Abstract
A relationship between resistance to methicillin and resistance to fluoroquinolones, rifampin, and mupirocin has been described for Staphylococcus aureus. Differences in resistance rates may be explainable by a higher spontaneous mutation rate (MR) or a faster development of resistance (DIFF) in methicillin-resistant S. aureus (MRSA). No differences in MR, DIFF, and mutations in grlA and gyrA were detected between methicillin-susceptible S. aureus and MRSA. The higher resistance rates in MRSA are not the result of hypermutability of target genes or a faster emergence of different mutations and may be the consequence of clonal spread of multiresistant MRSA.
Fluoroquinolone, rifampin, and low-level mupirocin resistances in Staphylococcus aureus are chromosomally encoded, based mainly on mutations in the gyrA and grlA genes (4, 5, 11, 13), the rpoB gene (1, 14), and, probably, the its gene (3), respectively.
A relationship between resistance to methicillin and resistance to fluoroquinolones, rifampin, and mupirocin has been described previously (9, C. L. C. Wielders, F.-J. Schmitz, J. Verhoef, A. C. Fluit, and the European SENTRY Participants Group, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1236, p. 162, 1999). However, the cause of these differences between methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates has not yet been elucidated. Besides the clonal spread of multiresistant MRSA, differences may be explainable by a higher spontaneous mutation rate (MR) and/or a faster accumulation of possibly different mutations resulting in a quicker development of resistance in MRSA isolates. Thus, this study tries to rule in or out the possibility that MRSA acquires resistance phenotypes as a result of the hypermutability of target genes (6, 8). The so-called mutator strains typically have mutations in genes that control DNA metabolism (6, 8). Hypermutability should be seen for a strain with each of the three antibiotics tested.
We investigated the MRs for 60 MRSA and MSSA isolates, respectively, exposed to the three compounds. In addition, the in vitro activities were measured for all 120 isolates, before and after 10 serial passages in antibiotic-containing medium, followed by sequencing of the target genes (gyrA, grlA, and rpoB) in 10 randomly selected MRSA and MSSA isolates, respectively.
All susceptible isolates were derived from patients at the University Hospital of Düsseldorf, Germany, and exhibited different pulsed-field gel electrophoresis types (10). Two isogenic S. aureus strains, BB270 and BB255, with mecA insertion-deletion, were also studied (strains kindly provided by B. Berger-Bächi) (2).
The MICs were determined using a broth microdilution method according to NCCLS criteria (7). MR was calculated as described before (12). In order to characterize the emergence of multistep resistance, bacteria were grown overnight in test tubes containing one of the antibiotics and brain heart infusion broth, respectively. Aliquots taken from the test tube containing the highest drug concentration that permitted visible growth (i.e., 0.5× the 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.
Sequencing of the resistance-determining region of grlA and gyrA (11) and of rpoB (1) was performed as described previously.
MR and development of resistance [as the difference of the log2(MIC) values before and after 10 passages (DIFF)] were statistically analyzed using two tests: a two-way analysis of variance (PROC GLM, SAS-PC 6.12) and a Bonferroni-Dunn a posteriori test for group differences. Both parameters were evaluated as means and 95% confidence intervals (CI). In order to screen for classic mutator strains, MRSA and MSSA isolates were compared with respect to the number of strains showing particularly high MRs and/or high DIFF values for all three drugs tested.
MRs were in the range of 10−5 to 10−7 for ciprofloxacin, 10−6 to 10−8 for rifampin, and 10−7 to 10−8 for mupirocin. MR remained unchanged irrespective of the resistance phenotype for methicillin. Analyzing statistical differences between MSSA and MRSA isolates with respect to MR using 60 isolates in each group aimed at detecting a critical difference of at least half a log unit (ratio = 3.16). For the worst case of a common standard deviation of 0.9 log units, the power for finding a significant difference (α = 0.05) was 85%, which is totally adequate for analyses.
Noteworthily, there were statistically significant differences (P < 0.05) between the antibiotics tested. The MR was highest for ciprofloxacin (mean, 8.75 × 10−6; CI, 5.97 × 10−6 and 1.28 × 10−5) followed by rifampin (mean, 4.74 × 10−7; CI, 3.27 × 10−7 and 6.68 × 10−7) and was lowest for mupirocin (mean, 1.55 × 10−7; CI, 1.19 × 10−7 and 2.02 × 10−7).
DIFF was also not statistically different between MRSA and MSSA isolates. The same argument concerning the significance and power calculations of statistical differences in MR applies equally well to the detection of differences in DIFF.
However, marked differences (P < 0.05) were detected again among the three antibiotics. DIFF was lowest for ciprofloxacin (mean, 9.82; CI, 9.48 and 10.16) followed by mupirocin (mean, 10.43; CI, 10.19 and 10.68) and was highest for rifampin (mean, 14.13; CI, 13.87 and 14.38).
Particularly high MRs for all three drugs were observed for only three MSSA and two MRSA isolates (ciprofloxacin, MR, >4.1 × 10−5; mupirocin, MR, >7.5 × 10−7; and rifampin, MR, >4.1 × 10−6). No differences concerning the incidences of mutator strains could be detected between MRSA and MSSA isolates. The incidences (3 to 5%) are similar to those described previously for Escherichia coli and Salmonella spp. (2.6%) (6).
High DIFF values for all three compounds in parallel were observed for four MSSA and five MRSA isolates (ciprofloxacin, DIFF of >12; mupirocin, DIFF of >11; rifampin, DIFF of >14). In no isolate were a high MR and a high DIFF similarly detected. Thus, DIFF is not exclusively dependent on MR but seems also to be dependent on other factors, e.g., efflux pump efficacy of the isolates or speed of accumulation and stability of mutations.
The development of ciprofloxacin resistance in S. aureus requires multiple mutations and is mainly associated with alterations at codons 80 and 84 of GrlA and 84 and 88 of GyrA (Table 1) (4, 11, 13).
TABLE 1.
Effect of ciprofloxacin on spontaneous MRs, MICs before and after 10 passages in ciprofloxacin-containing medium, and accumulation of mutations in the gyrA and grlA genes after 10 passages
| Strain | Inoculum (CFU/ml) | Spontaneous MR | MIC of the parent strain (μg/ml) | MIC after 10 passages (μg/ml) | Mutation(s) in gene after 10 passages
|
|
|---|---|---|---|---|---|---|
| gyrA | grlA | |||||
| MSSA1 | 2.0 × 1010 | 5.1 × 10−5 | 0.25 | 64 | Ser-84→Leu | Ser-80→Phe |
| MSSA2 | 1.2 × 109 | 4.9 × 10−6 | 0.25 | 128 | Glu-88→Lys | Ser-80→Tyr |
| MSSA3 | 5.3 × 109 | 1.6 × 10−7 | 0.12 | 128 | Ser-84→Leu | Ser-80→Phe |
| MSSA4 | 7.0 × 109 | 1.9 × 10−5 | 0.12 | 256 | Glu-88→Lys | Ser-80→Phe, Glu-84→Val |
| MSSA5 | 4.6 × 109 | 3.4 × 10−7 | 0.25 | 1,024 | Ser-84→Lys | Ser-80→Tyr, Glu-84→Val |
| MSSA6 | 1.2 × 1010 | 4.5 × 10−6 | 0.25 | 128 | Ser-84→Leu | Glu-84→Val |
| MSSA7 | 2.2 × 1010 | 1.3 × 10−5 | 0.06 | 64 | Glu-88→Val | Glu-84→Val |
| MSSA8 | 7.3 × 109 | 5.4 × 10−6 | 0.12 | >1,024 | Ser-84→Leu | Ser-80→Tyr, Glu-84→Val |
| MSSA9 | 6.5 × 109 | 9.0 × 10−7 | 0.25 | 128 | Ser-84→Lys | Ser-80→Phe |
| MSSA10 | 8.5 × 109 | 4.7 × 10−5 | 0.25 | 512 | Glu-88→Lys | Ser-80→Phe, Glu-84→Val |
| MRSA1 | 2.9 × 1010 | 7.2 × 10−6 | 0.12 | 64 | Ser-84→Leu | Glu-84→Val |
| MRSA2 | 9.2 × 109 | 8.5 × 10−6 | 0.25 | 1,024 | Ser-84→Lys | Ser-80→Phe, Glu-84→Val |
| MRSA3 | 7.3 × 109 | 3.6 × 10−7 | 0.12 | 256 | Glu-88→Lys | Ser-80→Phe, Glu-84→Val |
| MRSA4 | 6.5 × 109 | 4.6 × 10−5 | 0.25 | 512 | Ser-84→Leu | Ser-80→Tyr, Glu-84→Val |
| MRSA5 | 5.2 × 109 | 6.3 × 10−6 | 0.12 | 256 | Ser-84→Lys | Ser-80→Phe, Glu-84→Val |
| MRSA6 | 1.1 × 1010 | 7.8 × 10−7 | 0.06 | 128 | Glu-88→Val | Ser-80→Tyr |
| MRSA7 | 6.9 × 109 | 5.9 × 10−6 | 0.06 | 64 | Glu-88→Lys | Glu-84→Val |
| MRSA8 | 8.2 × 109 | 6.8 × 10−7 | 0.12 | 1,024 | Ser-84→Lys | Ser-80→Phe, Glu-84→Val |
| MRSA9 | 5.1 × 109 | 1.4 × 10−5 | 0.06 | 128 | Ser-84→Leu | Glu-84→Val |
| MRSA10 | 4.9 × 109 | 7.9 × 10−6 | 0.12 | >1,024 | Glu-88→Val | Ser-80→Phe, Glu-84→Val |
| BB255 | 4.8 × 109 | 3.8 × 10−6 | 0.25 | 256 | Ser-84→Lys | Ser-80→Phe, Glu-84→Val |
| BB270 | 5.7 × 109 | 4.1 × 10−6 | 0.25 | 512 | Ser-84→Leu | Ser-80→Tyr, Glu-84→Val |
In contrast, the development of rifampin resistance does not necessarily require multiple or stepwise mutations (1). Rifampin resistance in S. aureus is mainly associated with alterations at codons 486, 477, and 481 of RpoB (Table 2).
TABLE 2.
Effect of rifampin on spontaneous MRs, MICs before and after 10 passages in rifampin-containing medium, and accumulation of mutations in the rpoB gene after 10 passages
| Strain | Inoculum (CFU/ml) | Spontaneous MR (μg/ml) | MIC of the parent strain (μg/ml) | MIC after 10 passages (μg/ml) | Mutation(s) in rpoB after 10 passages |
|---|---|---|---|---|---|
| MSSA1 | 1.8 × 1010 | 4.8 × 10−7 | 0.03 | 512 | His-481→Asn |
| MSSA2 | 4.3 × 109 | 5.9 × 10−8 | 0.03 | >1,024 | Gln-468→Leu, Ala-477→Val, His-481→Tyr |
| MSSA3 | 6.4 × 109 | 2.3 × 10−6 | 0.06 | >1,024 | Gln-468→Arg, His-481→Tyr |
| MSSA4 | 5.3 × 109 | 4.8 × 10−7 | 0.015 | >1,024 | Gln-468→Lys, Ala-477→Thr, His-481→Asn |
| MSSA5 | 7.3 × 109 | 8.2 × 10−7 | 0.03 | 512 | Gln-468→Leu |
| MSSA6 | 2.4 × 1010 | 7.8 × 10−7 | 0.06 | 1,024 | Ala-477→Asp, His-481→Asn |
| MSSA7 | 4.3 × 1010 | 4.9 × 10−7 | 0.03 | 512 | Ser-486→Leu, Ser-529→Leu |
| MSSA8 | 3.2 × 109 | 2.3 × 10−8 | 0.03 | 256 | Gln-468→Arg, Ser-529→Leu |
| MSSA9 | 4.3 × 109 | 5.6 × 10−7 | 0.015 | 1,024 | Ala-477→Asp, His-481→Tyr |
| MSSA10 | 5.6 × 109 | 5.9 × 10−6 | 0.03 | 1,024 | Gln-468→Arg, His-481→Tyr |
| MRSA1 | 4.3 × 1010 | 7.3 × 10−7 | 0.06 | >1,024 | Ala-477→Asp, His-481→Asn, Ser- 529→Leu |
| MRSA2 | 6.6 × 109 | 6.6 × 10−8 | 0.03 | 1,024 | Ala-477→Asp, His-481→Tyr |
| MRSA3 | 6.0 × 109 | 7.3 × 10−7 | 0.015 | 256 | His-481→Tyr |
| MRSA4 | 5.1 × 109 | 5.8 × 10−6 | 0.03 | 512 | Gln-468→Leu, Ser-529→Leu |
| MRSA5 | 7.2 × 109 | 4.2 × 10−7 | 0.015 | >1,024 | Gln-468→Arg, Ala-477→Val, His-481→Tyr |
| MRSA6 | 4.3 × 1010 | 2.3 × 10−7 | 0.015 | 1,024 | Ala-477→Thr, His-481→Asn, Ser-529→Leu |
| MRSA7 | 6.5 × 109 | 3.9 × 10−8 | 0.03 | 512 | His-481→Tyr |
| MRSA8 | 8.3 × 109 | 7.9 × 10−7 | 0.03 | 1,024 | Ala-477→Asp, His-481→Tyr |
| MRSA9 | 5.6 × 109 | 6.5 × 10−8 | 0.015 | 512 | His-481→Asn |
| MRSA10 | 4.0 × 109 | 6.8 × 10−7 | 0.03 | 512 | Gln-468→Lys, Ser-529→Leu |
| BB255 | 4.7 × 109 | 6.1 × 10−7 | 0.015 | 512 | Ala-477→Val, His-481→Tyr |
| BB270 | 6.9 × 109 | 7.9 × 10−7 | 0.015 | 512 | Ala-477→Asp, His-481→Asn |
Notably, the observed mutations responsible for ciprofloxacin and rifampin resistances differed between the isogenic strains. This phenomenon, however, seems to be simply the result of randomly occurring mutational events and not to be related to resistance type.
In summary, no differences in MR and DIFF for ciprofloxacin, rifampin, and mupirocin were detected between MSSA and MRSA isolates, although considerable variations within each group of clinical isolates were observable. The results for the three antibiotics tested illustrate that the higher resistance rates in MRSA are not the result of hypermutability of target genes or a faster emergence of resistance or appearance of different mutations after serial passages with antibiotic pressure but might rather be the consequence of clonal spread of multiresistant MRSA.
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