The emergence of antimicrobial-resistant and virulent enterococci is a major public health concern. While enterococci are commonly found in food of animal origin, the knowledge on their zoonotic potential is limited. The aim of this study was to determine and compare the antimicrobial susceptibility and virulence traits ofEnterococcus faecalisandEnterococcus faeciumisolates from human clinical specimens and retail red meat in Slovenia. A total of 242 isolates were investigated: 101 from humans (71E. faecalis, 30E. faecium) and 141 from fresh beef and pork (120E. faecalis, 21E. faecium). The susceptibility to 12 antimicrobials was tested using a broth microdilution method, and the presence of seven common virulence genes was investigated using PCR. In both species, the distribution of several resistance phenotypes and virulence genes was disparate for isolates of different origin. All isolates were susceptible to daptomycin, linezolid, teicoplanin, and vancomycin. In both species, the susceptibility to antimicrobials was strongly associated with a food origin and the multidrug resistance, observed in 29.6% ofE. faecalisand 73.3%E. faeciumclinical isolates, with a clinical origin (Fisher’s exact test). Among meat isolates, in total 66.0% ofE. faecalisandE. faeciumisolates were susceptible to all antimicrobials tested and 32.6% were resistant to either one or two antimicrobials. InE. faecalis, several virulence genes were significantly associated with a clinical origin; the most common (31.0%) gene pattern included all the tested genes excepthyl. In meat isolates, the virulence genes were detected inE. faecalisonly and the most common pattern includedace,efaA, andgelE(32.5%), of whichgelEshowed a statistically significant association with a clinical origin. These results emphasize the importance ofE. faecalisin red meat as a reservoir of virulence genes involved in its persistence and human infections with reported severe outcomes.

Background: Listeria monocytogenesis the causative agent of listeriosis, a serious disease affecting both animals and humans. Here, multilocus sequence typing (MLST) was used to characterize the genetic diversity ofListeria monocytogenesstrains isolated from the natural environment and animal clinical cases in Europe. The prevalence of clonal complexes (CCs) obtained was compared according to (i) the origin of isolation – clinical cases vs. natural environment – and (ii) the clinical form of animal listeriosis – rhombencephalitis vs. abortion. To this aim, two datasets were constructed. The clinical dataset consisted of 350 animal clinical isolates originating from France and Slovenia and supplemented with isolates from Switzerland and Great Britain. The natural environment dataset consisted of 253 isolates from the natural environment originating from Slovenia and supplemented with isolates from nine other European countries.Results: For the clinical cases, CC1, CC4-CC217 and CC412 were the most prevalent in rhombencephalitis and CC1, CC37 and CC4-CC217 in abortion. The hypervirulent CC1 and CC4-CC217 prevailed in both datasets. These results indicated that livestock is constantly exposed to hypervirulent CCs. CC1 was significantly associated with a clinical origin, whereas CC9, CC29 and CC14 were associated with the natural environment. CC1 was predominant among rhombencephalitis cases both in cattle and small ruminants, and its prevalence did not differ significantly between these two groups. A novel association of CC37 and CC6 with abortion cases was revealed.Conclusions: Here, we show that CC1 and CC4-CC217 are prevalent in isolates of environmental and animal clinical origin, suggesting that ruminants are frequently exposed to hypervirulent CCs. The presence of CC4 in two mastitis cases calls for further attention due to direct threat to the consumer. We showed several associations between CCs and the origin of isolation or clinical form of listeriosis, e.g. CC37 and CC6 with abortion. This study improves our understanding of the population structure ofL. monocytogenesisolates from the natural environment and animal clinical cases. Moreover, it provides a basis for future studies aiming to determine the underlying mechanisms of phenotypic traits of interest.

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 (12523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1000 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.

Staphylococcus pseudintermedius is a common cause of skin and soft tissue infections in dogs but can also cause infections in cats and humans. The frequency of methicillin-resistant S. pseudintermedius (MRSP) strains is increasing worldwide. Here, we obtained 43 MRSP isolates from dogs (n = 41), one cat (n = 1) and the small animal clinic environment (n = 1) in Slovenia from the period 2008-2018, which underwent whole-genome sequencing (WGS) and antimicrobial susceptibility testing. Five sequence types (STs) were identified, with ST71 (32/43) and ST551 (8/43) being the predominant. In Slovenia, ST551 was first detected in 2016, whereas a decrease in the frequency of ST71 was observed after 2015. All isolates were multidrug-resistant and most antimicrobial-resistant phenotypes could be linked to acquisition of the corresponding resistance genes or gene mutations. Core-genome multilocus sequence typing (cgMLST) revealed several potential MRSP transmission routes: (i) between two veterinary clinics by a single MRSP-positive dog, (ii) between the environment of a veterinary clinic and a dog, and (iii) between a canine and a feline patient through the contaminated environment of a veterinary clinic. Of the six dogs that were additionally sampled from 14 days to five months after the initial sampling, each harbored the same MRSP strain, suggesting a limited within-host diversity of MRSP in symptomatic dogs. The present results highlight the importance of MRSP-positive dogs in the spread of veterinary care-associated MRSP infections and call for the implementation of strict control measures to reduce MRSP contamination in veterinary clinic environments originating from animal-contact surfaces.

Aims An extensive source investigation was conducted on a dairy farm with neurolisteriosis and subclinical mastitis cases to identify infection source and potential transmission routes of Listeria monocytogenes. Methods and Results A total of 36 L. monocytogenes isolates were obtained from animal clinical cases (neurolisteriosis and udder infection) and the farm environment (silage, feces, water). Isolates were typed using pulsed‐field gel electrophoresis (PFGE) and whole‐genome sequencing (WGS). Their virulence potential was assessed using the gentamicin protection assay and WGS‐based identification of virulence genes. PFGE and WGS revealed a high genetic diversity of L. monocytogenes. An epidemiological link was confirmed for isolates from (i) several subclinical mastitis cases, (ii) silage and subclinical mastitis cases and (iii) different water sources. The neurolisteriosis isolate belonged to clonal complex (CC) 1, but infection source was not identified. A high occurrence (9/47 cows; 19.1%) of subclinical mastitis was observed with isolates belonging to CC2, CC4 and CC11. Conclusions The dairy farm environment was contaminated with diverse L. monocytogenes strains, including genotypes associated with human disease. Several isolates harbored genetic determinants associated with increased infectious potential in humans. Significance and Impact of the Study Results suggest that subclinical listerial mastitis should not be neglected as a potential source of milk contamination. The presence of hypervirulent CCs in subclinical mastitis cases calls for the implementation of improved mastitis detection.

Six Helicobacter -like isolates were recovered from 15 gastric mucosa samples of red foxes (Vulpes vulpes) shot by hunters in the surroundings of Ljubljana, Slovenia. Gram-negative, tightly coiled, intensely motile, 7–15 µm long and ≤1 µm wide bacteria grew on the biphasic blood agar plates. By using a genus-specific polymerase chain reaction (PCR), all isolates were confirmed as Helicobacter sp. and subsequently subjected to whole-genome sequencing (WGS). Five isolates showed a genome-wide average nucleotide identity (ANI) value of <95 % to the previously described Helicobacter species and one isolate was classified as Helicobacter felis . In the five unidentified isolates, the 16S rRNA gene sequence similarity to the type strains of all Helicobacter species ranged from 98.6 to 98.9 %. Their taxonomic status was established using a polyphasic taxonomic approach comprising the core genome-based phylogeny, morphological and phenotypic characteristics, including an analysis of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra. Phylogeny revealed the existence of three novel and well-supported clusters, with Helicobacter bizzozeronii and Helicobacter baculiformis being the most closely related species. The isolates also differed from the previously described species in their MALDI-TOF profiles and some biochemical characteristics. In conclusion, the data presented herein indicate that the obtained isolates, excluding H. felis isolate, represent three novel Helicobacter species, for which the names Helicobacter labacensis sp. nov., Helicobacter mehlei sp. nov., and Helicobacter vulpis sp. nov. are proposed, with isolates L9^T (=DSM 108823^T=CRBIP 111719^T), L15^T (=DSM 108730^T=CCUG 72910^T) and L2^T (=DSM 108727^T=CCUG 72909^T) as type strains, respectively.