Detection, Identification and Diagnostic Characterization of the Staphylococcal Small Colony-Variant (SCV) Phenotype

Abstract

While modern molecular methods have decisively accelerated and improved microbiological diagnostics, phenotypic variants still pose a challenge for their detection, identification and characterization. This particularly applies if they are unstable and hard to detect, which is the case for the small-colony-variant (SCV) phenotype formed by staphylococci. On solid agar media, staphylococcal SCVs are characterized by tiny colonies with deviant colony morphology. Their reduced growth rate and fundamental metabolic changes are the result of their adaptation to an intracellular lifestyle, regularly leading to specific auxotrophies, such as for menadione, hemin or thymidine. These alterations make SCVs difficult to recognize and render physiological, biochemical and other growth-based methods such as antimicrobial susceptibility testing unreliable or unusable. Therefore, diagnostic procedures require prolonged incubation times and, if possible, confirmation by molecular methods. A special approach is needed for auxotrophy testing. However, standardized protocols for SCV diagnostics are missing. If available, SCVs and their putative parental isolates should be genotyped to determine clonality. Since their detection has significant implications for the treatment of the infection, which is usually chronic and relapsing, SCV findings should be specifically reported, commented on, and managed in close collaboration with the microbiological laboratory and the involved clinicians.

Keywords: Staphylococcus aureus; auxotrophy; chronic infection; coagulase-negative staphylococci; cultivation; diagnostics; identification; intracellular; relapse; small-colony-variant.

Figure 1

Flow scheme (dotted arrows) of the diagnostics of staphylococcal SCVs (exemplary for S. aureus). Subsequent to sample (e.g., tissue) collection (A) and inoculation on solid (B) and broth (C) media (to be striked out on agar plates after 48–72 h of incubation), agar plates should be inspected for the presence of SCV colonial morphotypes. In the case of mixed cultures, colonies displaying the normal phenotype (NP with yellowish pigmented colonies surrounded by a zone of hemolysis) and the SCV phenotype (colonies without pigmentation and hemolysis) should be isolated on solid media (D1,D2). Spontaneous reversion of the SCV phenotype into the NP should be noted (black arrow head, D2) throughout the complete diagnostic process. NP colonies (D1) should be used for usual diagnostic procedures (F), while SCV colonies (D2) should be identified by respective molecular approaches (E). Colonies of both types should be further processed for antimicrobial susceptibility testing (H1,H2), e.g., by disk diffusion test (DDT), which offers the advantage of revealing possible reversions, and for genotyping (G) to test the clonality of the different phenotypes. If possible, the SCV’s auxotrophism should also be determined on chemically defined media (CDM) agar plates (I) with disks supplemented with substances on which the growth of the SCV may specifically depend. Specific auxotrophism is visible as a growth zone around the respective disk (red arrowhead). All media for SCV cultivation should be incubated 48–72 h.

Figure 2

Columbia blood agar plate (with 5% sheep blood) showing Staphylococcus aureus after 24 h of incubation displaying the typical shapes of the normal S. aureus phenotype with grayish-pigmented colonies surrounded by a large hemolysis zone (solid arrows) and the S. aureus SCV phenotype with tiny, nonhemolytic, and nonpigmented colonies (dashed arrows). (Image has been processed to reduce irritating light reflections.)

Figure 3

Agar plate for auxotrophy testing of an S. aureus SCV after 48 h of incubation with disks impregnated with substances (A, B) on which the growth of an SCV may specifically depend. An SCV suspension was streaked on the entire agar surface. While substance A resulted in the growth of the SCV in the diffusion zone around the left disk with partial reconstitution of the normal phenotype (note size and pigmentation), no growth can be observed around the right disk impregnated with substance B.