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Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

Background: Clostridioides difficile is an important human and animal intestinal pathogen. Because of increasing indications of an association between C. difficile and food, in 2015, the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection (UVHVVR) included C. difficile in its national food surveillance. Aim: We aim to report the results and experience with a nationwide and longterm testing of food for C. difficile as a part of a regular national food surveillance programme. Methods: Retail minced meat and meat preparations (beef, pork and poultry) were sampled within a three-year period, 2015 to 2017. Selected raw retail vegetables, leaf salads and root vegetables, and ready-to-eat salads were only sampled during 2016 and 2017. Seafood was only sampled in 2017. Results: Altogether, 434 samples were tested, with 12 of 336 (3.6%) meat samples and 6 of 98 (6.1%) raw vegetables contaminated with C. difficile. Twelve of 18 recovered food isolates were toxigenic (toxinotypes 0, III, V, XII). The isolates belonged to 13 different PCR ribotypes, 001 being most common (5 isolates). Several food types with an increased potential of being contaminated with C. difficile were detected by surveillance. Conclusion: The three-year C. difficile testing within the national food surveillance revealed a low proportion of C. difficile-contaminated food and high genotype variability. Because the risk of C. difficile infection associated with C. difficile-contaminated food is unknown, no measures were recommended in the case of positive results.

Antibiotics have always appeared miraculous, saving innumerable lives. However, the unwise use of antimicrobial drugs has led to the appearance of resistant bacteria. The purpose of this study was to evaluate antimicrobial resistance in Escherichia coli (n =160) isolated from food of animal origin. The focus was on E. coli -producing extended-spectrum β-lactamases. E. coli was chosen because it is a part of the normal microbiota in mammals and can enter the food chain during slaughtering and food manipulation. Subsequently, its resistance genes can be transferred to pathogenic bacteria and human microbiota. Phenotypic and genotypic analyses of selected antimicrobial resistances were carried out together with a molecular analysis of virulence genes. E. coli isolates from food of animal origin were compared with clinical E. coli strains isolated from the human intestinal tract. Extended-spectrum β-lactamase-producing E. coli isolates were found in 9.4% of food isolates and in 1.8% of intestinal isolates. Phylogenetically, the majority of food (86.3%) and intestinal E. coli (58.1%) isolates were found to belong to the commensal phylogenetic groups A and B1. The distribution of 4 of 14 analyzed virulence factors was similar in the food and intestinal isolates. Strains isolated from food in Slovenia harbored resistance genes and virulence factors, which can constitute a problem for food safety if not handled properly.

Purpose The purpose of this paper is to study the microbiological quality of raw milk delivered by 17 vending machines (VM) owned by different Slovenian milk producers. Design/methodology/approach For the determination of hygiene-technical conditions of VM, an observation list that included criteria for estimation of hygiene-technical suitability was made. A total of 51 milk samples were collected in three different seasons. The swabs and the cleaning liquid (eluates) of dispensing nozzles and chambers were also sampled. The main groups of microorganisms were determined by colony count technique according to international standards in all collected samples. Findings The aerobic colony count was higher than 100,000 CFU/mL in 20 (39.2 per cent) of milk samples. Its mean value was 4.8 log10 CFU/mL. The mean values of Enterobacteriaceae, psychrotrophic microorganisms, lipolytes, proteolytes, yeasts and moulds together, coagulase-positive staphylococci and somatic cell count were 3.3 log10 CFU/mL, 4.1 log10 CFU/mL, 3.2 log10 CFU/mL, 3.9 log10 CFU/mL, 2.2 log10 CFU/mL, 2.8 log10 CFU/mL and 5.3 log10 cells/mL, respectively. E. coli was found in 33.3 per cent of milk samples, while Listeria monocytogenes and antibiotics were not detected. The inner surface contamination of the dispensing nozzles and chambers was estimated in the range from 1.8 log10 CFU to 6.0 log10 CFU/cm2. The presence of detergents and disinfectants in supply valve eluates was determined in more than one-third of the samples. The hygienic-technical conditions of observed VM show some deviations from specified hygienic-technical requirements which could influence the safety of raw milk. Research limitations/implications The data about construction and the cleaning practice of VM, included in the experiment, were not available during the inspection facility. Originality/value In the paper the pathogenic and also the spoilage microorganisms in milk in the combination with hygienic conditions of inside surfaces of VM were studied.