Land application of manure that contains antibiotics and resistant bacteria may facilitate the establishment of an environmental reservoir of antibiotic-resistant microbes, promoting their dissemination into agricultural and natural habitats. The main objective of this study was to search for acquired antibiotic resistance determinants in the gut microbiota of wild boar populations living in natural habitats. Gastrointestinal samples of free-living wild boars were collected in the Zemplén Mountains in Hungary and were characterised by culture-based, metagenomic, and molecular microbiological methods. Bioinformatic analysis of the faecal microbiome of a hunted wild boar from Japan was used for comparative studies. Also, shotgun metagenomic sequencing data of two untreated sewage wastewater samples from North Pest (Hungary) from 2016 were analysed by bioinformatic methods. Minimum spanning tree diagrams for seven-gene MLST profiles of 104 E. coli strains isolated in Europe from wild boars and domestic pigs were generated in Enterobase. In the ileum of a diarrhoeic boar, a dominant E. coli O112ab:H2 strain with intermediate resistance to gentamicin, tobramycin, and amikacin was identified, displaying sequence type ST388 and harbouring the EAST1 toxin astA gene. Metagenomic analyses of the colon and rectum digesta revealed the presence of the tetQ, tetW, tetO, and mefA antibiotic resistance genes that were also detected in the gut microbiome of four other wild boars from the mountains. Furthermore, the tetQ and cfxA genes were identified in the faecal microbiome of a hunted wild boar from Japan. The gastrointestinal microbiota of the free-living wild boars examined in this study carried acquired antibiotic resistance determinants that are highly prevalent among domestic livestock populations.

Introduction: The aim of the study was to determine microbiota in the cloacal samples of European herring gulls (Larus argentatus) and to compare a variety of genes encoding antimicrobial resistance in cultivable and non-cultivable bacteria. Material and Methods: Cloacal samples from European herring gulls were collected from a Kaunas city dump. Cultivable microbiota were isolated, their microbial susceptibility was tested, and genes encoding antimicrobial resistance were detected. Additionally, a metagenomic study was performed using Next-Generation Sequencing (NGS). Results: In total, 697 different operational taxonomic units at genus level were detected; however, only 63 taxonomic units were detected at the amount of ≥0.1% of the total number of DNA copies. Catellicoccus marimammalium was found to have the highest prevalence. The bacterial amount of other genera was up to 5% with the most highly prevalent being Psychrobacter (4.7%), Helicobacter (4.5%), unclassified Enterococcaceae (3.2%), Pseudomonas (2.9%), and Brachyspira (2.6%). Conclusions: C. marimammalium are predominant microbiota in the cloacal samples of Larus argentatus. This species of gulls is a reservoir of bacteria carrying a wide-spectrum of genes encoding antimicrobial resistance. The same genes were detected in both cultivable microbiota and in the total DNA of the samples.

High-throughput sequencing (HTS) identifies random viral fragments in environmental samples metagenomically. High reliability gains it broad application in virus evolution, host-virus interaction, and pathogenicity studies. Deep sequencing of field samples with content of host genetic material and bacteria often produces insufficient data for metagenomics and must be preceded by target enrichment. The main goal of the study was the evaluation of HTS for complete genome sequencing of field-case rabies viruses (RABVs). The material was 23 RABVs isolated mainly from red foxes and one European bat lyssavirus-1 isolate propagated in neuroblastoma cells. Three methods of RNA isolation were tested for the direct metagenomics and RABV-enriched approaches. Deep sequencing was performed with a MiSeq sequencer (Illumina) and reagent v3 kit. Bioinformatics data were evaluated by Kraken and Centrifuge software and de novo assembly was done with metaSPAdes. Testing RNA extraction procedures revealed the deep sequencing scope superiority of the combined TRIzol/column method. This HTS methodology made it possible to obtain complete genomes of all the RABV isolates collected in the field. Significantly greater rates of RABV genome coverages (over 5,900) were obtained with RABV enrichment. Direct metagenomic studies sequenced the full length of 6 out of 16 RABV isolates with a medium coverage between 1 and 71. Direct metagenomics gives the most realistic illustration of the field sample microbiome, but with low coverage. For deep characterisation of viruses, e.g. for spatial and temporal phylogeography during outbreaks, target enrichment is recommended as it covers sequences much more completely.

OBJECTIVE To evaluate erythema and number of CFUs on the skin of dogs with hair clipped by use of 2 sizes of clipper blades. ANIMALS 67 client-owned dogs receiving an epidural. PROCEDURES Hair was clipped with a No. 10 blade (approx hair length, 1.5 mm) on one half and a No. 40 blade (approx hair length, 0.25 mm) on the other half of each epidural site. Skin was surgically scrubbed with 2% chlorhexidine gluconate and 70% isopropyl alcohol. Samples were obtained immediately after clipping, after skin was scrubbed, and again 24 hours after clipping. Number of CFUs for both sides of the clipped areas, types of microorganisms, and growth on MacConkey agar were evaluated every 24 hours for 72 hours. Colonies were evaluated for bacterial morphology and Gram stain characteristics. Sites were evaluated 24 hours after clipping for evidence of erythema. RESULTS 24 hours after hair was clipped, there was a significantly higher incidence of erythema and higher number of Micrococcaceae bacteria for the side clipped with the No. 40 blade than the side clipped with the No. 10 blade. Number of CFUs did not differ significantly between size of clipper blades. CONCLUSIONS AND CLINICAL RELEVANCE Clipping hair with a No. 40 blade resulted in a significant increase in the incidence of erythema and higher number of Micrococcaceae bacteria, compared with results for clipping with a No. 10 blade. These results supported use of a No. 10 clipper blade to prevent erythema and reduce variation in the skin microbiome.

Bovine mastitis is an important microbial disease in the dairy industry. We investigated the frequencies of bacterial pathogens in 62 farms and pathogen antibiotic resistance from mastitis samples (n = 748). We tested the antimicrobial activity of chicken and duck egg white and lysozyme purified from chicken egg white. Moreover, we compared the microbiomes of normal and mastitic raw milk obtained by 16S rRNA gene pyrosequencing and culture methods. The results showed that the frequencies of Gram-positive pathogens (Enterococcus faecalis 37% and Staphylococcus aureus 36%) were higher than that of a Gram-negative pathogen (Escherichia coli 15%). Resistance frequencies to ampicillin and norfloxacin were lowest in Staphylococcus aureus (21%), Enterococcus faecalis (23%), and Escherichia coli (33%), and the antimicrobial activity of chicken egg white was higher than those of lysozyme and duck egg white. Pyrosequencing results revealed clear differences between the microbiomes of mastitic and normal raw milk samples and revealed a slightly similar, but clearly different, composition of pathogens compared to that from the culture method. Thus, pyrosequencing may be useful for elucidating changes in microbiomes during mastitis progression and treatment. A chicken egg white and antibiotic combination may help with mastitis treatment; however, further studies are needed.