Po-dghm12.p65

Physiology and Antibiotic Resistance of
Staphylococcus aureus Small Colony Variants
Nicole Baumert,1 Christof von Eiff,2 Franziska Schaaff,1 Richard A. Proctor,3 Georg Peters,2
and Hans-Georg Sahl 1
1Institut für Medizinische Mikrobiologie, Universität Bonn, Sigmund-Freud-Str. 25, 53105 Bonn2Institut für Medizinische Mikrobiologie, Universität Münster, Domagkstr. 10, 48149 Münster3Departments of Medicine and Medical Microbiology/ Immunology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706, USA.
INTRODUCTION:
Small colony variants (SCV) are slow growing subpopulations with altered represented by the membrane potential (∆Ψ). The reduced membrane potential physiology and reduced antibiotic susceptibility which, in case of Staphylococcus in turn is assumed to decrease the efficacy of cationic antimicrobial compounds.
aureus, can cause persisting and recurrent infections. Therapeutic complications We studied four SCVs, one clinical isolate, one menaquinone-deficient spontaneous arise from the frequent intracellular localization of SCVs and from increased mutant and two constructed mutants obtained by inactivation of hemB in S. aureus resistance to aminoglycosides. On the other hand, the occurrence of SCVs is 8325-4 and COL, respectively, as well as the corresponding wild type parent strains.
greately enhanced in the presence of antibiotics, particularly aminoglycosides.
Strains were grown in chemically defined medium over a period of 72 hours and Most clinical SCV isolates are auxotrophic for menadione, thiamine or hemin.
growth, pH as well as ∆Ψ were monitored as a direct indicator for the physiological While the latter is required for the synthesis of cytochromes, menadione and competence. For determination of the antibiotic suscebtibility, four strain pairs thiamine are involved in the synthesis of menaquinone. The impact on electron were tested against a panel of 10 clinically important antibiotics (Tab. 1). In an transport chains arising from cofactor deficiencies are supposed to interfere with additional experiment, the gentamicin susceptibility of the hemB deficient mutant the generation of an appropriate proton motive force which, at pH 7.5, is mainly of S. aureus 8325-4 (I10) at different physiological conditions was determined.
∆ψ [mV]
∆ψ [mV]
∆ψ [mV]
∆ψ [mV]
8325-4 SCV
8325-4 SCV
Fig 1: Growth of S. aureus strains in CDM under aerobic conditions. OD (u) and membrane
Fig 2: Determination of glucose (p), lactate (l) and pH (¯) in the medium of S. aureus strains
potential ∆Ψ (¡) of strains 8325-4 (A) and COL (B) and their corresponding hemin auxotrophic 8325-4 (A) and COL (B) and their corresponding hemin auxotrophic SCVs 8325-4 SCV and COL SCVs 8325-4 SCV and COL SCV, respectively. SCV, respectively, in CDM under aerobic conditions. Acetate concentration remained unchangedthroughout the experiments (data not shown). Table 1: Antibiotic susceptibility of S. aureus parental strains and their corresponding SCVs. Strains
Table 2: Gentamicin susceptibility of the hemB deficient mutant of S. aureus 8325-4 (I10) at different were tested in MH broth. MICs were determined after 24 hours of incubation for wild type strains physiological conditions. Three cultures were run in paralell and gentamicin was added to one each and after 72 hours in case of the SCVs. (1 The stable mutants S. aureus 8325-4 SCV and S. aureus after 1h (T1,-130 mV), 5h (T2,-60 mV) and 24 h (T3,-90mV). The viable counts were determined from COL SCV were generated by insertion of an ermB cassette into the hemin biosynthesis gene hemB, aliquots taken after the exposure times listed. Times and membrane potentials correspond to Fig. 1A. resulting in a resistance to erythromycin and a cross-resistance to clindamycin). SUMMARY:
With all four strain pairs we observed similar overall patterns of growth behaviour, although individual features and characteristic deviations particularly with the COL strain pair, were detected.
Fig. 1 and 2 illustrate representative experiments obtained with S. aureus 8325-4 and its hemB knock out mutant (S. aureus 8325-4 SCV) as well as the COL strain pair with its substantial differences. When the experiments were started with inoculation into fresh medium, growth was resumed quickly resulting in 3-5 fold increase in OD for SCVs and a 10-15 fold increase for wild-type strains. During an initial period of about 2-3 hours, glucose was consumed completely; And simultaneously, lactate was produced and the pH dropped by 0.2 to 0.3 pH units (Fig. 2). The ∆Ψ values recorded in the course of such experiments revealed interesting aspects of the physiology of SCVs. SCVs growing in chemically defined w A22616-5
medium with glucose limitation and enhanced buffering capacity were found to generate ∆Ψ of -120 to ¡ A22616-3
-140 mV which is comparable to the parent strains. However, glucose is consumed inefficiently via substrate phosphorylation with small growth yields. ∆Ψ dropped immediately to values below -100 mV when glucose expired and other nutrients did not allow for further growth. In contrast, in wild type strains ∆Ψ dropped only slowly along with acid production from glucose and then started to increase again, reaching maximum potentials of approximately -160 mV after 30 hours of incubation. Accordingly, the susceptibility of SCVs towards antibiotics known to be taken up through ∆Ψ, such as aminoglycosids,dropped 10 to 30 fold. The influence of ∆Ψ on gentamicin killing was demonstrated for the hemB Fig 3: Menadione auxotrophic strains. Membrane potential (∆Ψ) of S. aureus
strain 6850 and A22616-5 (u) and their corresponding SCVs JB-1 and
deficient mutant of S. aureus 8325-4 (I10) (see Table 2).
A22616-3 (¡) growing aerobically in CDM. (DIN A 4 Poster download: http://mibi03.meb.uni-bonn.de/~groups/sahl/Paper.html#Poster)

Source: http://mibi03.meb.uni-bonn.de/~groups/sahl/po-old28.pdf