Selection of Small-Colony Variants of Salmonella enterica Serovar Typhimurium in Nonphagocytic Eucaryotic Cells

Abstract

Salmonella enterica strains are enteropathogenic bacteria that survive and proliferate within vacuolar compartments of epithelial and phagocytic cells. Recently, it has been reported that fibroblast cells are capable of restricting S. enterica serovar Typhimurium intracellular growth. Here, we show that prolonged residence of bacteria in the intracellular environment of fibroblasts results in the appearance of genetically stable smallcolony variants (SCV). A total of 103 SCV isolates, obtained from four independent infections, were subjected to phenotypic analysis. The following phenotypes were observed: (i) ␦-aminolevulinic acid auxotrophy; (ii) requirement for acetate or succinate for growth in glucose minimal medium; (iii) auxotrophy for aromatic amino acids; and (iv) reduced growth rate under aerobic conditions not linked to nutrient auxotrophy. The exact mutations responsible for the SCV phenotype in three representative isolates were mapped in the lpd, hemL, and aroD genes, which code for dihydrolipoamide dehydrogenase, glutamate-1-semyaldehyde aminotransferase, and 3-dehydroquinate dehydratase, respectively. The lpd, hemL, and aroD mutants had intracellular persistence rates in fibroblasts that were 3 to 4 logs higher than that of the parental strain and decreased susceptibility to aminoglycoside antibiotics. All three of these SCV isolates were attenuated in the BALB/c murine typhoid model. Complementation with lpd ؉ , hem ؉ , and aroD ؉ genes restored the levels of intracellular persistence and antibiotic susceptibility to levels of the wild-type strain. However, virulence was not exhibited by any of the complemented strains. Altogether, our data demonstrate that similar to what it has been reported for SCV isolates of other pathogens, S. enterica SCV display enhanced intracellular persistence in eucaryotic cells and are impaired in the ability to cause overt disease. In addition, they also suggest that S. enterica SCV may be favored in vivo.

Listeria monocytogenes PerR Mutants Display a Small-Colony Phenotype, Increased Sensitivity to Hydrogen Peroxide, and Significantly Reduced Murine Virulence

“…This shows that environmental adaptation through PerR is essential for complete virulence in L. monocytogenes and that the true phenotype may be underrepresented in clones that have developed a secondary mutation (⌬perR lg ). It is likely that the ⌬perR sm mutant examined in this study differs from the general small-colony variants that have been isolated from infections caused by a variety of gramnegative and gram-positive organisms (9,44,45,48,49). These small-colony variants actually have a greater capacity to persist intracellularly than the wild type, whereas the ⌬perR sm mutant has a greatly reduced virulence potential.…”

Genetic and Phenotypic Identification of Fusidic Acid-Resistant Mutants with the Small-Colony-Variant Phenotype in Staphylococcus aureus

“…Thus, this FA-resistant mutant in S. enterica serovar Typhimurium shares with FA-resistant SCV mutants in S. aureus an auxotrophy in the heme biosynthesis pathway. It was earlier shown that mutations that directly affect heme biosynthesis, hemL in S. enterica serovar Typhimurium (16) or hemB in E. coli (49), result in SCVs that cause persistent and recurrent infections. In the case of fusA1, it is clear that the SCV-like phenotypes are indirect consequences of alterations in gene expression resulting from an aberrant EF-G-ribosome interaction.…”

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

“…Additional features of SCVs often found are decreased respiration, increased resistance to aminoglycosides, reduced fermentation of sugars and an unstable phenotype (Proctor et al, 2006). Since SCVs can persist in host cells (Cano et al, 2003;Balwit et al, 1994) and might be less susceptible to antibiotics (Chuard et al, 1997) they are often associated with persistent or recurrent infections of the bone marrow, joints, soft skin, endocardium and the respiratory tract (Proctor et al, 2006). Supplementation with specific compounds such as menadione, haemin or thiamine can cause reversal of the SCV phenotype to the parental phenotype (electron-transport-defective SCVs) (Proctor et al, 2006).…”