Characterization of Gentamicin‐Resistant Respiratory‐Deficient (Res⁻) Variant Strains of Staphylococcus aureus

Abstract

Exposure of sensitive cells of Staphylococcus aureus to concentrations of gentamicin higher than the minimal inhibitory concentration, results in the recovery of low level resistant strains with a greatly altered phenotype (variants). Because the phenotypic alteration in these strains is so great the expected diagnostic characterization of these variants as S. aureus is obscured. Starting with a geneticallymarked parent strain, a comprehensive cytological, physiological, morphological , genetic and biochemical analysis of the variants isolated from it was carried out. The genetic lineage of the variants to the parent was also established by DNA/DNA hybridization. Variants result from mutations in the hemin biosynthesis locus , the effect of which is to disrupt the synthesis of components of the electron transport system, lipid synthesis and selected nucleotide synthesis. Thus the strains are defective in aerobic and anaerobic respiration, (res-), in active transport of aminoglycosides (which confers low level resistance), export of characteristic exo-enzymes, and in cell wall composition and structure.

In Vitro Susceptibility of Clinical Staphylococcus aureus Small-Colony Variants to β-Lactam and Non-β-Lactam Antibiotics

“…In seven of 10 pairs, the SCV strains had slightly or considerably lower MICs for gentamicin than their corresponding NP strains after 18 h of incubation (see Table S1 in the supplemental material). This finding is in contrast to those of prior reports about the reduced susceptibility of SCVs to aminoglycosides (9,11,19,20) and may suggest that most clinical SCVs tested here exhibited an intact electron transport, which implies functional aminoglycoside uptake (21). However, we also found SCVs with higher gentamicin MICs than those of their NPs in two strain pairs, resulting in categorization as resistant, while the corresponding NPs were susceptible (Table S1).…”

A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly

“…aureus SCVs have been isolated from patients with persistent or antibiotic-resistant infectious diseases (35,36). The biochemical characterization of these strains suggested specific defects in electron transport (25,35,36,49). Specifically, the following findings are very likely linked to energy-dependent processes: (i) slow growth because cell wall synthesis requires large quantities of ATP, (ii) decreased pigment formation because carotenoid biosynthesis requires electron transport, (iii) resistance to aminoglycosides because their uptake requires the large membrane potential generated by electron transport, and (iv) absence of mannitol fermentation because utilization of mannitol (sugar alcohol) is decreased when electron transport is not used.…”

“…Biochemical characterization of the large majority of clinically isolated SCVs suggests specific defects in electron transport (25,35,36,49). Clinical and laboratory-generated S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds required in the biosynthesis of the electron transport chain components menaquinone and cytochromes, respectively (1,3,4,25,35,36,46). Recently, electron-transportdeficient S. aureus SCVs were found to persist inside cultured endothelial cells (4).…”

“…SCVs represent subpopulations of S. aureus that grow slowly on routine media and yield small, nonpigmented, nonhemolytic colonies (35,36,38,50). Biochemical characterization of the large majority of clinically isolated SCVs suggests specific defects in electron transport (25,35,36,49). Clinical and laboratory-generated S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds required in the biosynthesis of the electron transport chain components menaquinone and cytochromes, respectively (1,3,4,25,35,36,46).…”

Persistent Wound Infection after Herniotomy Associated with Small-Colony Variants of Staphylococcus aureus

“…Further phenotypic characteristics of SCVs include the absence of coagulase, decreased susceptibility to aminoglycosides and an auxotrophic requirement for hemin, menadione, thiamine, or CO 2 , which typically reverse the above phenotypes [2]. SCVs are found in patients with infections that persist despite appropriate antibiotic treatment or that recur after long disease-free intervals [3].…”