Small colony variants (SCV) are slow-growing subpopulations with altered metabolism and reduced antibiotic susceptibility which, in the case of Staphylococcus aureus, can cause persisting and recurrent infections. We studied four SCVs and their corresponding parent strains: one clinical strain pair, one menaquinone-deficient spontaneous mutant, and two constructed mutants obtained by inactivation of hemB in S. aureus 8325-4 and COL, respectively. SCVs growing in chemically defined medium (CDM) with glucose limitation and enhanced buffering capacity were found to generate deltapsi of -120 to -140 mV, which is comparable to the parent strains. However, glucose is consumed inefficiently with small growth yields. In contrast to wild-type strains, deltapsi dropped immediately to values below -100 mV when glucose expired and other nutrients such as acetate and lactate did not allow for further growth. Accordingly, the sensitivity of SCVs toward antibiotics known to be taken up through deltapsi, such as aminoglycosides, dropped 10- to 30-fold when compared to the parent strain under routine MIC determination conditions. When growing in CDM, the susceptibility of SCVs varied according to the magnitude of deltapsi.
The emergence of intermediate vancomycin resistance, mainly in methicillin-resistant Staphylococcus aureus strains, has become a great concern. Thorough characterization of clinical and laboratory vancomycinintermediately resistant S. aureus (VISA) strains identified multiple, resistance-associated changes most probably due to stepwise mutations. We hypothesized that an elevated mutation frequency as found, e.g., in mutator strains defective in DNA mismatch repair could allow rapid acquisition of adaptive mutations in the presence of vancomycin. We therefore subjected S. aureus RN4220 and its isogenic mutator strain, the mutS-knockout mutant RN4220⌬mutS, to a stepwise vancomycin selection procedure. Vancomycin resistance evolved much more quickly in the mutator background than in the wild type (5 versus 19 passages, respectively). In addition, a higher resistance level could be reached (MIC, 32 versus 4 g/ml, respectively). The susceptibility to other antibiotics with the exception of teicoplanin remained unchanged. Concomitantly with increasing vancomycin resistance, a loss of phage typeability and differences in growth behavior as well as an improved ability to regrow at high vancomycin concentrations were observed. In conclusion, an elevated mutation rate in S. aureus led to the rapid development of vancomycin resistance, indicating that a high mutation frequency could be one of the factors that favor the emergence of vancomycin resistance in S. aureus.Staphylococcus aureus causes severe community-acquired and nosocomial disease. The glycopeptide antibiotic vancomycin has long been reserved for treatment of infections with methicillin-resistant S. aureus (MRSA). However, over the last 10 years, vancomycin-intermediately resistant S. aureus strains, mainly MRSA strains, have emerged in many countries (5,11,14,15,25,35). According to the National Committee for Clinical Laboratory Standards (NCCLS), strains for which the MIC of vancomycin is Յ4 g/ml in Mueller-Hinton (MH) medium are considered susceptible whereas strains for which the MIC is Ͼ16 g/ml are considered resistant to vancomycin. For most clinical isolates that have been reported so far, MICs of vancomycin are 8 g/ml, and thus these isolates are considered intermediately resistant. Nevertheless, these strains are of clinical importance since treatment failure has been reported elsewhere (24,35,43). Many strains display a heterogeneous phenotype, i.e., although the overall MICs for them are Յ4 g/ml, subpopulations resistant to higher vancomycin concentrations are present at a frequency of at least 10 Ϫ6 (24). Higher MICs up to 100 g/ml have been obtained for strains that were selected by stepwise passage on vancomycin agar in the laboratory (7, 13, 40).The low-level resistance of clinical S. aureus isolates is not associated with one of the van gene clusters which mediate the high-level resistance in enterococci (21, 46). Up to now, the mechanism leading to vancomycin resistance in staphylococci has not been fully understood. The thorough characterizati...
Neurodegenerative disorders, including spin-ocerebellar ataxias (SCA), Huntington disease (HD) and dentatorubral-pallidoluysian atrophy (DRPLA), are associated with unstable CAG repeats. To investigate the mitotic stability of the repetitive element in the genes for SCA1, SCA3, HD, and DRPLA we extracted DNA from up to 13 tissue samples from four deceased individuals with progressive neurological disorders and neuropathological signs. Due to the formalin fixation of some tissues the genomic DNA was highly degraded and unsuitable for amplification of fragments longer than 150 bp. After size selection and primer extension preamplification, specific analyses could be performed even for expanded alleles. In all four patients the SCA1 mutation could be demonstrated, in one case with remarkable somatic heterogeneity of the elongated allele, whereas alleles of the normal range were stable in all tissues examined.
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