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.

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.

For almost a decade, the number of Salmonella enterica subspecies enterica serovar Infantis-positive broiler flocks has been steadily increasing in Slovenia, doubling the number of positive holdings in only a few years. Since multidrug resistant S. Infantis isolates are highly prevalent in the broiler meat industry and may represent a public health concern through the food chain, we aimed to investigate the antimicrobial susceptibility, genetic diversity, and biofilm-forming ability of S. Infantis from Slovenian broiler flocks. A total of 87 S. Infantis strains isolated from broiler faeces in the period between 2007 and 2013 were studied. The samples originated from 41 farms which were subcontractors of three major food business operators and from two autonomously operating holdings (farms). Isolates were phenotypically tested for their susceptibility to 14 antimicrobials from nine classes by determining the minimum inhibitory concentration with the microdilution method. Only 8% of the isolates were susceptible to all of the antimicrobial agents tested, while 88.5% of the isolates were multidrug resistant, with the most common resistance pattern CipNxSSuT (65.5%) followed by CipNxSuT (17.2%). Pulsed-field gel electrophoresis (PFGE) divided the strains into five clusters (A-E) comprising 16 distinct XbaI PFGE profiles. Sixty-five out of 87 isolates were grouped in clusters A and B, with the predominant PFGE profiles A1 and B1 encompassing 33 and 28 isolates, respectively. A vast majority of the isolates (75/87) showed >90% PFGE profile similarity. The biofilm-forming capacity of the tested isolates, determined with crystal violet assay in polystyrene microwell plates, was generally weak. The average biofilm formation for persistent strains was higher than for presumably nonpersistent strains; however, the difference was not significant. It seems that S. Infantis persistence on broiler farms is more related to its widespread occurrence in the broiler production chain and ineffective disinfection protocols than to its ability to form biofilm.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a fatal disease of honeybee brood. Here, we obtained 506 P. larvae isolates originating from honey or brood samples and from different geographic regions of Slovenia in the period 2017–2019. In the first part of the study, we conducted ERIC-PCR typing to assess the frequency of ERIC types in Slovenia. Capillary electrophoresis was used for the analysis of ERIC patterns, revealing good separation efficiency and enabling easy lane-to-lane comparisons. ERIC II was the predominant type (70.2%), followed by ERIC I (29.8%); two slightly altered ERIC I banding patterns were observed but were not considered relevant for the discrimination of ERIC types. No evident spatiotemporal clustering of ERIC types was observed. To assess the clonality of the outbreak-related P. larvae ERIC I isolates, 59 isolates of this type underwent whole-genome sequencing (WGS). Whole-genome multilocus sequence typing (wgMLST) revealed seven ERIC I-ST2 outbreak clusters (≤35 allele differences) with the median intra-outbreak diversity ranging from 7 to 27 allele differences. In all seven clusters, the transmission of P. larvae outbreak clone within a 3-km radius (AFB zone) was observed, which could be explained by the activity of honeybees. In three clusters, the transmission of the outbreak clone between geographically distant apiaries was revealed, which could be explained by the activities of beekeepers such as migratory beekeeping and trading of bee colonies. The present findings reinforce the importance of beekeeping activities in the transmission of P. larvae. WGS should be used as a reference typing method for the detection of P. larvae transmission clusters.