BackgroundListeria monocytogenes is 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 of Listeria monocytogenes strains 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.ResultsFor 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.ConclusionsHere, 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 of L. monocytogenes isolates 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.

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 of Enterococcus faecalis and Enterococcus faecium isolates from human clinical specimens and retail red meat in Slovenia. A total of 242 isolates were investigated: 101 from humans (71 E. faecalis, 30 E. faecium) and 141 from fresh beef and pork (120 E. faecalis, 21 E. 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% of E. faecalis and 73.3% E. faecium clinical isolates, with a clinical origin (Fisher’s exact test). Among meat isolates, in total 66.0% of E. faecalis and E. faecium isolates were susceptible to all antimicrobials tested and 32.6% were resistant to either one or two antimicrobials. In E. faecalis, several virulence genes were significantly associated with a clinical origin; the most common (31.0%) gene pattern included all the tested genes except hyl. In meat isolates, the virulence genes were detected in E. faecalis only and the most common pattern included ace, efaA, and gelE (32.5%), of which gelE showed a statistically significant association with a clinical origin. These results emphasize the importance of E. faecalis in red meat as a reservoir of virulence genes involved in its persistence and human infections with reported severe outcomes.

Paenibacillus larvae causes the American foulbrood (AFB), a highly contagious and devastating disease of honeybees. Whole-genome sequencing (WGS) has been increasingly used in bacterial pathogen typing, but rarely applied to study the epidemiology of P. larvae. To this end, we used 125 P. larvae genomes representative of a species-wide diversity to construct a stable whole-genome multilocus sequence typing (wgMLST) scheme consisting of 5745 loci. A total of 51 P. larvae isolates originating from AFB outbreaks in Slovenia were used to assess the epidemiological applicability of the developed wgMLST scheme. In addition, wgMLST was compared with the core-genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analyses. All three approaches successfully identified clusters of outbreak-associated strains, which were clearly separated from the epidemiologically unlinked isolates. High levels of backward comparability of WGS-based analyses with conventional typing methods (ERIC-PCR and MLST) were revealed; however, both conventional methods lacked sufficient discriminatory power to separate the outbreak clusters. The developed wgMLST scheme provides an improved understanding of the intra- and inter-outbreak genetic diversity of P. larvae and represents an important progress in unraveling the genomic epidemiology of this important honeybee pathogen.

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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.