Comparison of IncK-blaCMY-2 Plasmids in Extended-Spectrum Cephalosporin-Resistant Escherichia coli Isolated from Poultry and Humans in Denmark, Finland, and Germany.

Escherichia coli carrying IncK-blaCMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-blaCMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the blaCMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the blaCMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0-60 and 13-90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-blaCMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-blaCMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-blaCMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.

Effect of feeding dairy calves with milk fermented with selected probiotic strains on occurrence of diarrhoea, carriage of pathogenic and zoonotic microorganisms and growth performance.

Calf-diarrhoea is a major health problem in dairy calves and a primary reason for use of antimicrobials. We aimed to investigate the effect of feeding milk fermented with a combination of four probiotic bacterial strains to young-calves on; occurrence of diarrhoea and associated-pathogens (bacteria, virus and parasites), shedding of Salmonella Dublin and Campylobacter, occurrence of virulence genes linked to Clostridium perfringens, Enterotoxigenic Escherichia coli and shiga-toxin producing E. coli (STEC), as well as growth performance. For this, 143 new-born calves from three Danish dairy-farms were allocated into Treatment- (fed the fermented milk for the first 8-weeks-of-life) and Control-groups (fed regular farm-milk). Diarrhoea was observed in 18.6 % (Farm 1), 22.4 % (Farm 2) and 15.7 % (Farm 3) of the total registrations mainly within the first 3-weeks-of-life. C. perfringens was the most frequently detected pathogen. The treatment did not affect the occurrence of virulence genes linked to STEC and C. perfringens and, overall, their detection levels were very low/undetected. The statistical model applied found no significant effect of the treatment on prevalence of early-diarrhoea (≤ 3 weeks), late-diarrhoea (>3 weeks), occurrence of C. perfringens and Cryptosporidium parvum or levels of Campylobacter spp. Limited detection of the other pathogens and associated virulence-genes under study, did not allow for assessment of the impact of the treatment on their occurrence. Notably, the feeding-approach showed a significant detrimental effect on daily-weight-gain. The inefficacy of the treatment may be associated with the complexity of influencing factors under field conditions including management practices.

Infectious potential of human derived uropathogenic Escherichia coli UTI89 in the reproductive tract of laying hens.

Avian pathogenic E. coli (APEC) and human uropathogenic E. coli (UPEC) harbour common virulence factors in spite of being associated with disease in different hosts. APEC strains have been shown to have zoonotic potential. In contrast, it is not known whether UPEC strains can cause infection in immunologically competent hens. The objective of the current study was to compare the ability of the well-characterized UPEC strain, UTI89, and the APEC strain, F149H1S2, to infect human and avian cells in culture and to cause salpingitis in an infection model in adult laying hens. In vitro characterization showed that the strains grew equally well in human urine, and both were able to infect human intestinal (Int407) and bladder (J82) epithelial cell lines, and they survived in avian macrophages (HD11) to the same extent. Groups of adult birds were inoculated with 108 bacteria directly into the oviduct using a surgical procedure. After an infection period of 48 h, bacterial load in the oviduct was determined by dilution series, and pathology was determined based on gross lesions and histological observations. Similar counts of UPEC UTI89 (ST95) and the APEC strain F149H1S2 (ST117) were obtained from tissues of infected birds, and salpingitis as evaluated by clinical score and histopathology was observed to a similar extent after infection with the two strains. Together, the results showed that UPEC UTI89 and APEC F149H1S2 have a similar potential for causing salpingitis in laying hens in the model used. No infection differences were observed between the UPEC UTI89 wild type and a mutant strain with knock-out of the well-known virulence gene, fimH, (UPEC UTI89ΔfimH), showing that the salpingitis model is not suitable for the detection of all UPEC virulence factors.

Dynamics and Outcome of Macrophage Interaction Between Salmonella Gallinarum, Salmonella Typhimurium, and Salmonella Dublin and Macrophages From Chicken and Cattle.

Salmonella Gallinarum only infects avian species, where it causes a severe systemic infection in birds of all ages. It is generally accepted that interaction with phagocytic cells plays an important role in the development of systemic, host-specific Salmonella infections. The current study detailed the interaction of S. Gallinarum with macrophages derived from chicken (HD11) and cattle (Bomac) compared to interaction of the broad host range serovar, Salmonella Typhimurium and the cattle adapted serovar Salmonella Dublin. Results showed a weaker invading ability of S. Gallinarum in both kinds of macrophages, regardless whether the bacteria were opsonized or not before infections. However, opsonization of S. Gallinarum by chicken serum increased its intracellular survival rate in chicken macrophages. No significant induction of nitrogen oxide was observed in the infected HD11 cells within the first 6 h, and levels of reactive oxygen species (ROS) were similar among the three serovars. S. Gallinarum infection was associated with low cell deaths in both chicken and cattle macrophages, whereas S. Dublin only induced a comparable high level of cell death in chicken macrophages, but not in macrophages of its preferred host species (Bomac) compared to host generalist S. Typhimurium. S. Gallinarum-infected HD11 macrophages exhibited low induction of pro-inflammation genes [interleukin (IL)1ß, CXCLi1, and CXCLi2] compared to the two other serovars, and contrary to the other serovars, it did not induce significant downregulation of Toll-like receptor (TLR)2, TLR4, and TLR5. In in vivo infection of 1-week-old chicken, a significant upregulation of the TLR4 and TLR5 genes in the spleen was observed in S. Gallinarum-infected chickens, but not in S. Typhimurium-infected chicken at 5 days post-infections. Taken together, results show that S. Gallinarum infection of macrophages was characterized by low uptake and low cytotoxicity, possibly allowing long-term persistence in the intracellular environment, and it caused a low induction of pro-inflammatory responses.