Characterization of Clinical Enterococcus faecalis Small-Colony Variants

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.Small-colony variants (SCVs) constitute a slow-growing subpopulation of bacteria with distinctive phenotypic and pathogenic characteristics. Since their first description in 1910, SCVs have been described in a wide range of gram-positive and gram-negative bacterial species, including Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Burkholderia cepacia (23). Generally, SCVs display a small colony size, a low growth rate, and atypical colony morphology (16). Biochemical characteristics of SCVs include a deficiency in electron transport or in thymidine biosynthesis (4, 23). SCVs are specialized for intracellular persistence in host cells and have been shown to be associated with persistent and recurrent infections, such as osteomyelitis, arthritis, abscesses, and respiratory infections, in humans (23). Reduced susceptibility to antimicrobial substances relative to that of their normal counterparts (26), which are often isolated simultaneously, restrict the therapeutic options for SCV infections. While SCVs of staphylococci, especially those of S. aureus, are intensively studied for their morphological, ultrastructural, metabolic, and growth characteristics (3,4,(15)(16)(17)22), little is known about the SCVs of Enterococcus species. Their occurrence in a patient with endocarditis (13) an...

Characterization of an Enterococcus faecium small-colony variant isolated from blood culture

“…By contrast, growth of E. faecium SCV was only visible around discs containing NAD + or thymidine after 24-48 h, but not around discs containing menadione, thiamine or haemin indicating that growth of E. faecium SCV was dependent on NAD + and thymidine but not menadione, thiamine or haemin. This is in contrast to a previously reported SCV phenotype which was dependent on external haemin (Wellinghausen et al, 2009). Overnight incubation on Columbia blood agar in a 5% CO 2 atmosphere did also not enhance growth of the E. faecium SCV phenotype when compared to aerobic incubation.…”

“…2A). Altogether, these changes are very similar to that observed in E. faecalis SCV (Wellinghausen et al, 2009) and in S. aureus SCV (Kahl et al, 2003). By contrast, E. faecium cells of normal phenotype exhibited regular cell division with single cross walls in dividing cells (Fig.…”

“…There are only two reports describing E. faecalis SCVs. One deals with infections of joints in chickens (Petersen et al, 2008), the other reports on a patient with endocarditis due to infection of a prosthetic aortic valve (Wellinghausen et al, 2009).…”

Enterococcus faecalis Infection Activates Phosphatidylinositol 3-Kinase Signaling To Block Apoptotic Cell Death in Macrophages

“…Do these bacteria coordinate the expression of genes specifically required for intracellular survival, likely genes that are on the E. faecalis core genome, or do they represent genetic variants of the parent strain that are better adapted to survive inside the host cell? Although the transcriptional changes occurring in the E. faecalis genome in response to the intramacrophage environment has not been reported, SCVs of E. faecalis isolated from clinical specimens have been described (45). The SCV phenotype has been characterized as cells of different sizes with aberrant shapes forming smaller colonies than the clonally related normal strain on culture media, growing with an extended lag phase with delayed entry into stationary phase, failing to grow on simple media without the addition of blood, and showing changes in metabolic pathways.…”

Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection

“…Subpopulations of SCVs have been found in a wide variety of bacteria (Haussler et al, 1999;Proctor et al, 2006;Wellinghausen et al, 2009), but they have been most extensively studied in S. aureus. In general, SCVs form small colonies on agar plates (about 10 times smaller than the parent strain) due to their slow growth rate and reduced metabolism, which also explains their decreased susceptibility to a variety of antibiotics (Chuard et al, 1997;von Eiff, 2008).…”