2. Universidad Cardenal Herrera-CEU

Listeria monocytogenes is a major human and animal foodborne patho-gen. However, data from environmental reservoirs remain scarce. Here, we usedwhole-genome sequencing to characterize Listeria species isolates recovered over 1year from wild animals in their natural habitats in Spain. Three different Listeria spp.(L. monocytogenes [n = 19], Listeria ivanovii subsp. londoniensis [n = 4], and Listeriainnocua [n = 3]) were detected in 23 animal tonsils (9 deer, 14 wild boars) and 2feeding troughs. No Listeria species was detected in feces. L. monocytogenes wasdetected in tonsils of 44.4% (8 out of 18) of deer and 40.7% (11 out of 27) of wildboars. L. monocytogenes isolates belonged to 3 different core genome multilocussequence typing (cgMLST) types (CTs) of 3 distinct sublineages (SL1, SL387, andSL155) from lineages I and II. While cgMLST type L1-SL1-ST1-CT5279 (IVb; clonalcomplex 1 [CC1]) occurred only in one animal, types L1-SL387-ST388-CT5239 (IVb;CC388) and L2-SL155-ST155-CT1170 (IIa; CC155) were retrieved from multiple ani-mals. In addition, L1-SL387-ST388-CT5239 (IVb; CC388) isolates were collected 1 yearapart, revealing their long-term occurrence within the animal population and/orenvironmental reservoir. The presence of identical L. monocytogenes strains in deerand wild boars suggests contamination from a common food or environmentalsource, although interhost transmission cannot be excluded. Pathogenicity islandsLIPI-1, LIPI-3, and LIPI-4 were present in 100%, 5%, and 79% of the L. monocytogenesisolates, respectively, and all L. monocytogenes lineage II isolates (n = 3) carried SSI-1stress islands. This study highlights the need for monitoring L. monocytogenes envi-ronmental contamination and the importance of tonsils as a possible L. monocyto-genes intrahost reservoir.

Background: Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis, an infectious disease in animals and people, with pigs acting as asymptomatic reservoirs. In August 2019 an outbreak associated with the consumption of pork meat caused 222 human cases of listeriosis in Spain. Determining the diversity as well as the virulence potential of strains from pigs is important to public health. Methods: The behaviour of 23 L monocytogenes strains recovered from pig tonsils, meat and skin was compared by studying (1) internalin A, internalin B, listeriolysin O, actin assembly-inducing protein and PrfA expression levels, and (2) their invasion and intracellular growth in eukaryotic cells. Results: Marked differences were found in the expression of the selected virulence factors and the invasion and intracellular replication phenotypes of L monocytogenes strains. Strains obtained from meat samples and belonging to serotype 1/2a did not have internalin A anchored to the peptidoglycan. Some strains expressed higher levels of the studied virulence factors and invaded and replicated intracellularly more efficiently than an epidemic L monocytogenes reference strain (F2365). Conclusion: This study demonstrates the presence of virulent L monocytogenes strains with virulent potential in pigs, with valuable implications in veterinary medicine and food safety.

Listeriosis is a zoonotic disease caused by Listeria monocytogenes and Listeria ivanovii. The genus Listeria currently includes 27 recognized species and is found throughout the environment. The number of systematic studies on antimicrobial resistance in L. monocytogenes isolates from domestic farms using antimicrobial substances is limited. Importantly, dairy ruminant farms are reservoir of hypervirulent lineage I L. monocytogenes isolates, previously associated with human clinical cases. Considering that the classes of antibiotics used in food-producing domestic animals are frequently the same or closely related to those used in human medicine, studies about the impact of antibiotic use on the acquisition of antibiotic resistance in Listeria spp. in domestic animal farms are, therefore, of high importance. Here, susceptibility to 25 antibiotics was determined. Eighty-one animal-related, 35 food and 21 human pathogenic Listeria spp. isolates and 114 animal-related non-pathogenic Listeria spp. isolates were tested. Whole genome sequencing data was used for molecular characterization. Regarding L. monocytogenes, 2 strains from the clinical-associated linage I showed resistance to erythromycin, both related to dairy ruminants. Acquired resistance to one antibiotic was exhibited in 1.5% of L. monocytogenes isolates compared with 14% of non-pathogenic Listeria spp. isolates. Resistance to tetracycline (7.9%), doxycycline (7.9%), penicillin (4.4%), and ampicillin (4.4%) were the most frequently observed in non-pathogenic Listeria spp. While resistance to two or more antibiotics (5.6%) was most common in Listeria spp., isolates, resistance to one antibiotic was also observed (1.6%). The present results show that non-pathogenic Listeria spp. harbour antimicrobial resistance genes.