Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2000, p. 3229-3231, Vol. 44, No. 11
Institute for Medical Microbiology and
Virology1 and Institute for Pharmacology
and Clinical Pharmacology,3 Heinrich-Heine
Universität Düsseldorf, Düsseldorf, and
Institute of Medical Microbiology, Westfälische
Wilhelms-Universität, Münster,4
Germany, and Eijkman-Winkler Institute for Medical
Microbiology, University Medical Center, Utrecht, The
Netherlands2
Received 3 March 2000/Returned for modification 3 May 2000/Accepted 11 August 2000
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 Noteworthily, there were statistically significant differences
(P < 0.05) between the antibiotics tested. The MR was
highest for ciprofloxacin (mean, 8.75 × 10 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 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).
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development of Resistance to Ciprofloxacin,
Rifampin, and Mupirocin in Methicillin-Susceptible and -Resistant
Staphylococcus aureus Isolates
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
5 to 107 for
ciprofloxacin, 106 to 108 for rifampin, and
107 to 108 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 (
= 0.05) was 85%,
which is totally adequate for analyses.
6; CI,
5.97 × 106 and 1.28 × 105)
followed by rifampin (mean, 4.74 × 107; CI,
3.27 × 107 and 6.68 × 107) and
was lowest for mupirocin (mean, 1.55 × 107; CI,
1.19 × 107 and 2.02 × 107).
5; mupirocin, MR, >7.5 × 107; and
rifampin, MR, >4.1 × 106). 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).
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
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).
|
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.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institute for Medical Microbiology and Virology, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, Geb. 22.21, D-40225 Düsseldorf, Germany. Phone and fax: 49-2132-72040. E-mail: schmitfj{at}uni-duesseldorf.de.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Aubry-Damon, H.,
C. J. Soussy, and P. Courvalin.
1998.
Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus.
Antimicrob. Agents Chemother.
42:2590-2594 |
| 2. |
Berger-Bächi, B.
1983.
Insertional inactivation of staphylococcal methicillin resistance by Tn551.
J. Bacteriol.
154:479-487 |
| 3. |
Cookson, B.
1998.
The emergence of mupirocin resistance: a challenge to infection control and antibiotic prescribing practice.
J. Antimicrob. Chemother.
41:11-18 |
| 4. | Ferrero, L., B. Cameron, and J. Crouzet. 1995. Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. Antimicrob. Agents Chemother. 39:1554-1558[Abstract]. |
| 5. | Hooper, D. C. 1999. Mechanisms of fluoroquinolone resistance. Drug Resist. Updates 2:38-55[CrossRef][Medline]. |
| 6. |
LeClerc, J. E.,
B. Li,
W. L. Payne, and T. E. Gebula.
1996.
High mutation frequencies among Escherichia coli and Salmonella pathogens.
Science
274:1208-1211 |
| 7. | National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial tests for bacteria that grow aerobically. Approved standard M7-A4. National Committee for Laboratory Standards, Wayne, Pa. |
| 8. |
Oliver, A.,
R. Canton,
P. Campo,
F. Baquero, and J. Blazquez.
2000.
High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection.
Science
288:1251-1253 |
| 9. |
Schmitz, F.-J.,
E. Lindenlauf,
B. Hofmann,
A. C. Fluit,
J. Verhoef,
H.-P. Heinz, and M. E. Jones.
1998.
The prevalence of low and high-level mupirocin-resistance in staphylococci from 19 European hospitals.
J. Antimicrob. Chemother.
42:489-495 |
| 10. |
Schmitz, F.-J.,
M. Steiert,
H.-V. Tichy,
B. Hofmann,
J. Verhoef,
H. P. Heinz,
K. Köhrer, and M. E. Jones.
1998.
Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods.
J. Med. Microbiol.
47:341-351 |
| 11. |
Schmitz, F.-J.,
B. Hofmann,
B. Hansen,
S. Scheuring,
M. Lückefahr,
M. Klootwijk,
J. Verhoef,
A. Fluit,
H. P. Heinz,
K. Köhrer, and M. E. Jones.
1998.
Relationship between ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin MICs and with mutations in grlA, grlB, gyrA and gyrB in 116 unrelated clinical isolates of Staphylococcus aureus.
J. Antimicrob. Chemother.
41:481-484 |
| 12. | Schmitz, F.-J., C. von Eiff, M. Gondolf, A. C. Fluit, J. Verhoef, G. Peters, U. Hadding, H.-P. Heinz, and M. E. Jones. 1999. Staphylococcus aureus small colony variants: rate of selection and MIC-values compared to wild-type strains, using ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin. Clin. Microbiol. Infect. 5:376-378[Medline]. |
| 13. | Schmitz, F.-J., A. C. Fluit, S. Brisse, J. Verhoef, K. Köhrer, and D. Milatovic. 1999. Molecular epidemiology of quinolone resistance and comparative in-vitro activities of new quinolones against European Staphylococcus aureus isolates. FEMS Immunol. Med. Microbiol. 26:281-287[CrossRef][Medline]. |
| 14. |
Wichelhaus, T. A.,
V. Schäfer,
V. Brade, and B. Böddinghaus.
1999.
Molecular characterization of rpoB mutations conferring cross-resistance to rifamycins on methicillin-resistant Staphylococcus aureus.
Antimicrob. Agents Chemother.
43:2813-2816 |
Antimicrobial Agents and Chemotherapy, November 2000, p. 3229-3231, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Lannergard, J., von Eiff, C., Sander, G., Cordes, T., Seggewiss, J., Peters, G., Proctor, R. A., Becker, K., Hughes, D.
(2008). Identification of the Genetic Basis for Clinical Menadione-Auxotrophic Small-Colony Variant Isolates of Staphylococcus aureus. Antimicrob. Agents Chemother.
52: 4017-4022
[Abstract] [Full Text] -
Credito, K., Lin, G., Appelbaum, P. C.
(2007). Antistaphylococcal Activity of DX-619 Alone and in Combination with Vancomycin, Teicoplanin, and Linezolid Assessed by Time-Kill Synergy Testing. Antimicrob. Agents Chemother.
51: 1508-1511
[Abstract] [Full Text] -
Pankuch, G. A., Lin, G., Hoellman, D. B., Good, C. E., Jacobs, M. R., Appelbaum, P. C.
(2006). Activity of Retapamulin against Streptococcus pyogenes and Staphylococcus aureus Evaluated by Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methodologies.. Antimicrob. Agents Chemother.
50: 1727-1730
[Abstract] [Full Text] -
Murphy, C. K., Mullin, S., Osburne, M. S., van Duzer, J., Siedlecki, J., Yu, X., Kerstein, K., Cynamon, M., Rothstein, D. M.
(2006). In Vitro Activity of Novel Rifamycins against Rifamycin-Resistant Staphylococcus aureus. Antimicrob. Agents Chemother.
50: 827-834
[Abstract] [Full Text] -
Bogdanovich, T., Ednie, L. M., Shapiro, S., Appelbaum, P. C.
(2005). Antistaphylococcal Activity of Ceftobiprole, a New Broad-Spectrum Cephalosporin. Antimicrob. Agents Chemother.
49: 4210-4219
[Abstract] [Full Text] -
Bogdanovich, T., Esel, D., Kelly, L. M., Bozdogan, B., Credito, K., Lin, G., Smith, K., Ednie, L. M., Hoellman, D. B., Appelbaum, P. C.
(2005). Antistaphylococcal Activity of DX-619, a New Des-F(6)-Quinolone, Compared to Those of Other Agents. Antimicrob. Agents Chemother.
49: 3325-3333
[Abstract] [Full Text] -
Prunier, A.-L., Leclercq, R.
(2005). Role of mutS and mutL Genes in Hypermutability and Recombination in Staphylococcus aureus. J. Bacteriol.
187: 3455-3464
[Abstract] [Full Text] -
NORAZAH, A., LIM, V.K. E., KOH, Y.T., ROHANI, M.Y., ZURIDAH, H., SPENCER, K., NG, P.P., KAMEL, A.G. M.
(2002). Molecular fingerprinting of fusidic acid- and rifampicin-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) from Malaysian hospitals. J Med Microbiol
51: 1113-1116
[Abstract] [Full Text] -
O'Neill, A. J., Chopra, I.
(2002). Insertional inactivation of mutS in Staphylococcus aureus reveals potential for elevated mutation frequencies, although the prevalence of mutators in clinical isolates is low. J Antimicrob Chemother
50: 161-169
[Abstract] [Full Text] -
Venezia, R. A., Domaracki, B. E., Evans, A. M., Preston, K. E., Graffunder, E. M.
(2001). Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolone exposure. J Antimicrob Chemother
48: 375-381
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»