This study aimed to characterize in silico Enterotoxigenic E. coli F4- and F18-positive-isolates (n=90) causing swine post-weaning diarrhea, including pathogenic potential, phylogenetic relationship, antimicrobial and biocide resistance (R), prophage content and metal tolerance rates. F4-strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 MLST-types. F18-strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23 and ST42. The highest rates of antimicrobial-R were found against streptomycin, sulfamethoxazole, tetracycline, trimethoprim and ampicillin. No resistance was found towards ciprofloxacin, cefotaxime, ceftiofur and colistin. Genes conferring tolerance to copper (showing highest diversity), cadmium, silver and zinc were predicted in all genomes. Enterotoxins (ltcA; 100 % F4, 62 % F18, astA; 100 % F4, 38.1 % F18, sta;18.8 % F4, 38.1 % F18, stb; 100 % F4, 76.2 % F18) and fimbriae encoding genes typed as F4ac and F18ac were detected in all the strains, in addition to up to 16 other virulence genes in individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each strain. High diversity of plasmid replicons was shown; IncFII, IncFIB and IncFIC were prevalent among F4-isolates while IncI1, and IncX1 were dominant among F18-strains. Interestingly, F4-isolates from the early 90′s belonged to the same clonal group detected for most of the F4-strains from 2018-2019 (ONT:H10-A-ST100-CH27-0). The low number of SNPs differences between the oldest and recent F4-ST100-isolates suggests a relative stable genome. Overall, the isolates analyzed in this study showed remarkable different genetic traits depending on the fimbria type. IMPORTANCE Diarrhea in the post-weaning period due to Enterotoxigenic E. coli (ETEC) is an economically relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by Zinc-oxide administration (discontinued by 2022). Besides, a breeding program has been implemented aiming to reduce this illness. Treatment with antimicrobials contributes to the problem of antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand the genetic population structure and variation among isolates diarrhea-associated by whole genome sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds and showed high similarity to ETEC-ST100-isolates from China, USA and Spain. High rates of AMR and high diversity of virulence genes were detected. Characterization of ETEC diarrhea-related is important for understanding the disease epidemiology and pathogenesis, and for implementation of new strategies aiming to reduce the impact of the disease in pig production.

Multidrug-resistant Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02–03/002 (Δst313-td) followed by complementation (02-03/002-C). Δst313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n = 82) and 100% of S. Dublin strains (n = 50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n = 82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313.

This is the first report on the genetic diversity of commensal E. coli from pigs reared in an antibiotic free production system and belonging to different age groups. The study investigated the genetic diversity and relationship of 900 randomly collected commensal E. coli strains from non-antimicrobial treated pigs assigned to five different age groups in a Danish farm. Fifty-two unique REP profiles were detected suggesting a high degree of diversity. The number of strains per pig ranged from two to 13. The highest and the lowest degree of diversity were found in the early weaners group (Shannon diversity index, H' of 2.22) and piglets (H' of 1.46) respectively. The REP profiles, R1, R7 and R28, were the most frequently observed in all age groups. E. coli strains representing each REP profile and additional strains associated with the dominant profiles were subjected to PFGE and were assigned to 67 different genotypes. Whole genome sequence analysis of 52 isolates leading to unique REP profiles identified a high level of sequence variation. Six and six strains were assigned to sequence type ST10 and sequence type ST58, respectively. Virulence and antimicrobial resistance genes, as well as, genes associated with mobile genetic elements were commonly found among these commensal E. coli strains. Interestingly, strains yielding the three most common REP profiles clustered together in the SNPs phylogenetic tree, and such strains may represent the archetypal commensal E. coli in Danish pigs.

In the current study, we identified plasmids carrying antimicrobial resistance genes in draft whole genome sequences of 16 selected Salmonella enterica isolates representing six different serovars from humans in Ghana. The plasmids and the location of resistance genes in the genomes were predicted using a combination of PlasmidFinder, ResFinder, plasmidSPAdes and BLAST genomic analysis tools. Subsequently, S1-PFGE was employed for analysis of plasmid profiles. Whole genome sequencing confirmed the presence of antimicrobial resistance genes in Salmonella isolates showing multidrug resistance phenotypically. ESBL, either bla_TEM52−B or bla_CTX−M15 were present in two cephalosporin resistant isolates of S. Virchow and S. Poona, respectively. The systematic genome analysis revealed the presence of different plasmids in different serovars, with or without insertion of antimicrobial resistance genes. In S. Enteritidis, resistance genes were carried predominantly on plasmids of IncN type, in S. Typhimurium on plasmids of IncFII(S)/IncFIB(S)/IncQ1 type. In S. Virchow and in S. Poona, resistance genes were detected on plasmids of IncX1 and TrfA/IncHI2/IncHI2A type, respectively. The latter two plasmids were described for the first time in these serovars. The combination of genomic analytical tools allowed nearly full mapping of the resistance plasmids in all Salmonella strains analyzed. The results suggest that the improved analytical approach used in the current study may be used to identify plasmids that are specifically associated with resistance phenotypes in whole genome sequences. Such knowledge would allow the development of rapid multidrug resistance tracking tools in Salmonella populations using WGS.