Characterization of clinical Enterococcus faecalis small-colony variants - PubMed (original) (raw)

Case Reports

. 2009 Sep;47(9):2802-11.

doi: 10.1128/JCM.00485-09. Epub 2009 Jul 15.

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Case Reports

Characterization of clinical Enterococcus faecalis small-colony variants

Nele Wellinghausen et al. J Clin Microbiol. 2009 Sep.

Abstract

In this report, we present a clinical case of chronic aortic valve endocarditis caused by Enterococcus faecalis small-colony variants (SCVs), with ensuing characterization of the SCV phenotype in comparison to the clonally related normal phenotype with respect to alterations in microscopic and ultrastructural morphology, growth behavior, and metabolic pathways. In contrast to the normal phenotype, light and electron microscopy of the Enterococcus SCVs demonstrated the presence of heterogeneous cells of different sizes with aberrant shapes. Furthermore, SCVs showed excessive production of an intercellular substance and alterations in cell division displayed by a thick, coarse cell wall and incomplete, branched, and multiple cross walls without obvious cell separation. In addition, empty "ghost" cells were visible. In growth experiments, SCVs displayed an extended lag phase with delayed entrance into the stationary phase. Interestingly, SCV cells growing under aerobic conditions did not attain the growth and viability of the normal phenotype or those of SCVs growing under microaerobic conditions, suggesting impaired growth behavior and enhanced vulnerability in the presence of oxygen. By metabolite analysis, SCVs failed to produce significant amounts of acetate or lactate under aerobic growth conditions but were able to produce lactate under microaerobic growth conditions, implicating the induction of a fermentative metabolism. In conclusion, the observed structural alterations and changes in the cellular growth and metabolic pathways facilitated the survival of Enterococcus SCVs under microaerobic conditions in vitro and thus presumably in vivo during endocarditis.

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Figures

FIG. 1.

FIG. 1.

Colony morphologies of E. faecalis wild-type and SCV phenotypes on CASO blood agar after 24 h of incubation.

FIG. 2.

FIG. 2.

PFGE of E. faecalis wild-type and SCV clinical isolates after SmaI restriction. Lanes 1 and 5, 48.5- to 970-kb molecular weight marker (lambda λ_c_1857_S_am7; Bio-Rad, Germany); lane 2, wild-type phenotype; lane 3, SCV phenotype; lane 4, E. faecalis reference strain ATCC 29212.

FIG. 3.

FIG. 3.

Gram staining of SCV (A) and wild-type (B) cells after 24 h of incubation on CASO blood agar.

FIG. 4.

FIG. 4.

SEM of SCV (A) and wild-type (B) cells. Magnification, ×20,000.

FIG. 5.

FIG. 5.

TEM of SCV (A to D) and wild-type (E and F) cells. White arrows point to coarse surfaces of SCV cells; black arrows indicate “ghost,” or empty, cells. Insets in panels D and F show details of cell wall structure at the same magnification (bars, 100 nm).

FIG. 6.

FIG. 6.

(A) Growth curves (OD600) of E. faecalis normal and SCV strains were determined in THY medium under aerobic and microaerobic conditions. Data are means ± standard errors of the means of values obtained in three independent experiments. **, P ≤ 0.005 for comparison to normal cells growing under microaerobic conditions; ***, P < 0.005 for comparison to SCV cells growing under microaerobic conditions (t test). (B) Viability assay for determination of the CFU counts (CFU ml−1) of E. faecalis normal and SCV strains. At different intervals, aliquots were removed, and CFU ml−1 was determined in duplicate. Data are means ± standard deviations of values obtained in two independent experiments.

FIG. 7.

FIG. 7.

Determination of glucose concentrations and levels of metabolites (acetate, ammonia, and lactate) in culture supernatants under aerobic and microaerobic growth conditions. At different times, supernatants of E. faecalis normal and SCV strains, cultivated in THY medium, were analyzed for concentrations of glucose (A), acetate (B), ammonia (C), and lactate (D). Values are representative results of at least two independent experiments.

FIG. 8.

FIG. 8.

Intracellular replication of SCV and wild-type bacteria in MM6 cells. MM6 cells were infected at an MOI of 1:10, and cells were lysed 2, 4, 8, and 24 h after infection. The mean, minimal, and maximal values for four independent experiments are presented.

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