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Enterococcus faecium is an opportunistic pathogen able to colonize the intestines of hospitalized patients. This initial colonization is an important step in the downstream pathogenesis, which includes outgrowth of the intestinal microbiota and potential infection of the host. The impact of intestinal overgrowth on host–enterococcal interactions is not well understood. We therefore applied a RNAseq approach in order to unravel the transcriptional dynamics of E. faecium upon co-culturing with human derived colonic epithelium. Co-cultures of colonic epithelium with a hospital-associated vancomycin resistant (vanA-type) E. faecium (VRE) showed that VRE resided on top of the colonic epithelium when analyzed by microscopy. RNAseq revealed that exposure to the colonic epithelium resulted in upregulation of 238 VRE genes compared to the control condition, including genes implicated in pili expression, conjugation (plasmid_2), genes related to sugar uptake, and biofilm formation (chromosome). In total, 260 were downregulated, including the vanA operon located on plasmid_3. Pathway analysis revealed an overall switch in metabolism to amino acid scavenging and reduction. In summary, our study demonstrates that co-culturing of VRE with human colonic epithelium promotes an elaborate gene response in VRE, enhancing our insight in host– E. faecium interactions, which might facilitate the design of novel anti-infectivity strategies. [ABSTRACT FROM AUTHOR]

Various species of the intestinal microbiota have been associated with the development of colorectal cancer1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5 and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island. Organoids derived from human intestinal cells that are co-cultured with bacteria carrying the genotoxic pks+ island develop a distinct mutational signature associated with colorectal cancer. [ABSTRACT FROM AUTHOR]

The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. A fundamental part of research into E. faecium biology relies on the ability to generate targeted mutants but this process is currently labour-intensive and time-consuming, taking 4 to 5 weeks per mutant. In this report, we describe a method relying on the high recombination rates of E. faecium and the application of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 genome editing tool to more efficiently generate targeted mutants in the E. faecium chromosome. Using this tool and the multi-drug resistant clinical E. faecium strain E745, we generated a deletion mutant in the lacL gene, which encodes the large subunit of the E. faecium β -galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl- β -D-galactopyranoside (X-gal) could be used to distinguish between the wild-type and lacL deletion mutant. We also inserted two copies of gfp into the intrinsic E. faecium macrolide resistance gene msrC to generate stable green fluorescent cells. We conclude that CRISPR-Cas9 can be used to generate targeted genome modifications in E. faecium in 3 weeks, with limited hands-on time. This method can potentially be implemented in other Gram-positive bacteria with high intrinsic recombination rates. [ABSTRACT FROM AUTHOR]