Enterotoxigenic and Antimicrobic Susceptibility Profile of Staphylococcus aureus Isolates from Fresh Cheese in Croatia.

Certain Staphylococcus aureus strains harbour staphylococcal enterotoxin genes and hence can produce enterotoxin during their growth in food. Therefore, food can be a source of staphylococcal food poisoning, one of the most common food-borne diseases worldwide. Epidemiological data show that S. aureus is often present in raw milk cheeses, and consequently, cheeses are often the source of staphylococcal food poisoning outbreaks. The aim of this study was to determine the phenotypic characteristics of S. aureus isolates from fresh cheese, including antibiotic susceptibility; the presence of classical sea-see enterotoxin genes through molecular methods; and the isolate's ability to produce SEA-SEE enterotoxins in vitro through reversed passive latex agglutination. A total of 180 coagulase-positive staphylococci were isolated from 18 out of 30 cheese samples, and 175 were confirmed as S. aureus through latex agglutination and API STAPH tests. All isolates possessed phenotypic characteristics typical for S. aureus, with certain variations in the egg yolk reaction (18.3% of the isolates showed a weak reaction and 28% no reaction at all) and haemolysis pattern (36.6% of the isolates produced double-haemolysis and 4.6% were non-haemolytic). Antibiotic resistance was observed in 1.1% of the isolates and to mupirocin only. Real-time PCR detected the sec gene in 34 (19.4%) isolates, but most isolates (80.6%) were not enterotoxigenic. For all 34 (19.4%) strains that carried the sec gene, the RPLA method detected the production of the SEC enterotoxin in vitro. For those enterotoxigenic strains, the possibility of enterotoxin production in fresh cheese could not be ruled out.

Tracing of Listeria monocytogenes Contamination Routes in Fermented Sausage Production Chain by Pulsed-Field Gel Electrophoresis Typing.

In this study, the presence of Listeria monocytogenes was assessed along the production process of fermented sausages in a small-scale facility. Following the isolation of the pathogen from the final product (ISO 11290-1), retrospective sampling was performed during the production of a new batch of sausages, including raw materials, casings, additives, sausage mixtures, sausages during fermentation, and environmental samples. L. monocytogenes was recovered from the following sampling points: the defrosting room and the cuttering, mixing, stuffing, and fermentation phases. Ten strains were isolated, molecularly confirmed as L. monocytogenes by means of a molecular detection system, and subjected to pulsed-field gel electrophoresis (PFGE) typing. On the basis of an unweighted pair group method with arithmetic mean (UPGMA) dendrogram from Ascl pulsotypes, the strains were indistinguishable (no band difference). The same pulsotypes of strains present in both batches of sausages, as well as in environmental samples, indicated the persistence of L. monocytogenes in the sausage production unit.

The impact of frozen storage duration on physical, chemical and microbiological properties of pork.

The aim of the present study was to evaluate the effect of frozen storage duration (3, 6, 9, 12, 15, 18 months) on physical, chemical and microbial properties of pork in three cuts (loin, ham, and belly rib). During frozen storage, significant alterations in physical and chemical parameters were observed through the increase of the total exudate, pH and lightness (L*), and a decrease of shear force and yellowness (b*). Water content in ham samples decreased, while protein content increased. The lipid oxidation (TBA) values in loin and ham samples increased for up to fifteen months of frozen storage, after which they decreased. The proportion of saturated fatty acids in frozen samples was significantly higher than in fresh meat. The total amount of Enterobacteriaceae, S. aureus and Pseudomonas spp. decreased during frozen storage, indicating that freezing may reduce the number of bacteria found in meat. Frozen pork microflora did not change after eighteen months of storage.

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

Microbial and physicochemical succession in fermented sausages produced with bacteriocinogenic culture of Lactobacillus sakei and semi-purified bacteriocin mesenterocin Y.

The influence of the bacteriocinogenic culture Lactobacillus sakei (10(5)/g) and semi-purified bacteriocin mesenterocin Y (2560AU/kg) on the safety and quality of traditional Croatian fermented sausages was investigated. The addition of Lb. sakei and/or mesenterocin Y reduced microbial counts (P<0.05) in the final products. After 28 days of ripening, coagulase-negative cocci decreased 1.5-2.0log, yeasts 1.2-1.4log and enterococci 1.7-2.7log. In the case of the addition of Lb. sakei, the lactic acid bacteria count was significantly (P<0.05) higher at day 7 of ripening, and was accompanied by a lower pH and a higher amount of lactic acid (P<0.05). In the final product the amount of acetic acid was significantly lower. More intensive proteolysis and an increase in ammonia content were found at the beginning of fermentation, and in the second phase of ripening in the control samples, respectively. The free fatty acid concentration was significantly lower during the entire ripening process compared to the control (P<0.05). Semi-purified mesenterocin Y did not affect the sensory properties of the sausages, whilst the addition of Lb. sakei enhanced them.