A Genomic Virulence Reference Map of Enterococcus faecalis Reveals an Important Contribution of Phage03-Like Elements in Nosocomial Genetic Lineages to Pathogenicity in a Caenorhabditis elegans Infection Model

Abstract

eIn the present study, the commensal and pathogenic host-microbe interaction of Enterococcus faecalis was explored using a Caenorhabditis elegans model system. The virulence of 28 E. faecalis isolates representing 24 multilocus sequence types (MLSTs), including human commensal and clinical isolates as well as isolates from animals and of insect origin, was investigated using C. elegans strain glp-4 (bn2ts); sek-1 (km4). This revealed that 6 E. faecalis isolates behaved in a commensal manner with no nematocidal effect, while the remaining strains showed a time to 50% lethality ranging from 47 to 120 h. Principal component analysis showed that the difference in nematocidal activity explained 94% of the variance in the data. Assessment of known virulence traits revealed that gelatinase and cytolysin production accounted for 40.8% and 36.5% of the observed pathogenicity, respectively. However, coproduction of gelatinase and cytolysin did not increase virulence additively, accounting for 50.6% of the pathogenicity and therefore indicating a significant (26.7%) saturation effect. We employed a comparative genomic analysis approach using the 28 isolates comprising a collection of 82,356 annotated coding sequences (CDS) to identify 2,325 patterns of presence or absence among the investigated strains. Univariate statistical analysis of variance (ANOVA) established that individual patterns positively correlated (n ‫؍‬ 61) with virulence. The patterns were investigated to identify potential new virulence traits, among which we found five patterns consisting of the phage03-like gene clusters. Strains harboring phage03 showed, on average, 17% higher killing of C. elegans (P ‫؍‬ 4.4e ؊6 ). The phage03 gene cluster was also present in gelatinase-and-cytolysinnegative strain E. faecalis JH2-2. Deletion of this phage element from the JH2-2 clinical strain rendered the mutant apathogenic in C. elegans, and a similar mutant of the nosocomial V583 isolate showed significantly attenuated virulence. Bioinformatics investigation indicated that, unlike other E. faecalis virulence traits, phage03-like elements were found at a higher frequency among nosocomial isolates. In conclusion, our report provides a valuable virulence map that explains enhancement in E. faecalis virulence and contributes to a deeper comprehension of the genetic mechanism leading to the transition from commensalism to a pathogenic lifestyle. Enterococcus faecalis is a Gram-positive commensal bacterium of the mammalian gastrointestinal (GI) tract (1) and a leading cause of nosocomial infections worldwide (2). In humans, the normal abundance of E. faecalis in the intestinal lumen ranges between 10 5 and 10 8 CFU/g of feces without causing any obvious deleterious effects on the host (3, 4). However, if either perturbations of the host/commensal balance that weaken the host immune system or environmental factors such as use of antibiotics that inadvertently facilitate outgrowth of resistant E. faecalis occur, life-threatening infections might arise. Moreover, the intri...

Global Emergence and Dissemination of Enterococci as Nosocomial Pathogens: Attack of the Clones?

“…As mentioned above the population structure of E. faecalis seems to be significantly different from E. faecium as no clearly defined E. faecalis ecotypes appear to exist. Correspondingly, previous studies have reported shared antibiotic resistance and virulence genes, such as the E. faecalis pathogenicity island, esp , capsule polysaccharide genes, and genes encoding for gelatinase, aggregation substance, cytolysin, and Ace, among E. faecalis isolates from a wide variety of different niches like the hospital, animals, food products and the environment ( Eaton and Gasson, 2001 ; Creti et al, 2004 ; McBride et al, 2007 ; Freitas et al, 2009 ; Solheim et al, 2009 ; La Rosa et al, 2015 ). Note that the absence of clearly defined ecotypes in E. faecalis can be interpreted as a high multiplicity of closely related fine-grained ecotypes, none of them reaching predominance.…”

Comparative genomics of global optrA-carrying Enterococcus faecalis uncovers a common chromosomal hotspot for optrA acquisition within a diversity of core and accessory genomes

“…Intact or relics of prophages highly identical to sequences from common enterococcal prophages such as phiL1A and vB_IME197 were identified in most strains, but the exact phage(s) present in each genome cannot be ruled out with accuracy. The phiL1A and vB_IME197 phages are widespread in E. faecalis from variable sources [31] and were originally described in clinical settings in UK and China, respectively [54,55] [58]. In this study, this phage was putatively identified in three related ST476 strains from hospitalized patients, farm and retail chicken in China and Tunisia (Table S6), once more enphasizing the lack of a demarcation between clinical and non-clinical genomes in relevant mobile genetic elements (MGEs).…”

Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF

“…Many studies of enterococcal virulence and pathogenesis have understandably focused on gene products encoded by mobile elements identified in clinical isolates from infections ( 7 , 8 ). In contrast, we and others have identified loci within the conserved core genome that are also required for virulence ( 9 – 14 ).…”