a Institute for Medical Microbiology and Virology, Heinrich-Heine Universität Düsseldorf, Germany; b Eijkman-Winkler Institute for Medical Microbiology, University Medical Center Utrecht, The Netherlands; c Institute of Medical Microbiology, Westfälische Wilhelms-Universität, Münster, Germany
Sir,
Small-colony variants (SCVs) represent subpopulations of Staphylococcus aureus and are frequently auxotrophic for either menadione or haemin, compounds required in the biosynthesis of menaquinone and cytochromes, which are components of the electron transport chain. The decrease in electron transport activity seen in these auxotrophic SCVs may provide a mechanism of persistence within host tissues and may account for their resistance in vitro to a variety of antibiotics.1
Fluoroquinolone, rifampicin and low-level mupirocin resistance in S. aureus is chromosomally encoded, based mainly on mutations in the gyrA and grlA genes,2 the rpoB gene3 and, probably, the its gene,4 respectively.
In order to test whether SCVs acquire such chromosomally encoded resistance phenotypes differently from parent strains with normal phenotype, we investigated the mutation rates and the accumulation of mutations in the target genes (gyrA, grlA, rpoB) of isolates exposed to ciprofloxacin, rifampicin and mupirocin. The in vitro activities of these three compounds were measured in SCVs and their corresponding parent strains before and after 10 serial passages in antibiotic-containing medium, followed by sequencing of the target genes.
The MICs of the three antibiotics were determined using a broth microdilution method and an inoculum of 105 cfu/mL. The results were analysed after 48 h of incubation according to NCCLS criteria.5
We studied unrelated S. aureus SCVs derived from clinical isolates, which had large and small colony types, from three different patients. These SCVs and their corresponding parent strains with normal phenotype were found to be clonal despite large differences in their colony phenotype. Two strain pairs [(i) 22616/5 with normal phenotype (NP), subsequently referred to as NP1, and 22616/3 with SCV phenotype and auxotrophic for menadione, subsequently referred to as SCV1; and (ii) 8629/2 (NP2) and 8628/1 (SCV2), which are auxotrophic for haemin] were isolated from patients with osteomyelitis who had previously been treated with gentamicin beads. One pair [F2/5 (NP3) and F1/3 (SCV3), auxotrophic for haemin] were isolated from a patient with Darier's disease in whom both S. aureus strains with normal colony size and SCVs were associated with recurrent infections of the skin. A genetically defined hemB mutant exhibiting the small colony phenotype and a corresponding plasmid-complemented mutant (PCM) with normal phenotype were also used in this study. The SCV mutant was constructed by interrupting hemB, a gene for haemin biosynthesis, by inserting an ermB cassette. S. aureus 8325-4 was used as the parent strain to generate the hemB mutant.6
Mutant colonies with MICs higher than those of the parent strains were acquired by spreading 1091010 cfu/mL of each isolate over agar plates containing 4 x MIC of each of the compounds tested. Trypticase soy agar (TSA) plates (supplemented with erythromycin 2.5 mg/L and chloramphenicol 10 mg/L to select for the hemB mutant and PCM) were used. The frequency of the appearance of mutants with increased MICs was calculated as the number of mutants that developed after overnight incubation divided by the number of cfu originally inoculated. This experiment was repeated four times with each of the three compounds tested.
In order to characterize the emergence of multistep resistance, bacteria were grown overnight in test tubes containing antibiotic and TSA broth. Aliquots taken from the test tube containing the highest drug concentration that permitted visible growth (i.e. 0.5 x MIC) were used to inoculate the second set of serial drug dilutions. Following overnight incubation, bacteria were transferred again over a period of 10 days. Afterwards, sequencing of the resistance-determining region of grlA and gyrA and of rpoB was performed.
All isolates tested became resistant to ciprofloxacin, rifampicin and mupirocin. Rates of appearance of colonies with higher MICs were in the range 105 to 106 for ciprofloxacin, 106 to 107 for rifampicin and 107 for mupirocin (Table). No differences in mutation rates or MICs were detected between SCVs exhibiting different auxotrophisms and their clonally identical parent strains with normal phenotype (Table
), indicating that this phenotype does not affect the development of ciprofloxacin, rifampicin or (low-level) mupirocin resistance in S. aureus.
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In summary, SCVs acquire resistance to ciprofloxacin, rifampicin and (low-level) mupirocin in a manner similar to that of the parent strains with normal phenotype. Mutation rates and the accumulation of mutations in the target genes (gyrA, grlA and rpoB) were comparable. However, in some pairs, the observed alterations differed between the SCVs and their corresponding parent strains.
Notes
J Antimicrob Chemother 2001; 47: 113115
* Correspondence address. Institute for Medical Microbiology and Virology, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, Geb. 22.21, 40225 Düsseldorf, Germany. Tel and Fax: +49-2132-72040; E-mail: schmitfj{at}uni-duesseldorf.de
References
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