Vincenzo Spanu

Antibiotic Resistance in Staphylococcus aureus and Coagulase Negative Staphylococci Isolated from Goats with Subclinical Mastitis

Antimicrobial resistance patterns and gene coding for methicillin resistance (mecA) were determined in 25 S. aureus and 75 Coagulase Negative Staphylococci (CNS) strains isolates from half-udder milk samples collected from goats with subclinical mastitis. Fourteen (56.0%) S. aureus and thirty-one (41.3%) CNS isolates were resistant to one or more antimicrobial agents. S. aureus showed the highest resistance rate against kanamycin (28.0%), oxytetracycline (16.0%), and ampicillin (12.0%). The CNS tested were more frequently resistant to ampicillin (36.0%) and kanamycin (6.7%). Multiple antimicrobial resistance was observed in eight isolates, and one Staphylococcus epidermidis was found to be resistant to six antibiotics. The mecA gene was not found in any of the tested isolates. Single resistance against β-lactamics or aminoglicosides is the most common trait observed while multiresistance is less frequent.

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.

A survey on Aflatoxin M1 content in sheep and goat milk produced in Sardinia Region, Italy (2005-2013)

In the present work the results of a survey conducted in Sardinia Region on Aflatoxin M1 (AFM1) contamination in milk of small ruminants from 2005 to 2013 are reported. A total of 517 sheep and 88 goat milk samples from bulk tank, tank trucks and silo tank milk were collected. Analyses were performed by the Regional Farmers Association laboratory using high-performance liquid chromatography following the ISO 14501:1998 standard. None of the sheep milk samples analysed during 2005-2012 showed AFM1 contamination. In sheep milk samples collected in 2013, 8 out of 172 (4.6%) were contaminated by AFM1 with a concentration (mean±SD) of 12.59±14.05 ng/L. In one bulk tank milk sample 58.82 ng/L AFM1 was detected, exceeding the EU limit. In none of goat milk samples analysed from 2010 to 2012 AFM1 was detected. In 2013, 9 out of 66 goat milk samples (13.6%) showed an AFM1 concentration of 47.21±19.58 ng/L. Two of these samples exceeded the EU limit, with concentrations of 62.09 and 138.6 ng/L. Higher contamination frequency and concentration rates were detected in bulk tank milk samples collected at farm than in bulk milk truck or silo samples, showing a dilution effect on AFM1 milk content along small ruminants supply chain. The rate and levels of AFM1 contamination in sheep and goat milk samples were lower than other countries. However, the small number of milk samples analysed for AFM1 in Sardinia Region in 2005-2013 give evidence that food business operators check programmes should be improved to ensure an adequate monitoring of AFM1 contamination in small ruminant dairy chain.

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.

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.

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.

PREVALENCE AND IDENTIFICATION OF VIBRIO SPP. ISOLATED ON AQUACULTURED GILTHEAD SEA BREAM

SUMMARY The aim of the study was to investigate the prevalence of Vibrio spp isolated from gilthead sea bream ( Sparus aurata ) farmed on sea cages and to identify and characterize the pathogen by molecular techniques. Eighty fish were collected from two hatcheries located on the North+Est Sardinian Mediterranean coast, and microbiological analysis were performed on different body parts such as skin, gills, muscle and intestinal tract. Subsequently 100 pure colonies with typical morphology and phenotypic characteristics were selected and submitted to the molecular identification. The analysis on the prevalence of Vibrio spp showed the effect of the hatchery rearing system ( P<0.001), of the date of sampling ( P<0.001), and of the body part ( P<0.001). All the strains selected were confirmed to be members of the genus Vibrio spp by the molecular method/techinique/identification, whereas the rpo A gene sequence analyses allowed to identify 89 strains belonging to the species Vibrio harveyi, 6 to V. diabolicus, 2 to V. parahaemolyticus and 1 to V. mediterranei .