title: 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: In 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-cytolysin-negative 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.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Laboratory of Microbial Gene Technology and Food Microbiology, Norwegian University of Life Sciences, Ås, Norway Department of Internal Medicine, Division of Infectious Diseases, University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Sabina Leanti
authors.initials: SL
authors.lastname: La Rosa
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Section for Biostatistics, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: Lars-Gustav
authors.initials: LG
authors.lastname: Snipen
authors.affiliation: Department of Internal Medicine, Division of Infectious Diseases, University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Murray
authors.affiliation: Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Rob J L
authors.initials: RJ
authors.lastname: Willems
authors.affiliation: Departments of Ophthalmology, Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Gilmore
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Laboratory of Microbial Gene Technology and Food Microbiology, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: Dzung B
authors.initials: DB
authors.lastname: Diep
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Laboratory of Microbial Gene Technology and Food Microbiology, Norwegian University of Life Sciences, Ås, Norway.
authors.forename: Ingolf F
authors.initials: IF
authors.lastname: Nes
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Laboratory of Microbial Gene Technology and Food Microbiology, Norwegian University of Life Sciences, Ås, Norway dag.anders.brede@nmbu.no.
authors.forename: Dag Anders
authors.initials: DA
authors.lastname: Brede
mesh_terms: D000328:Adult; D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D017173:Caenorhabditis elegans / Q000382:microbiology* / Q000502:physiology*; D004195:Disease Models, Animal; D013293:Enterococcus faecalis / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification / Q000821:virology; D016680:Genome, Bacterial; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology; D006801:Humans; D007223:Infant; D007313:Insecta / Q000382:microbiology; D058885:Multilocus Sequence Typing; D039002:Prophages / Q000235:genetics*; D016019:Survival Analysis; D013559:Symbiosis; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics*
chemical_list: D037521:Virulence Factors