Genomic analyses of Enterococcus faecium: vancomycin-resistant isolates from Dallas, Texas and occurance of CRISPR-Cas


The Gram-positive bacterium Enterococcus faecium is among the most prominent causes of hospital-acquired infections (HAI). Multi-drug resistant (MDR) strains have emerged, including those resistant to last-resort antibiotics like vancomycin. Therefore, infections caused by MDR strains leave very few treatment options and impose a major health problem. The vancomycin resistance genes and many other antibiotic resistance genes in E. faecium are encoded on mobile genetic elements (MGEs) such as plasmids or transposons that can be rapidly disseminated. The MGEs themselves continue to evolve through mutation and recombination, changing their mobility, maintenance, and host range. Therefore, surveillance of MDR strains, and specifically their MGEs, is critical. The genome defense mechanism, CRISPR-Cas, is an established system of defense against MGE acquisition in many bacteria but has not been explored in E. faecium extensively. The primary focus of the research presented here was the genomics analysis of vancomycin-resistant E. faecium strains (VREfm) obtained during routine fecal surveillance of high-risk patients from a Dallas, Texas area hospital system, with an emphasis on their plasmids and the transposon Tn1546, which are responsible for dissemination of vancomycin resistance genes. High-quality genome sequences were achieved for 47 E. faecium isolates through the hybrid assemblies of long (Oxford Nanopore Technology) and short (Illumina) sequence reads. A total of 251 closed plasmid sequences were generated and were assigned to 12 previously described and 9 newly defined rep family types. New variants of the Tn1546 were described that harbored a combination of 7 insertion sequences (IS) including 3 novel IS elements reported in this study. Overall, it was concluded that the VREfm isolates analyzed in our collection possess novel arrangements of the Tn1546-like elements and the vanA operon, which have evolved independently of the pRUM-like backbone carrying them. A second part of the research analyzed the occurrence of a previously reported Type II CRISPR-Cas system among E. faecium genomes and experimentally assessed its activity against plasmid acquisition. Publicly available genome sequences of 817 E. faecium strains were screened. Only 2.9% of the strains were identified to harbor the Type II EfmCRISPR-Cas system, which is characterized by the cas9 gene. Of the 61 non-redundant CRISPR spacers identified, 32.8% (20 out of 61) had sequence identity with bacteriophage genomes, indicating the hypothetical anti-phage role of CRISPR-Cas in E. faecium. The activity of the EfmCRISPR-Cas system was experimentally determined in the E. faecium strain 1,141,733. A cas9-dependent significant decrease in the acquisition of a small mobilizable plasmid was observed. Overall, this study identified and classified diverse plasmids harbored by Dallas, Texas, VREfm and demonstrated that the EfmCRISPR-Cas is able to defend E. faecium against plasmid acquisition, thus this system could be engineered to prevent antibiotic resistance dissemination among E. faecium strains.



Drug resistance in microorganisms, Enterococcus, CRISPR (Genetics)