Carlo Pala

Antibiotic Resistance Traits and Molecular Subtyping of Staphylococcus aureus Isolated from Raw Sheep Milk Cheese

UNLABELLED The main objective of the present research was to evaluate the antibiotic resistance profiles of Staphylococcus aureus isolated from raw sheep milk cheese. A total of 150 strains were isolated from curd cheese samples and identified as S. aureus. The survey on antibiotic resistance was carried out on 47 strains, selected among isolates showing differences in the banding pattern after Pulsed Field Gel Electrophoresis (PFGE) screening or, belonging at the same pulsotype but isolated from different cheese samples. On selected strains antimicrobial resistance against ampicillin, penicillin, cloxacillin, tetracycline, erythromycin, and vancomycin was assessed by broth microdilution method. The presence of the genes coding for antibiotic resistance and virulence factors (agr alleles, sea-see, and tst) was also investigated by PCR. Thirty-one isolates belonging to agrI and agrIII groups carried at least one gene coding for enterotoxins or toxic shock syndrome toxin. Approximately 60% of the selected strains were susceptible to the tested antibiotics. Twelve of 47 isolates showed multiple resistance against ampicillin and penicillin. Only 1 strain, represented by a unique PFGE profile showed simultaneous resistance to ampicillin, penicillin and cloxacillin. Single resistance against tetracycline was found in 5 isolates belonging to 2 different pulsotypes. The results of this study suggest that the recovery of S. aureus resistant strains in raw milk cheese samples is quite common but it is limited to few antibiotic classes, mainly β-lactams and tetracyclines. None of the strains showed resistance to erythromycin and vancomycin. PRACTICAL APPLICATION The present research contributes to increase the knowledge on the diffusion of antibiotic resistant S. aureus strains isolated from raw sheep milk cheeses. These can be regarded as a vehicle for the introduction of strains of animal origin to humans through food.

Microbiological challenge testing for Listeria monocytogenes in ready-to-eat food: a practical approach

Food business operators (FBOs) are the primary responsible for the safety of food they place on the market. The definition and validation of the product’s shelf-life is an essential part for ensuring microbiological safety of food and health of consumers. In the frame of the Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs, FBOs shall conduct shelf-life studies in order to assure that their food does not exceed the food safety criteria throughout the defined shelf-life. In particular this is required for ready-to-eat (RTE) food that supports the growth of Listeria monocytogenes. Among other studies, FBOs can rely on the conclusion drawn by microbiological challenge tests. A microbiological challenge test consists in the artificial contamination of a food with a pathogen microorganism and aims at simulating its behaviour during processing and distribution under the foreseen storage and handling conditions. A number of documents published by international health authorities and research institutions describes how to conduct challenge studies. The authors reviewed the existing literature and described the methodology for implementing such laboratory studies. All the main aspects for the conduction of L. monocytogenes microbiological challenge tests were considered, from the selection of the strains, preparation and choice of the inoculum level and method of contamination, to the experimental design and data interpretation. The objective of the present document is to provide an exhaustive and practical guideline for laboratories that want to implement L. monocytogenes challenge testing on RTE food.

Breast Nodular Fasciitis: A Comprehensive Review

Background: This article describes the demographic, clinical, pathological and prognostic features of breast nodular fasciitis through a comprehensive review of the cases reported in modern literature. Material and Methods: English-language articles published from January 1970 to October 2015 and related to breast nodular fasciitis were non-systematically retrieved using the PubMed database. Titles, abstracts and references were evaluated in order to include the most relevant studies. The demographic characteristics of the patients and the signs and symptoms of the disease were reviewed, as well as the results of the radiological and diagnostic procedures employed and of the treatments adopted. Results: 26 papers reporting on 28 cases were enrolled; however, manuscripts published before 1990 were excluded because of old methodologies, technologies and clinical approaches. Thus, 22 cases included in 20 papers were analyzed in detail. Conclusion: The rarity and the unspecific clinical and radiological characteristics of breast nodular fasciitis make the differential diagnosis and management challenging. Knowledge of the clinical, pathological and prognostic aspects of this condition is crucial for breast care specialists in order to improve their diagnostic and therapeutic interventions.

Axillary lymph node dissection in patients with breast cancer and sentinel lymph node micrometastasis or isolated tumor cells: is it necessary?

AIMS AND BACKGROUND Sentinel lymph node biopsy is the standard method for axillary lymph node staging in patients with early stage breast cancer. The aim of the study was to evaluate the necessity of axillary lymph node dissection in patients with breast cancer and sentinel lymph node micrometastasis or isolated tumor cells. METHODS Sentinel lymph node biopsy was performed in 136 patients for breast cancer staging: 16 of them (11.7%) were found to have micrometastasis or isolated tumor cells and underwent axillary lymph node dissection. Micrometastases were considered when tumor invasion was ≤ 2 mm and >0.2 mm in diameter and isolated tumor cells when detected alone or in clusters of <0.2 mm in diameter. The dissection of the three axillary lymph node levels of Berg was performed in all cases. RESULTS Two patients (12.5%) presented isolated tumor cells and 14 (87.5%) micrometastasis in the sentinel lymph node. Among them, 25% presented nonsentinel axillary lymph node tumor invasion, whereas 75% had no further nodal involvement. CONCLUSIONS Results suggest that micrometastasis or isolated tumor cells of the sentinel lymph node represent the only site of cancer involvement of the axilla, especially in patients with early breast tumors, and that axillary lymph node dissection may be unnecessary in these cases and represent an overtreatment.

Listeria spp. and Listeria monocytogenes contamination in ready-to-eat sandwiches collected from vending machines

Ready-to-eat (RTE) food is characterised by a long shelf-life at refrigerated temperature and can be consumed as such, without any treatment. The aim of the work was to evaluate the presence of Listeria spp. and Listeria monocytogenes in RTEs collected from refrigerated vending machines placed in hospital environment and accessible to the hospitalised patients. In 4 different sampling, 55 RTEs were collected from vending machines of six hospitals located in different areas of Sardinia region. All the samples were characterised by similar manufacturing process, such as the use of modified atmosphere packaging and belonged to 5 different producers. Listeria spp. was not countable using the enumeration method in all of the analysed samples. Using the detection method, Listeria spp. was recovered from 9 sandwich samples. Interestingly, 3 of these samples (5.5%) made by the manufacturer, were positive for L. monocytogenes contamination. The risk related to the L. monocytogenes presence in RTEs proportionally increases when food is introduced in susceptible environments, such as hospitals and consumed by susceptible people. Although the RTEs analysed showed values that complied with the European microbiological criteria for foodstuffs, the availability of these products in a susceptible environment should be carefully checked. Therefore, in order to limit the possible exposition to L. monocytogenes, more information on the risk related to RTE consumption should be provided to the hospitalised patients.

Inactivation of Listeria monocytogenes using Water Bath Heat Treatment in Vacuum Packed Ricotta Salata Cheese Wedges

UNLABELLED Ricotta salata cheese is frequently contaminated on the surface with Listeria monocytogenes. Water bath heat treatment in vacuum packed whole ricotta salata cheese wheels demonstrated to be effective in inactivating L. monocytogenes. However, the risk of cross-contamination in ricotta salata wedges is increased during cheese cutting. Therefore, the effectiveness of heat treatment in ricotta salata wedges has to be demonstrated conducting a new validation study. In this study, 9 different time temperature combinations, 75, 85, and 90 °C applied for 10, 20, and 30 min each, were tested on artificially contaminated ricotta salata cheese wedges. The extent of the lethal effect on L. monocytogenes was assessed 1 and 30 d after the application of the hot water bath treatment. Five of 9 combinations, 75 °C for 30 min, 85 °C for 20, and 30 min, and 90°C for 20 and 30 min, demonstrated to meet the process criteria of at least 5 log reduction. Sensory analyses were also conducted in order to account for the potential impact on sensory features of ricotta salata wedges, which showed no significant differences between treatments. PRACTICAL APPLICATION This study allowed to select water bath heat treatments of vacuum packed ricotta salata wedges effective to reduce L. monocytogenes contamination. Such treatments can be successfully applied by food business operator to meet compliance with microbiological criteria through the designated shelf-life.

Proteomic analysis of human plasma and peripheral blood mononuclear cells in Systemic Lupus Erythematosus patients

Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease with a broad spectrum of clinical presentations and incompletely understood pathogenesis. This autoimmune disease is characterized by alterations in both the innate and adaptive immune system that lead to the loss of immunologic tolerance. In autoimmune diseases particularly in SLE, early diagnosis, flare or remission phases can be difficult to identify. Proteomics can help to find new therapeutic targets and it also could help to better understand the cellular mechanisms. The aim of this study was to observe the variations in plasma and Peripheral Blood Mononuclear Cells (PBMCs) proteome in order to increase our knowledge about pathogenesis and to find possible diagnostic markers and/or therapeutic targets for improving diagnosis and treatment. The comparative proteomic analyses showed that several proteins were differentially expressed in the PBMCs from SLE patients. Among these, PRDX2 may be used as candidate biomarker or target protein for further investigations. In plasma, we showed that plasma clusterin levels increased in SLE patients compared to healthy controls, but this increase is not statistically significant. These proteomic results provide suggestions for understanding the molecular mechanisms of SLE, as well as the physiological changes correlated with SLE disease.

Evolution of the microbiological profile of vacuum-packed ricotta salata cheese during shelf-life

Ricotta salata cheese is a salted variety of ricotta traditionally made in Sardinia (Italy) from the whey remaining after the production of Pecorino Romano protected designation of origin or other sheep milk cheeses. Ricotta salata cheese is very critical for the possible growth of pathogenic and spoilage microorganisms. Sporadic cases of listeriosis associated with ricotta salata cheese have been reported over recent years. The objective of the present study was to assess the evolution of spoilage and pathogen microorganism of vacuum-packed ricotta salata cheese during the entire product shelf-life. The durability study was conducted on 18 vacuum-packed ricotta salata cheese samples analysed at the beginning of the shelf-life and after 60 and 90 days of refrigerated storage. Pathogens as Listeria monocytogenes and Bacillus cereus were never detected. During shelf-life total bacterial counts ranged between 7.90±0.64 and 9.19±0.58 CFU g-1 on the rind and between 2.95±0.68 and 4.27±1.10 CFU g-1 in the inner paste, while Enterobacteriaceae ranged between 4.22±0.66 and 5.30±0.73 CFU g-1 on the rind and 3.13±1.80 and 2.80±0.88 CFU g-1 in the inner paste. By considering the technology used, the intrinsic properties and the almost total absence of competing microflora, ricotta salata cheese can support the growth of spoilage and pathogen microorganisms originating from the processing environment. The high level of total bacterial counts and Enterobacteriaceae observed both on the rind and in the inner paste suggests contamination of the product from the processing environment. Therefore, a strict implementation of hygiene during processing is essential in order to reduce the load of environmental contaminants that may grow during refrigerated storage.

Radiofrequency Ablation in the Management of Advanced Stage Thymomas: A Case Report on a Novel Multidisciplinary Therapeutic Approach

We describe in this report a case of successful radiofrequency ablation of an unresectable stage III-type B3 thymoma, and we discuss the role of this novel approach in the management of patients with advanced stage thymoma. The patient, a 59-year-old Caucasian male underwent neoadjuvant chemotherapy with only a slight reduction of the mass. Subsequently, an explorative sternotomy and debulking were performed; before closing the thorax, radiofrequency ablation of the residual tumor was carried out and a partial necrosis of the mass was achieved. A further percutaneous radiofrequency ablation was performed subsequently, obtaining complete necrosis of the lesion. Successively, the patient underwent adjuvant radiotherapy. As a result of this multidisciplinary treatment, complete and stable response was obtained. It is hard to say which of the single treatments had the major impact on cure; nevertheless, the results obtained suggest that radiofrequency ablation must be taken into account for the treatment of advanced stage thymomas, and its effectiveness must be further assessed in future studies.

Shelf life evaluation of ricotta fresca sheep cheese in modified atmosphere packaging

Ricotta fresca cheese is the product of Sardinian dairy industry most exposed to microbial post-process contamination. Due to its technological characteristics, intrinsic parameters, pH (6.10-6.80) and water activity (0.974-0.991), it represents an excellent substrate for the growth of spoilage and pathogenic microorganisms, which are usually resident in cheese-making plants environments. Generally, ricotta fresca has a shelf life of 5-7 days. For this reason, at industrial level, modified atmosphere packaging (MAP) is used to extend the durability of the product. However, few investigations have been conducted to validate the use of MAP in ricotta fresca. The aim of this work is to evaluate the shelf life of ricotta fresca under MAP. A total of 108 samples were collected from three Sardinian industrial cheese-making plants and analysed within 24 h after packaging and after 7, 14 and 21 days of refrigerated storage. Aerobic mesophilic bacteria, mesophilic and thermophilic cocci and lactobacilli, Enterobacteriaceae and E. coli, L. monocytogenes, Pseudomonas spp, Bacillus cereus, yeasts and moulds, and the chemical-physical parameters and composition of the product were determined. At the end of the shelf life, Pseudomonas spp. and Enterobacteriaceae reached high concentrations, 5 to 7 and 3 to 6 log10 colony forming unit g–1, respectively. The presence of environmental contaminants indicates that the use of MAP without the appropriate implementation of prerequisite programmes is not sufficient to extend the durability of ricotta fresca. Gas mixture and packaging material should be selected only on the basis of scientific evidence of their effectiveness.