Fate of CMY-2-Encoding Plasmids Introduced into the Human Fecal Microbiota by Exogenous Escherichia coli.

The gut is a hot spot for transfer of antibiotic resistance genes from ingested exogenous bacteria to the indigenous microbiota. The objective of this study was to determine the fate of two nearly identical blaCMY-2-harboring plasmids introduced into the human fecal microbiota by two Escherichia coli strains isolated from a human and from poultry meat. The chromosome and the CMY-2-encoding plasmid of both strains were labeled with distinct fluorescent markers (mCherry and green fluorescent protein [GFP]), allowing fluorescence-activated cell sorting (FACS)-based tracking of the strain and the resident bacteria that have acquired its plasmid. Each strain was introduced into an established in vitro gut model (CoMiniGut) inoculated with individual feces from ten healthy volunteers. Fecal samples collected 2, 6, and 24 h after strain inoculation were analyzed by FACS and plate counts. Although the human strain survived better than the poultry meat strain, both strains transferred their plasmids to the fecal microbiota at concentrations as low as 102 CFU/ml. Strain survival and plasmid transfer varied significantly depending on inoculum concentration and individual fecal microbiota. Identification of transconjugants by 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that the plasmids were predominantly acquired by Enterobacteriaceae species, such as E. coli and Hafnia alvei Our experimental data demonstrate that exogenous E. coli of human or animal origin can readily transfer CMY-2-encoding IncI1 plasmids to the human fecal microbiota. Small amounts of the exogenous strain are sufficient to ensure plasmid transfer if the strain is able to survive the gastric environment.

Engineered phage with antibacterial CRISPR-Cas selectively reduce E. coli burden in mice.

Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR-Cas machinery to specifically target E. coli. We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli, which may cause fatal infections in hematological cancer patients.

Mixed effect modeling of tetracycline resistance levels in Danish slaughter pigs.

Mathematical and statistical modeling can be a very useful tool in understanding and fighting antimicrobial resistance (AMR). Here we present investigations of mixed effect models of varying complexity in order to identify and address possible management factors affecting the tetracycline AMR levels in Danish pig farms. Besides antimicrobial exposure during pigs life cycle, the type of production seems to also have an influence. The results concludes that not only fully integrated farms (CHR integrated) but also farms in a production network with a single ownership (CVR integrated) might have a preventive effect on levels of tetracycline AMR compared to more complex trading patterns.

New insights into the biodiversity of coliphages in the intestine of poultry.

Despite phages' ubiquitous presence and great importance in shaping microbial communities, little is known about the diversity of specific phages in different ecological niches. Here, we isolated, sequenced, and characterized 38 Escherichia coli-infecting phages (coliphages) from poultry faeces to gain a better understanding of the coliphage diversity in the poultry intestine. All phages belonged to either the Siphoviridae or Myoviridae family and their genomes ranged between 44,324 and 173,384 bp, with a G+C content between 35.5 and 46.4%. Phylogenetic analysis was performed based on single "marker" genes; the terminase large subunit, portal protein, and exonucleases, as well as the full draft genomes. Single gene analysis resulted in six distinct clusters. Only minor differences were observed between the different phylogenetic analyses, including branch lengths and additional duplicate or triplicate subclustering. Cluster formation was according to genome size, G+C content and phage subfamily. Phylogenetic analysis based on the full genomes supported these clusters. Moreover, several of our Siphoviridae phages might represent a novel unclassified phage genus. This study allowed for identification of several novel coliphages and provides new insights to the coliphage diversity in the intestine of poultry. Great diversity was observed amongst the phages, while they were isolated from an otherwise similar ecosystem.