Whole-genome sequencing-based antimicrobial resistance and shedding dynamics of Escherichia coli isolated from calves before and after antimicrobial group treatments.

The fattening of calves is often associated with high antimicrobial use and the selection of antimicrobial resistance (AMR). The objective of this observational longitudinal study was to describe the AMR and strain dynamics, using whole-genome sequencing (WGS), of fecal Escherichia coli in a cohort of 22 calves. All calves received antimicrobial group treatments on Day (D) 1 (oxytetracycline, intramuscularly) and on D4 through D12 (doxycycline, in-feed). Additionally, eight calves received individual parenteral treatments between D7 and D59, including florfenicol, amoxicillin, marbofloxacin, and gamithromycin. Rectal swabs were collected from all calves on D1 (prior to treatment), D2, D9, and D82. The swabs were spread onto Enterobacterales-selective agar, and three E. coli colonies per plate were subjected to WGS. Out of 264 isolates across all calves and sampling times, 80 unique strains were identified, a majority of which harbored genes conferring resistance to tetracyclines, streptomycin, and sulfonamides. The diversity of strains decreased during the in-feed antimicrobial group treatment of the calves. On D82, 90% of isolates were strains that were not isolated at previous sampling times, and the median number per strain of AMR determinants to tetracyclines, florfenicol, ß-lactams, quinolones, or macrolides decreased compared to D9. Additionally, clonal dissemination of some strains represented the main transmission route of AMR determinants. In this study, WGS revealed important variations in strain diversity and genotypic AMR of fecal E. coli over time in calves subjected to group antimicrobial treatments. IMPORTANCE: The continued emergence and spread of antimicrobial resistance (AMR) determinants are serious global concerns. The dynamics of AMR spread and persistence in bacterial and animal host populations are complex and not solely driven by antimicrobial selection pressure. In calf fattening, both antimicrobial use and carriage prevalence of antimicrobial-resistant bacteria are generally recognized as high. This study provides new insights into the short-term, within-farm dynamics and transmission of AMR determinants in Escherichia coli from the dominant fecal flora of calves subjected to antimicrobial group treatments during the rearing period. The diversity of E. coli strains decreased over time, although, in contrast to previous observations in extended-spectrum ß-lactamase-producing Enterobacterales, the predominance of a few clones was not observed. The spread of AMR determinants occurred through the dissemination of clonal strains among calves. The median number per strain of AMR determinants conferring resistance to selected antimicrobials decreased toward the end of the rearing period.

Whole-Genome Sequences of a Lactobacillus melliventris Strain and Its Myovirus Temperate Phage, phIBH004, Isolated from the Digestive Tract of Apis mellifera in Switzerland.

The complete genome sequence of Lactobacillus melliventris strain IBH004, isolated from the gut of a honeybee worker (Apis mellifera) and containing two plasmids and a temperate phage, was determined using hybrid assembly of Oxford Nanopore and Illumina reads. Phage-sequence relationships were identified from the coding sequences, and a proteomic tree was constructed.

Intra- and Interspecies Spread of a Novel Conjugative Multidrug Resistance IncC Plasmid Coharboring blaOXA-181 and armA in a Cystic Fibrosis Patient.

A novel multidrug resistance conjugative 177,859-bp IncC plasmid pJEF1-OXA-181 coharboring the carbapenemase-coding blaOXA181 and the aminoglycoside resistance 16S rRNA methyltransferase-coding armA genes was detected in two unrelated Escherichia coli gut isolates of ST196 and ST648, as well as two ST35 Klebsiella pneumoniae gut and sputum isolates of a cystic fibrosis patient. The armA gene was located within the antimicrobial resistance island ARI-A and the blaOXA181 gene, which was preceded by IS903 and ISEcp1Δ was inserted within the transfer genes region without affecting conjugation ability. Comparative plasmid analysis with other related IncC plasmids showed the presence of blaOXA181, as well as its integration site, are thus far unique for these types of plasmids. This study illustrates the potential of a promiscuous multidrug resistance plasmid to acquire antibiotic resistance genes and to disseminate in the gut of the same host. IMPORTANCE Colocalization of carbapenemases and aminoglycoside resistance 16S rRNA methylases on a multidrug resistance conjugative plasmid poses a serious threat to public health. Here, we describe the novel IncC plasmid pJEF1-OXA-181 cocarrying blaOXA-181 and armA as well as several other antimicrobial resistance genes (ARGs) in different Enterobacterales isolates of the sputum and gut microbiota of a cystic fibrosis patient. IncC plasmids are conjugative, promiscuous elements which can incorporate accessory antimicrobial resistance islands making them key players in ARGs spread. This plasmid was thus far unique among IncC plasmids to contain a blaOXA-181 which was integrated in the transfer gene region without affecting its conjugation ability. This study highlights that new plasmids may be introduced into a hospital through different species hosted in one single patient. It further emphasizes the need of continuous surveillance of multidrug-resistant bacteria in patients at risk to avoid spread of such plasmids in the health care system.

Brucella spp. Lumazine Synthase Induces a TLR4-Mediated Protective Response against B16 Melanoma in Mice.

Brucella Lumazine Synthase (BLS) is a highly immunogenic decameric protein which can accept the fusion of foreign proteins at its ten N-termini. These chimeras are very efficient to elicit systemic and oral immunity without adjuvants. BLS signaling via Toll-Like Receptor 4 (TLR4) regulates innate and adaptive immune responses, inducing dendritic cell maturation and CD8(+) T-cell cytotoxicity. In this work we study the effect induced by BLS in TLR4-expressing B16 melanoma. In order to evaluate the effectiveness of BLS as a preventive vaccine, C57BL/6J mice were immunized with BLS or BLS-OVA, and 35 days later were subcutaneously inoculated with B16-OVA melanoma. BLS or BLS-OVA induced a significant inhibition of tumor growth, and 50% of mice immunized with the highest dose of BLS did not develop visible tumors. This effect was not observed in TLR4-deficient mice. For treatment experiments, mice were injected with BLS or BLS-OVA 2 days after the inoculation of B16 cells. Both treatments induced significant and equal tumor growth delay and increased survival. Moreover, BLS and BLS-OVA stimulation were also effective in TLR4-deficient mice. In order to study whether BLS has a direct effect on tumor cells, B16 cells were preincubated with BLS, and after 48h, cells were inoculated. Tumors induced by BLS-stimulated cells had inhibited growth and survival was increased. In the BLS group, 40% of mice did not develop tumors. This effect was abolished by the addition of TLR4/MD2 blocking antibody to cells before BLS stimulation. Our work demonstrates that BLS immunization induces a preventive antitumor response that depends on mice TLR4. We also show that BLS generates a therapeutic effect in mice inoculated with B16 cells. Our results show that BLS acts directly in cultured tumor cells via TLR4, highly suggesting that BLS elicits its therapeutic effects acting on the TLR4 from B16 melanoma cells.