ABSTRACT
Enterococcus faecalis is a common commensal bacterium in animal gastrointestinal (GI) tracts and a leading cause of opportunistic infections of humans in the modern health care setting. E. faecalis OG1RF is a plasmid-free strain that contains few mobile elements yet retains the robust survival characteristics, intrinsic antibiotic resistance, and virulence traits characteristic of most E. faecalis genotypes. To facilitate interrogation of the core enterococcal genetic determinants for competitive fitness in the GI tract, biofilm formation, intrinsic antimicrobial resistance, and survival in the environment, we generated an arrayed, sequence-defined set of chromosomal transposon insertions in OG1RF. We used an orthogonal pooling strategy in conjunction with Illumina sequencing to identify a set of mutants with unique, single Himar-based transposon insertions. The mutants contained insertions in 1,926 of 2,651 (72.6%) annotated open reading frames and in the majority of hypothetical protein-encoding genes and intergenic regions greater than 100 bp in length, which could encode small RNAs. As proof of principle of the usefulness of this arrayed transposon library, we created a minimal input pool containing 6,829 mutants chosen for maximal genomic coverage and used an approach that we term SMarT (sequence-defined mariner technology) transposon sequencing (TnSeq) to identify numerous genetic determinants of bile resistance in E. faecalis OG1RF. These included several genes previously associated with bile acid resistance as well as new loci. Our arrayed library allows functional screening of a large percentage of the genome with a relatively small number of mutants, reducing potential effects of bottlenecking, and enables immediate recovery of mutants following competitions. IMPORTANCE The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest.
ABSTRACT
Bacterial biofilms are intrinsically resistant to antimicrobial treatment, which contributes to microbial persistence in clinical infections. Enterococcus faecalis is an opportunistic pathogen that readily forms biofilms and is the most prevalent enterococcal species identified in healthcare-associated infections. Since intrinsic resistance to multiple antibiotics is common for enterococci, and antibiotic resistance is elevated in biofilm populations, it is imperative to understand the mechanisms involved. Previously, we identified two glycosyltransferase genes whose disruption resulted in impaired nascent biofilm formation in the presence of antibiotic concentrations subinhibitory for parent growth and biofilm formation. The glycosyltransferases are involved in synthesis of the cell-wall-associated rhamnopolysaccharide Epa. Here we examined the effect of epa mutations on the temporal development of E. faecalis biofilms, and on the effects of antibiotics on pre-formed biofilms using scanning electron microscopy. We show that ΔepaOX mutant cells arrange into complex multidimensional biofilms independent of antibiotic exposure, while parent cells form biofilms that are monolayers in the absence of antibiotics. Remarkably, upon exposure to antibiotics parent biofilm cells restructure into complex three-dimensional biofilms resembling those of the ΔepaOX mutant without antibiotics. All biofilms exhibiting complex cellular architectures were less structurally stable than monolayer biofilms, with the biofilm cells exhibiting increased detachment. Our results indicate that E. faecalis biofilms restructure in response to cellular stress whether induced by antibiotics in the case of parent cells, or by deficiencies in Epa composition for the ΔepaOX strain. The data demonstrate a link between cellular architecture and antibiotic resistance of E. faecalis biofilms.