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.

Validation according to ISO 16140:2003 of a commercial real-time PCR-based method for detecting Campylobacter jejuni, C. coli, and C. lari in foods.

Campylobacteriosis was the most frequently reported zoonosis in the European Union (EU) in 2010, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari as the most frequently reported species in foodborne outbreaks (FBOs). Relatively sensitive to environmental factors, these species may be present in low numbers. In line with EU policy for food control and FBO detection and in view of the need to reduce response time, we validated an alternative molecular method according to ISO 16140:2003 which establishes the general principle and technical protocol for the validation of alternative methods in the microbiological analysis of food. We used a qualitative real-time PCR commercial kit for the detection of C. jejuni, C. coli, and C. lari in two food categories "fruit and vegetable-based products" and "dairy products". The validation protocol comprises two phases: the first is a method comparison study of the alternative method against the reference method, and the second is an interlaboratory study of each of the two methods. In the first step, ISO 16140:2003 validation examines the following parameters: limit of detection (LOD); relative accuracy, relative specificity and sensitivity; relative detection level (RDL); and inclusivity and exclusivity. Except for LOD, inclusivity and exclusivity, the other steps were performed against the reference method (ISO 10272:2006). The LOD of the real-time PCR method was set at 4CFU/25g or mL for both food categories. Relative accuracy (98.33%), specificity (96.77%), and sensitivity (100%) were recorded for the food category "fruit and vegetable-based products" and 93.3%, 88.24%, 100%, respectively, for "dairy products". The RDL according to Fisher's exact test was p=1 for both food categories, for each level, and each food/strain combination. The interlaboratory study results showed correct identification of all 24 blind samples with both methods by all the participating laboratories. The results show that this commercial kit is suitable for the rapid detection of C. jejuni, C. coli, and C. lari on fruit, vegetables and dairy products and may aid in official controls. In conclusion, the use of alternative methods is recommended for the rapid identification of positive samples and the identification of the possible bacterial source in a FBO within 48 h.

Molecular characterization of Pseudomonas fluorescens isolates involved in the Italian "blue mozzarella" event.

Between June and September 2010, widespread Italian consumer reports of unusual blue spoilage on fresh dairy products were publicized, resulting in the so-called blue mozzarella event. An inordinately high number of samples from mozzarella and whey cheese products of Italian and German production subsequently tested positive for Pseudomonas fluorescens. The aim of this study was to verify whether a selected P. fluorescens strain was responsible for this apparently unusual event. Molecular characterization of 181 isolated P. fluorescens strains was conducted using a newly optimized pulsed-field gel electrophoresis protocol. Although a high number of pulsotypes was found (132), only four pulsotypes were associated with more than one production plant, and only one German isolate had the same pulsotype as was detected in two Italian plants. This is the only evidence of possible cross-contamination among cheeses from the two countries. The overall results did not support the spread of contamination from German to Italian plants or the presence of one environmental strain that spread in both countries.

Nocardia otitidiscaviarum pneumonia in an Alpine chamois (Rupicapra rupicapra rupicapra).

Nocardia otitidiscaviarum was cultured from the lung of an Alpine chamois (Rupicapra rupicapra rupicapra) with suppurative bronchopneumonia. This is the first report of both nocardiosis and Nocardia otitidiscaviarum in this wild ungulate species.

Coagulase-positive Staphylococci and Staphylococcus aureus in food products marketed in Italy.

Staphylococcus aureus is a very common organism capable of producing several enterotoxins (SEs) that cause intoxication symptoms of varying intensity in humans when ingested through contaminated food. This paper reports the results of an investigation on the presence of Coagulase-Positive Staphylococci (CPS) and S. aureus in several food products marketed in Italy and on food contact surface swabs sampled from the food industry. A total of 11,384 samples were examined and 1971 of them (17.3%) were found to contain CPS. The assays performed on 541 CPS strains led to the identification of 537 S. aureus strains on which characterization of type A, B, C and D staphylococcal enterotoxins (SEA, SEB, SEC and SED) was performed. A total of 298 S. aureus strains (55.5%) produced one or more SEs: 33.9% of the strains produced SEC, 26.5% SEA, 20.5% SEA+SED, 13.4% SED, 2.7% SEB, 1.7% SEA+SEB, 0.7% SEC+SED and 0.3% produced SEA+SEC and SEB+SEC. The investigation highlighted that these organisms are very common and constitute a potential risk for consumers' health.

Physical and genetic map of Streptococcus mutans GS-5 and localization of five rRNA operons.

The physical map of the 2.1 megabase chromosome of Streptococcus mutans GS-5 has been refined by including all ApaI and SmaI fragments of 5 kbp or greater, and by positioning the fragments generated by the endonuclease I-CeuI. Sixty-three new genetic loci have been added to the map, so that it now contains 90 loci. The new loci include those for 35 cloned streptococcal genes of established function and for 23 S. mutans genes of putative function. In addition, five rrn operons were identified and placed on the map of the chromosome. The presence of a SmaI site in each of the rrn operons allowed the direction of transcription of each operon to be deduced. The orientation of the rrn loci indicates that their transcription is directed away from a small region of the chromosome, identifying a possible region for the initiation of chromosome replication.

Ribosomal RNA gene (rrn) organization in enterococci.

A cloned 1.8-kb probe containing the 3' end of 16S ribosomal RNA and the 5' end of 23S ribosomal RNA from Enterococcus hirae was used to analyze various endonuclease digests of enterococci. In the ATCC strains tested we observed a remarkable conservation of the ApaI sites in the rrn operons, and a partial conservation of EcoRI sites. Using a number of other endonuclease digestions with the ApaI rrn probe, we estimate the number of rrn operons in enterococci to be between five and six.