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Antimicrobial Agents and Chemotherapy, May 2001, p. 1553-1557, Vol. 45, No. 5
Institute for Medical Microbiology and
Hygiene, University of Regensburg, Regensburg, Germany
Received 31 October 2000/Returned for modification 18 December
2000/Accepted 30 January 2001
Of 94 clinical isolates of Staphylococcus aureus
(n = 51) and coagulase-negative staphylococci (CNS)
(n = 43), mutations in the quinolone
resistance-determining region of topoisomerases GrlA, GrlB, GyrA, and
GyrB together with MICs of six quinolones were analyzed. Amino acid
substitutions at identical residues (GrlA residues 80 and 84; GyrA
residues 84 and 88) were found in S. aureus and CNS. Active
efflux, as suggested by blocking by reserpine, contributed
substantially to the resistance phenotype in some strains. Among
ciprofloxacin, clinafloxacin, levofloxacin, nalidixic acid,
trovafloxacin, and sparfloxacin, a 0.5-µg/ml concentration of
sparfloxacin discriminated best between strains with two or three
mutations and those with no mutations.
Considerable information about the
mechanisms of quinolone resistance is available for
Staphylococcus aureus (2, 5, 7, 12, 16, 17, 20,
23); however, less is known for Staphylococcus
epidermidis (9, 19) and other coagulase-negative staphylococci (CNS) (21, 27). In the present study, we
analyzed 94 unique clinical isolates with regard to the MICs of various quinolones and to the combinations of ciprofloxacin (CIP)-reserpine (RES) and trovafloxacin (TVA)-RES for these isolates. The MICs were
correlated with mutations in the quinolone-resistance determining region (QRDR) of the grlA and gyrA genes (all
strains) and grlB and gyrB genes (S. aureus and S. epidermidis).
All strains (except methicillin-resistant [Metr] S. aureus) were consecutive isolates collected from individual
patients at the Institute for Medical Microbiology, University of
Regensburg, between 1995 and 1998 and included 27 methicillin-susceptible (Mets) S. aureus
isolates, 24 (Metr) S. aureus isolates
(each with a unique pattern in pulsed-field gel electrophoresis
[24]), 12 Mets S. epidermidis
isolates, 19 Metr S. epidermidis isolates, 8 Mets CNS (1 Staphylococcus haemolyticus isolate,
5 Staphylococcus hominis isolates, and 2 Staphylococcus capitis isolates), and 4 Metr CNS
(3 S. haemolyticus isolates and 1 Staphylococcus
simulans isolate). CNS were isolated from normally sterile sites.
Isolates were identified by a latex agglutination test (Slidex
Staph-Kit; bioMérieux sa, Marcy-l'Étoile, France) and
by biochemical reactions (ID 32 STAPH; bioMérieux sa).
Antimicrobial agents were provided by the manufacturers: CIP (Bayer AG,
Leverkusen, Germany), clinafloxacin (Parke-Davis Pharmaceutical
Research, Freiburg, Germany), levofloxacin (Hoechst Marion Roussel,
Frankfurt, Germany), sparfloxacin (SPX) (Rhone-Poulenc-Rohrer,
Köln, Germany), and TVA (Pfizer, Karlsruhe, Germany). Nalidixic
acid was purchased from Sigma (Deisenhofen, Germany) (catalog no.
N8878). MICs were determined by the agar dilution method on
Mueller-Hinton agar (Oxoid, Wesel, Germany) according to NCCLS
guidelines (13). In two determinations the effect of RES
(catalog no. R0875; Sigma) on MICs of TVA and CIP was evaluated on
Mueller-Hinton agar plates with and without RES (20 µg/ml) and Etest
strips (AB BIODISK, Solna, Sweden) and was expressed as the change of
dilution steps. If the MIC exceeded the maximum concentration on the
strip, double the concentration was arbitrarily used for further
calculations. Protocols for the amplification of grlA, grlB,
gyrA, and gyrB of S. aureus and
grlA of CNS as published previously were used (3, 21,
26). Primers and PCR conditions for amplification of grlB,
gyrA, and gyrB genes of CNS are listed in Table
1. Three units of Expand High Fidelity Taq polymerase (precast solution; Roche Molecular
Biochemicals, Mannheim, Germany) was used for all amplifications. The
PCR products were purified with a PCR purification kit (QIAQuick;
Qiagen, Hilden, Germany). Complementary strands were sequenced on a 310 DNA sequencer (Perkin-Elmer, Foster City, Calif.) using PCR primers (6 µmol). Sequences were compared with published wild-type sequences of S. aureus (gyrA and gyrB:
GenBank accession number M86227; grlA and
grlB: GenBank accession number D67075). SPSS 10.0 for
Windows was used for calculation of the chi-square and Mann-Whitney U
test results. The partial sequences of the grlA gene of
S. haemolyticus, S. hominis, S. capitis, and S. simulans and of the gyrA gene of S. hominis, S. capitis, and S. simulans appear in the GenBank nucleotide sequence database under accession numbers
AF159150, AF159151, AF159152, AF159153, AF159154, AF159155, and
AF159156, respectively. Partial sequences of the grlB and gyrB genes of S. epidermidis are listed under
accession numbers AF314403 and AF314404, respectively.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1553-1557.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Vitro Activities of Six Quinolones and Mechanisms of
Resistance in Staphylococcus aureus and
Coagulase-Negative Staphylococci
ABSTRACT
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TABLE 1.
Primers and PCR conditions used in this study
The MICs at which 50 and 90% of S. aureus and CNS strains
tested were inhibited were comparable to those found in previous studies (1, 4, 8, 18). The distribution of the MICs for
the different groups of staphylococci is shown in Fig.
1. In both S. aureus and CNS,
resistance to methicillin and to quinolones was highly correlated
(P < 0.0001 [chi-square test]).
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In S. aureus, resistance to quinolones was correlated with the number of point mutations in grlA and gyrA, leading to amino acid changes in residues Ser80 and/or Glu84 of GrlA and Ser84 and/or Glu88 of GyrA. The association of the GrlB432 alteration with resistance is unclear (seen in two strains; MICs of SPX 8 and 16 µg/ml), because the MICs for strains with identical alterations in GyrA and GrlA ranged from 4 to 32 µg/ml (20, 22). Amino acid exchanges at position Ile45 or Pro144 of GrlA did not appear to affect MICs. No mutations in gyrB of S. aureus were observed.
The degree of similarity in nucleotide and amino acid sequences in the
QRDRs of GrlA, GrlB, GyrA, and GyrB between quinolone-susceptible S. aureus and CNS is shown in Table
2. In comparison to S. aureus, all quinolone-susceptible CNS had an aspartate residue at position 84 instead of the glutamate present in S. aureus. This has also been reported for S. epidermidis by Li et al.
(9). In the QRDR of GyrA, S. epidermidis, S. capitis, and S. simulans had amino acid sequences
identical to that of S. aureus. Both S. haemolyticus and S. hominis differ from the other
CNS at codon 88 in that they have a conservative change from glutamate
to aspartate (21). MICs of SPX for these strains were 0.06 to 0.25 µg/ml, indicating no apparent effect of the Glu88Asp change
in GyrA of S. haemolyticus or S. hominis.
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Identical to S. aureus, elevated MICs for CNS were found in strains with amino acid changes in residues Ser80 and Asp84 of GrlA and residues Ser84 and Glu88 of GyrA. In GrlA of S. epidermidis only Ser80Phe or Ser80Tyr changes were found by Li et al. (9) and in the present study, while S. hominis and S. haemolyticus had Ser80Val or Ser80Leu amino acid exchanges (21). No mutations in gyrB or grlB in any strain of S. epidermidis were found. In CNS other than S. epidermidis, only the grlA and gyrA genes were analyzed. Different primers designed for amplification of the grlB and gyrB genes of S. aureus or S. epidermidis, tested under various nonstringent conditions, did not yield any product in S. capitis, S. hominis, S. haemolyticus, and S. simulans.
In strains with identical amino acid changes but different MICs,
additional resistance mechanisms may be active. We investigated whether
inhibition of efflux pump systems (presumably NorA [11, 14; F. J. Schmitz, B. Hertel, B. Hoffmann, S. Scheuring, J. Verhoef, A. C. Fluit, H. P. Heinz, K. Kohrer, and M. E. Jones, Letter, J. Antimicrob. Chemother., 42:561-563,
1998]) by the alkaloid RES would abolish such differences or whether
efflux-mediated resistance was associated with certain species or
strains subgrouped according to quinolone resistance. RES decreased
MICs of CIP by a median of 1.0 dilution step (range, 
4 to 6 [not
significant]), and TVA MICs by a median of 1.4 dilution steps (range,
3 to 4.4 [not significant]). This indicates a major contribution of
efflux pumps to the resistance phenotype in some strains. Similar
findings have been reported for S. aureus and pneumococci
(10, 15). In CIP-susceptible (CIP-S) S. aureus
the decrease was a median of 3.0 dilution steps (P < 0.001 [Mann-Whitney test] compared to CIP-resistant [CIP-R]
S. aureus) when testing CIP plus RES. This indicates a basic
efflux activity in wild-type S. aureus. In CIP-R S. aureus the observation may be lost because of the high level of
resistance conferred by topoisomerase mutations. Unexpectedly, the
combination of RES and CIP produced an increase of MIC in nine strains,
both S. aureus (n = 2) and CNS (n = 7), ranging from 1 to 4 dilution steps. We have no explanation
for this; however, RES may function as an inducer of efflux pumps in
these strains. Also, RES affected the change of MIC differently for CIP
and TVA (Table 3), since the changes of
MIC induced by CIP-RES and TVA-RES did not correlate, except for CIP-R
CNS (chi-square test, r2 = 0.87). No
correlation was found between the RES-inhibitable mechanism(s) and MICs
for strains with identical amino acid changes in GrlA or GyrA (data not
shown). Therefore, other efflux systems or other, as yet unknown
mechanisms of resistance are likely to exist (17, 20, 25).
Ince and Hooper (6) have recently suggested the extension
of the range of the QRDR of grlA of S. aureus.
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From an epidemiological rather than from a clinical point of view,
susceptibility testing not only should indicate whether a substance is
suitable for treatment but also should identify strains that have the
potential to become resistant. Since resistance to quinolones is
acquired in a stepwise fashion, detection of the first step might be
important. The distribution of MICs for staphylococci with different
numbers of amino acid alterations in the QRDRs of GrlA and GyrA is
shown graphically in Fig. 1. MICs of CIP of >2 µg/ml (gap of 2 dilution steps), MICs of TVA of >0.5 µg/ml (gap of 1 dilution step),
MICs of levofloxacin of >2 µg/ml, and MICs of SPX of >1 µg/ml
separated all strains with two or three alterations in GrlA or GyrA
from strains without alterations. According to NCCLS criteria TVA
classified 26 strains with two or three amino acid changes as
susceptible. SPX at 0.5 µg/ml separated all strains with no changes
in topoisomerases from those with two or more changes and could
therefore act as a predictor of quinolone resistance mechanisms, in the
same way that penicillin and methicillin are used in the prediction of susceptibility to other 
-lactam antibiotics.
In conclusion, similar resistance mechanisms were found in S. aureus and CNS. Since in staphylococcal infections the use of highly active quinolone agents is likely to increase, prudent use may be guided by the prediction of resistance mechanisms rather than MIC data.
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ACKNOWLEDGMENTS |
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The results of typing Metr S. aureus by pulsed-field gel electrophoresis were kindly provided by Michaela Metz. We thank Markus Bollwein and Christine Irtenkauf for excellent technical assistance. Nucleotide sequence determination was performed by Holger Melzl and Josef Köstler.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, Franz-Josef-Strauss-Allee 11, D-93049 Regensburg, Germany. Phone: 49-941- 944-6461. Fax: 49-941-944-6402. E-mail: hans-joerg.linde{at}klinik.uni-regensburg.de.
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Antimicrobial Agents and Chemotherapy, May 2001, p. 1553-1557, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1553-1557.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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