Okadaic acid as a major problem for the seafood safety (Mytilus galloprovincialis) and the dynamics of toxic phytoplankton in the Slovenian coastal sea (Gulf of Trieste, Adriatic Sea).

This article presents the first results on shellfish toxicity in the Slovenian sea (Gulf of Trieste, Adriatic Sea) since the analytical methods for the detection of biotoxins (PSP, ASP, DSP and other lipophilic toxins) in bivalve molluscs were included in the national monitoring program in 2013. In addition to toxins, the composition and abundance of toxic phytoplankton and general environmental characteristics of the seawater (surface temperature and salinity) were also monitored. During the 2014-2019 study period, only lipophilic toxins were detected (78 positive tests out of 446 runs), of which okadaic acid (OA) predominated in 97 % of cases, while dinophysistoxin-2 and yessotoxins only gave a positive result in one sampling event each. The number of samples that did not comply with the EC Regulation for the OA group was 17 or 3.8 % of all tests performed, all of which took place from September to November, while a few positive OA tests were also recorded in December, April, and May. This toxicity pattern was consistent with the occurrence pattern of the five most common DSP-producing dinoflagellates, which was supported by the development of warm and thermohaline stratified waters: Dinophysis caudata, D. fortii, D. sacculus, D. tripos and Phalacroma rotundatum. The strong correlation (r = 0.611, p < 0.001) between D. fortii, reaching abundances of up to 950 cells L-1, and OA suggests that D. fortii is the main cause of OA production in Slovenian waters. Strong interannual variations in OA and phytoplankton dynamics, exacerbated by the effects of anthropogenic impacts in this coastal ecosystem, reduce the predictability of toxicity events and require continuous and efficient monitoring. Our results also show that the introduction of the LC-MS/MS method for lipophilic toxins has improved the management of aquaculture activities, which was not as accurate based on mouse bioassays.

Clostridioides difficile in national food surveillance, Slovenia, 2015 to 2017.

BackgroundClostridioides 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.AimWe aim to report the results and experience with a nationwide and long-term testing of food for C. difficile as a part of a regular national food surveillance programme.MethodsRetail 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.ResultsAltogether, 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.ConclusionThe 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.

Development and Validation of a New Protocol for Detecting and Recovering Clostridium difficile from Meat Samples.

There is no recommended protocol for detecting and isolating Clostridium difficile present in food samples. Here, we have evaluated the recovery of C. difficile in meat samples after incubating them in various enrichment broths. The media were as follows: cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme; cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme; and cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, C. difficile moxalactam norfloxacin selective supplement, and lysozyme. Samples were inoculated with various strains and quantities of C. difficile and then enriched in the different broths for 1, 4, and 7 days. C. difficile was isolated on agar plates and detected with quantitative real-time PCR (qPCR). The procedure using enrichment in cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme and incubation for 4 days for qPCR detection and 7 days for isolation (plating on C. difficile agar base with added C. difficile selective supplement and 7% [v/v] defibrinated horse blood after alcoholic shock and centrifugation) was validated. Samples of different kinds of meat and meat preparation were contaminated and used for validation of the chosen protocol. The sensitivity of detection with qPCR was 100%, and the sensitivity of the isolation method was 96%.

New Approaches on Quantification of Campylobacter jejuni in Poultry Samples: The Use of Digital PCR and Real-time PCR against the ISO Standard Plate Count Method.

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.

Antimicrobial Resistance and Molecular Characterization of Extended-Spectrum ß-Lactamases and Other Escherichia coli Isolated from Food of Animal Origin and Human Intestinal Isolates.

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 ß-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 ß-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.

Molecular characterisation of noroviruses detected in mussels (Mytilus galloprovincialis) from harvesting areas in Slovenia.

Noroviruses are a leading cause of viral gastroenteritis in humans and are responsible for many outbreaks worldwide. Mussels are one of the most important foodstuffs connected with norovirus outbreaks, also resulting in multinational dimensions. Two hundred and thirty-eight (238) samples of mussels (Mytilus galloprovincialis) were collected in periods between the years 2006-2008 and 2010-2012 to study the prevalence of noroviruses (NoVs) from harvesting areas along the Adriatic coast of Slovenia. Between 2006 and 2008, 9.1% to 24.6% of mussel samples tested by specific GI and/or GII real-time RT-PCR methods were found to be positive for NoVs while between 2010 and 2012 the percentage of NoV positive samples varied from 12.5% to 22.2%. At the nucleotide level within the RdRp gene fragment the genetic diversity of NoVs detected in mussels ranged between 78.8-81.0% nucleotide identity among GII strains (92.1-99.6% within the GII.P4 genotype), 100% nucleotide identity among GI and 58.4-60.2% among GI and GII strains. Nine of the NoV strains detected from mussels were genotyped as GII.4, while two samples were within GI.P2 and one was a positive sample within genotype GII.P21. This study confirmed that mussels are a potential source of the NoV infection. The detected NoVs share the same topology on the phylogenetic tree within the NoV strains detected in water samples and human patients, not only from Slovenia but also from many different countries worldwide. We can assume that mussels in harvesting areas are not only contaminated from the surrounding area but also by contaminated water and sewage from large transport ships, which are regularly present in the area.

Campylobacter jejuni contamination of broiler carcasses: Population dynamics and genetic profiles at slaughterhouse level.

Six slaughter batches deriving from six typical industrial broiler flocks were examined for the presence, quantity and genetic characteristics of contaminating Campylobacter jejuni (C. jejuni) during various stages of slaughtering and carcass processing. To assess the contamination dynamics of the carcasses, the analyses were always conducted on neck-skin samples from the same pre-selected and carefully marked carcasses in each batch. The skin samples were taken sequentially at three successive slaughter-line locations in the evisceration room, after three-day refrigeration and after three-day freezing procedure. Caecal samples from the same animals were also tested, as well as samples from the slaughterhouse environment before and after slaughtering. The samples were analysed by the ISO10272 isolation method; campylobacters from neck-skin samples were also quantified. Isolates were species-identified and genotyped by pulsed-field gel electrophoresis (PFGE). On average, the highest C. jejuni skin contamination was detected at the first sampling point (post-plucking), suggesting that the majority of Campylobacter contamination actually occurs before the entrance to the eviscerating room, probably during the preceding plucking stage. In two out of five positive batches, an additional increase in contamination was recorded after the evisceration step. An evident trend of increasing contamination level was detected when successive batches were compared at each of two initial sampling sites in the evisceration room, indicating an accumulation of contaminating C. jejuni at some point before the evisceration room. Three-day refrigeration and three-day freezing caused a 4.5- and 142-fold drop in mean C. jejuni CFU counts, respectively. All pre-slaughtering samples from the slaughterhouse environment were negative and all post-slaughtering samples, except water from the scalding tank, were positive. Pulsotypes were limited: altogether five different types were detected, typically one type per batch. The PFGE results from the slaughterhouse environment isolates indicate that cross-contamination is possible (multiple pulsotypes detected in e.g. eviscerating machine). Nevertheless, this was not confirmed in carcasses: analyses of neck-skin isolates suggest that carcasses are contaminated by their own caecal/farm/flock pulsotype.

Detection of Vibrio parahaemolyticus in Mediterranean mussels (Mytilus galloprovincialis) in Slovenia.

The aim of this study was to determine the prevalence of Vibrio parahaemolyticus in shellfish samples harvested along the Slovenian coast. Shellfish samples of Mediterranean mussels (Mytilus galloprovincialis) were collected along the Slovenian coast at four locations (Seca, Piran, Strunjan and Debeli Rtic) between 2006 and 2008. Samples were examined and analysed for the presence of V. parahaemolyticus by conventional and molecular methods. The presence of Vibrio in the samples was examined by conventional methods on plate grown bacterial cells before and after enrichment in alkaline saline peptone water (ASPW). PCR methods were used for the detection of V. parahaemolyticus-specific toxR and tlh genes and of the virulence-associated tdh and trh genes. Out of 168 samples examined, 24 were positive for toxR and tlh genes by PCR from enrichment broth. Five out of 62 (8.1%), 4 out of 32 (12.5%) and 15 out of 74 (20.2%) samples were positive in 2006, 2007 and 2008, respectively. Colonies of V. parahaemolyticus were isolated from only one sample positive for V. parahaemolyticus by PCR.