Behavior of Escherichia coli O157:H7 during the manufacture and ripening of Fontina Protected Designation of Origin cheese.

This study was conducted to describe the cheese-making procedure of Fontina Protected Designation of Origin (PDO) cheese and to evaluate the behavior of Escherichia coli O157:H7 during cheese manufacture and ripening. The study was divided into 2 phases: the production of Fontina PDO cheese was monitored at 3 different dairies in the Aosta Valley and an E. coli O157 challenge was conducted at a fourth dairy. The dairies employ different commercial starter cultures for cheese making. The growth of lactic acid bacilli (LAB) and the decrease in pH were slower in the first hours and the LAB concentrations were overall higher in dairy A than in the other 2 dairies. The pH remained substantially unchanged during ripening (range 5.2 to 5.4) in all dairies. Water activity remained constant at around 0.98 until d 21, when it decreased to around 0.97 until d 80 in dairies A and B and 0.95 in dairy E. Whole raw cow milk was used for making Fontina cheese according to the standard procedure. For the experimental production, the milk was inoculated with E. coli O157:H7 at a concentration of approximately 5 log10 cfu/mL and commercial starter cultures were used according to the Fontina PDO regulation. An increase of 2.0 log10 cfu/g in E. coli O157:H7 was observed during the first 9.5 h of cheese making, followed by a decrease at 46 h when pH decreased to 5.4 in all trials. Fresh cheeses were salted and held at 10°C for ripening for 80 d. Water activity was decreased to 0.952 at the end of the ripening stage. The LAB concentrations declined gradually; this trend was more marked for the lactobacilli than either the thermophilic or the mesophilic lactococci. The increase in LAB count and the decrease in pH in the first hours did not seem to affect E. coli O157 growth. Ripening was found to inhibit pathogen survival, however, as seen in the reduction of 3 log10 from the maximum concentration measured during the earlier stages of production.

Short communication: Characterization of Staphylococcus aureus isolated along the raw milk cheese production process in artisan dairies in Italy.

Staphylococcus aureus is a common cause of food-borne intoxications. Several staphylococcal food poisoning outbreaks have been linked to consumption of raw milk cheeses and artisanal cheese production. However, information on Staph. aureus isolated from artisanal raw milk cheeses and small-scale dairy production environments is very limited. Therefore, we aimed to characterize Staph. aureus isolated along the artisanal raw milk production chain by determining (1) the population structure, and (2) the presence/absence of enterotoxin genes, mecA/C, and pvl. We collected 276 samples from different production stages (raw milk, whey, curd, brine, drying worktops, and cheese) at 36 artisan dairies in Italy. A total of 102 samples from 25 dairies tested positive for Staph. aureus, with 80% positive samples among the tested artisan cheeses. All isolates were further characterized by spa typing and PCR screening for staphylococcal enterotoxin genes, the mecA/mecC genes characteristic for methicillin-resistant Staph. aureus, and the pvl gene encoding Panton-Valentine leukocidin. The 102 isolates represented 15 different spa types and were assigned to 32 different Staph. aureus strains. The spa type most frequently detected was t2953 (30%), which is associated with genotype B strains causing high within-herd levels of bovine mastitis. In addition, 3 novel spa types (t13269, t13277, and t13278) were identified. Although none of the strains harbored mecA/mecC or pvl, 55% of the isolates exhibited at least one enterotoxin gene. Many strains were present in samples from multiple dairies from different regions and years, highlighting the spread of Staph. aureus in small-scale cheese production plants. Our findings demonstrate that enterotoxigenic Staph. aureus and in particular t2953 (genotype B) isolates commonly occur in artisanal dairies and raw milk cheeses in Italy. It is particularly alarming that 80% of the artisan cheeses sampled in our study were positive for Staph. aureus. These findings stress the need for effective measures preventing staphylococcal food poisoning by limiting Staph. aureus growth and enterotoxin formation along the production chain and in the final product.

Risk-based control of food-borne pathogens Listeria monocytogenes and Salmonella enterica in the Italian fermented sausages Cacciatore and Felino.

Fermentation is the most important killing step during production of fermented meats to eliminate food-borne pathogens. The objective was to evaluate whether the food-borne pathogens Listeria monocytogenes and Salmonella enterica may survive during the production of two Italian fermented sausages. Sausage batter was inoculated with five strains of L. monocytogenes or S. enterica (ca. 10(5)-10(6) cfu/g) and their kinetic behavior was monitored during production. Both pathogens survived relatively well (in Cacciatore L. monocytogenes and S. enterica inactivation was ca. 0.38±0.23 and 1.10±0.24 log cfu/g, respectively; in Felino was ca. 0.39±0.25 and 1.62±0.38 log cfu/g, respectively) due to the conditions prevailing during production (slow dehydration rate, small reduction of water activity and fermentation temperature mainly below 20 °C during the first 48 h of fermentation). Quantitative analysis of data originating from challenge tests provide critical information on which combinations of the process parameters would potentially lead to better control of the pathogens.

Differential gene expression profiling of Listeria monocytogenes in Cacciatore and Felino salami to reveal potential stress resistance biomarkers.

The current study reports a) the in situ transcriptional profiles of Listeria monocytogenes in response to fermented sausage stress and b) an approach in which in situ RT-qPCR data have been combined with advanced statistical techniques to discover potential stress resistance or cell viability biomarkers. Gene expression profiling of the pathogen has been investigated using RT-qPCR to understand how L. monocytogenes responds to the conditions encountered during the fermentation and ripening of sausages. A cocktail of five L. monocytogenes strains was inoculated into the batter of Cacciatore and Felino sausages. The RT-qPCR data showed that the acidic and osmotic stress-related genes were up-regulated. The transcripts of the lmo0669 gene increased during the fermentation and ripening of Cacciatore, whereas gbuA and lmo1421 were up-regulated during the ripening of Felino and Cacciatore, respectively. sigB expression was induced in both sausages throughout the whole process. Finally, the virulence-related gene prfA was down-regulated during the fermentation of Cacciatore. The multivariate gene expression profiling analysis suggested that sigB and lmo1421 or sigB and gbuA could be used as different types of stress resistance biomarkers to track, for example, stress resistance or cell viability in fermented sausages with short (Cacciatore) or long (Felino) maturation times, respectively.

Reproducibility study for the detection of Staphylococcal enterotoxins in dairy products between official Italian national laboratories.

Staphylococcal food poisoning is a common foodborne disease caused by the ingestion of staphylococcal enterotoxins (SEs) produced mainly by enterotoxigenic strains of Staphylococcus aureus. To date, 21 SEs and/or enterotoxin-like types have been identified, several of which represent a potential hazard for consumers. To protect consumer health and to reduce the amount of SE-contaminated food entering the market, European Union legislation regulating food safety requires testing for SEs. The Italian National Reference Laboratory organized a ring trial to test technical and analytical proficiency in the national network of official food laboratories. Twenty-four laboratories took part, and each received and analyzed 24 blind dairy samples. Reproducibility of the results from the laboratories was assessed by the Cohen k index, and accuracy (sensitivity and specificity) was evaluated according to the International Organization for Standardization definition (ISO 16140:2003). Trial results revealed partially satisfactory agreement: 254 of 276 possible paired participants (92%) reached a k value >0.60, which is conventionally recognized as satisfactory. Accuracy was deemed satisfactory; 100% sensitivity and 100% specificity were achieved by 22 and 18 of the 24 laboratories, respectively.

Enterotoxin gene profiles of Staphylococcus aureus isolated from milk and dairy products in Italy.

UNLABELLED: Staphylococcal foodborne intoxication, occurring after consumption of staphylococcal enterotoxins (SEs) in food, is considered one of the most common forms of bacterial foodborne outbreaks worldwide. Milk and dairy products account for 5% of all the incriminated foods in staphylococcal outbreaks, referring to Europe. The distribution of genes encoding for enterotoxins in Staphylococcus aureus strains is highly variable, with some carried on stable regions of the chromosome and others carried on mobile genetic elements. The aim of this study was to analyse the distribution of genes encoding for SEs in Staph. aureus strains isolated from milk and dairy products. In the period from January 2010 to June 2011, a total of 1245 dairy samples (848 of raw milk and 397 of dairy products) were collected and analysed for detection of genes encoding for 11 SEs and SEls (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SER SElJ and SElP) according to the procedures of the Italian National Reference Laboratory for coagulase-positive Staphylococci including Staph. aureus. Staphylococcus aureus strains were isolated in 481 (39%) samples. Of the 481 isolates of Staph. aureus tested, 255 (53%) were positive for one or more SE genes, and thirty-five different enterotoxin gene profiles were distinguished among the isolates. ser gene, found in 134 (28%) of the isolates, was the most frequent, followed by sed (25%) and selj genes (25%). The identification of new SEs increased the isolation frequency of enterotoxigenic staphylococci, thus suggesting that the pathogenic potential of Staph. aureus may be of greater importance than previously thought. Further studies are needed to quantify the expression of these new enterotoxins, and to assess their contribution to foodborne disease burden. SIGNIFICANCE AND IMPACT OF THE STUDY: The analyses targeted 11 staphylococcal enterotoxins genes and 35 different enterotoxin gene profiles were distinguished among the isolates. A total of 255 Staph. aureus isolates were positive for one or more SE genes while ser gene was the most prevalent. In 93% of the isolates bearing genes located on the enterotoxin gene cluster (n = 89), both seg and sei genes were present.

Staphylococcal poisoning foodborne outbreak: epidemiological investigation and strain genotyping.

In June 2011, an outbreak of Staphylococcus aureus enterotoxin food poisoning gastroenteritis occurred in Turin, Italy, following a catered dinner party at a private home. Within a few hours, 26 of the 47 guests experienced gastrointestinal illness, and 9 were hospitalized. A retrospective cohort study using a standardized questionnaire was carried out, and the risk ratios for each food item were calculated. The analysis indicated consumption of seafood salad as the most probable cause of the outbreak (risk ratio = 11.72; 95 % confidence interval, 1.75 to 78.54). Biological samples were collected from four of the hospitalized guests (stool and vomit), nasal mucosa swabs from three food handlers employed with the caterer, and available food residuals. All stool and vomit samples tested positive for enterotoxigenic S. aureus. As residues of the seafood salad were no longer available for sampling, suspected contamination could not be verified. However, no other food was found contaminated by S. aureus or its enterotoxins. All isolates from the biological samples were characterized at the genomic level by means of two multiplex PCR protocols to determine the presence of genes encoding staphylococcal enterotoxins, pulsed-field gel electrophoresis and staphylococcal protein A gene (spa) typing to describe their genetic profiles. All the isolates presented genes encoding SEA and SEI; the pulsed-field gel electrophoresis genetic profiles revealed the same pulsotype in the microorganism isolated from the hospitalized guests as in one of the isolates from a food handler's nasal mucosa, and the spa typing analysis reported two closely related spa types (t701 and t267), implicating the food handler as the most likely outbreak source.