically by two different assays: the Congo Red Agar plates (CRA) and the Microtitre Plate test (MtP) and genetically by the amplification of icaA and icaD genes. Three virulence factors genes coding for bicomponent leukocidin and enterotoxins (luk-I, seC, se-int) were searched. Twenty-three strains revealed a value of MIC for the methicillin greater than 128 µg/mL. The 83% were mecA-positive and 86% resulted blaZ-positive; all the strains positive for mecA were also positive for blaZ. All SP strains resulted biofilm-producers by MtP assay and classified as weakly producers (4.7%), moderate producers (47.6 %) and strongly producers (47.6 %). In contrast, only 35.7% of all strains were considered biofilm-producers by CRA method. The amplification of icaA and icaD gene occurred respectively in 66.6% and 97.6%; only one strain was negative for both genes. Almost all strains were positive for luk-I (95%), seC (74%) and se-int (84%). Our data reveal the pathogenicity potential of SP strains from dogs, suggesting that they could be considered zoonotic potential agents and confirming other previous studies [3-5]. Moreover could be observed a clear linkage between antibiotic-resistance and ability to produce biofilm

Deliverable 2 # A3.13.8

We examine possibilities to delineate the boundaries between near-field and far-field radiation of seismic waves. Near-field (NF), intermediate-field (IF) and far-field (FF) terms represent different properties of the seismic wave-field: the near-source motions are sensitive to the spatio-temporal details of the rupture process, while far-field terms tend to carry the overall signature of the rupture. Due to the longer propagation path of far-field waves through complex Earth structure, their waveform properties also depend more strongly on media properties (scattering; intrinsic attenuation), than it is the case for the NF-wavefield