CO33 | DETECTION AND CHARACTERIZATION OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI SEROGROUP O103 FROM OVINE RAW MILK CHEESES AT DIFFERENT STAGES OF RIPENING

Aim. The aim of this study was to evaluate the presence of the main Shiga-toxin-producing Escherichia coli (STEC) serogroups in differently ripened ovine raw milk cheese samples. Methods. During the period between May and December 2025, a total of 168 ovine raw milk cheese samples, obtained from bulk milk and ripened at different stages, were collected from 7 different small and medium-sized food enterprises (SME1-7) located in the provinces of L’Aquila, Teramo and Pescara (Abruzzo region, Italy). Each SME was visited 8 times during the study period, and at each visit, one cheese sample (75.0 g), from the same cheese wheels, was aseptically collected for each ripening interval: day 3 (t3), 15 (t15), and 30 (t30). This resulted in 56 samples for each ripening interval, gaining a total of 168 samples across all standardized commercial productions. The experimental design adopted a culture dependent approach primarily aimed at STEC detection, also including quantitative microbiological analyses as hygienic indicators. Selective STEC detection was performed according to the ISO 13136:2013. For each sample, 25.0 g of cheese (including rind, medium and core) were homogenized in 225 mL of buffered peptone water (BPW) and incubated at 37.0°C for 24 hours. Following enrichment, a loopful was streaked onto Tryptone Bile X-glucuronide (TBX) agar and incubated at 44.0 °C for 24 hours. Suspected positive blue/green colonies were subcultured on nutrient agar and subjected to preliminary biochemical identification using the Vitek 2 system. DNA was extracted from isolates and initially screened by multiplex PCR for virulence genes (eae, stx1 and stx2). These qualitative results were followed by the real-time PCR assays targeting both virulence genes and O-antigen genes specific for STEC serogroups [rfbE (O157), wbdl (O111), wzx (026), ihp1 (O145), and wzx (O103)] (expressed as genome copies per millilitre – GC/mL), as described in the Annex E - ISO 13136:2012. Amplicons were sequenced and a phylogenetic tree was constructed. Additionally, quantitative microbiological parameters, such as Enterobacteriaceae (EBC) and E. coli (ECC), were assessed in parallel as hygienic quality indicators. Results. At stage t3, 9/56 (16.07%) samples presented typical blue-green colonies on TBX agar plates coming from 6 SME (SME1-4, SME6-7). At stage t15, 4/56 (7.14%) still grew suspected positive ones (SME1, SME3-4, SME7), and at stage t30, 1/56 (1.78%) was still positive from SME4. Seven out of nine (77.78%) strains harbored the stx1, stx2 and eae virulence genes with different GC/mL amounts (average values: 104 GC/mL for stx1, 101 stx2, and 104 GC/mL eae), according to the real time PCR assays. All the E. coli belonged to the O103 serogroup. In summary, 7/168 (4.17%) of raw milk cheese were positive for STEC. From a ripening perspective, 4/7 (57.14%) STEC were isolated at t3 from SME1, SME3, SME4, and SME7, 2/7 (28.57%) at t15 from SME3 and SME4, and 1/7 (14.28%) at t30 from SME4. The EBC, in the screened raw milk cheese samples, ranged between 4.0 to 7.0 log10 CFU/g, with a mean value of 5.2 log10 CFU/g, while the ECC ranged between 2.0 – 6.0 log10 CFU/g with an average value of 2.9 log10 CFU/g. Conclusions. These results emphasize the food safety risks associated with STEC O103 contamination in raw ovine milk cheese. The detection and suboptimal hygienic indicators reinforce the need for strict microbiological surveillance to ensure consumer protection.