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Antimicrobial Agents and Chemotherapy, May 1998, p. 1249-1252, Vol. 42, No. 5
Institute for Medical Microbiology and
Virology1 and
Molekularbiologisches
Zentrallabor im Biologisch-Medizinischen
Forschungszentrum,3 Heinrich-Heine University
Düsseldorf, Düsseldorf, Germany, and
Eijkman-Winkler Institute for Medical Microbiology, Utrecht
University, Utrecht, The Netherlands2
Received 25 August 1997/Returned for modification 18 December
1997/Accepted 25 February 1998
One hundred sixteen unrelated clinical isolates of
Staphylococcus aureus (70 ciprofloxacin resistant and 46 ciprofloxacin susceptible) from eight countries were studied for the
presence of mutations in the grlA, grlB,
gyrA, and gyrB gene loci. Two mutations within
grlA (located at codons 80 and 84) and two mutations within
gyrA (located at codons 84 and 88) were clearly associated with ciprofloxacin resistance, although other mutations detected within the four genes studied may also contribute to decreased susceptibility.
Infections caused by
methicillin-resistant Staphylococcus aureus (MRSA) are
a therapeutic challenge due to multiple antibiotic resistance
(10). Fluoroquinolones (FQs), of which ciprofloxacin is the
most widely used, are broad-spectrum antibiotics with good activity
against gram-positive organisms, including both methicillin-sensitive S. aureus and MRSA. The widespread use of FQs has led
to the emergence of FQ-resistant S. aureus, especially
among MRSA strains (5). Mutations within norA
(20), gyrA, gyrB (6, 8,
18), and grlA (3, 13, 19) have been shown
to be associated with FQ resistance in S. aureus.
norA encodes a membrane protein which acts as a efflux pump
(9, 14). gyrA and gyrB encode subunits of DNA gyrase (2, 11). Strains with mutations in
gyrA and gyrB, but without grlA
mutations, which confer high-level FQ resistance, can be isolated by
single-step selection with FQs for Escherichia coli but not
for S. aureus (3), although gyrA
mutations have been detected in FQ-resistant clinical isolates of
S. aureus (6, 8, 18). grlA and
grlB encode the structural proteins of DNA topoisomerase IV
(4), and mutations in the grlA gene, with or
without gyrA mutations, have been described for FQ-resistant S. aureus strains (3, 17). Genetic and
biochemical evidence suggests that the primary target site of
ciprofloxacin, and probably of other FQs, in S. aureus
is DNA topoisomerase IV (1, 7, 13) and not DNA gyrase, as in
E. coli and Neisseria gonorrhoeae.
This study aimed to characterize mutations in grlA,
grlB, gyrA, and gyrB of 116 unrelated
S. aureus isolates derived from eight countries and to
correlate the effects of mutations or combinations of mutations within
these genes with ciprofloxacin MICs.
Ninety-three MRSA isolates (67 ciprofloxacin resistant and 26 ciprofloxacin susceptible) and 23 methicillin susceptible S. aureus (MSSA) isolates (3 ciprofloxacin resistant and 20 ciprofloxacin susceptible) were included in this study. Eighty
S. aureus isolates from patients residing in Germany,
collected between 1990 and 1995, and 36 S. aureus
isolates from seven other countries (8 from Japan, 8 from Brazil, 6 from Switzerland, 4 from Sri Lanka, 4 from Spain, 3 from the
United Kingdom, and 3 from Hungary), collected between 1983 and 1989, were tested. All 116 clinical isolates from different
patients were screened for the presence of the mecA and
coa genes by multiplex PCR (16). All isolates were selected on the basis of belonging to different pulsed-field gel
electrophoresis types (15).
Ciprofloxacin MICs were derived by using a broth microdilution
method according to guidelines recommended by the National Committee
for Clinical Laboratory Standards (12).
Based on published sequences for grlA and grlB
(19) and gyrA and gyrB
(8), the appropriate oligonucleotide primers were selected as follows: for grlA, the 5' primer
2402-ACTTGAAGATGTTTTAGGTGAT-2423 and the 3' primer
2942-TTAGGAAATCTTGATGGCAA-2961; for grlB, the 5'
primer 1520-CGATTAAAGCACAACAAGCAAG-1541 and the 3' primer
1874-CATCAGTCATAATAATTACTC-1894; for gyrA, the 5'
primer 2311-AATGAACAAGGTATGACACC-2330 and the 3'
primer 2514-TACGCGCTTCAGTATAACGC-2533; and for
gyrB, the 5' primer
1400-CAGCGTTAGATGTAGCAAGC-1419 and the 3' primer
1631-CCGATTCCTGTACCAAATGC-1650.
Four independent PCR amplifications were carried out with a GeneAmp PCR
System 2400 (Perkin-Elmer, Weiterstadt, Germany), and all reagents
(GeneAmp deoxynucleoside triphosphates, high-fidelity Taq DNA polymerase, and 10 × PCR buffer) were
purchased from Perkin-Elmer or Boehringer Mannheim (Mannheim,
Germany). To prepare cell lysates for use as template DNA in PCR,
approximately 1/10 of a single bacterial colony was picked with a
pipette tip and mixed in the PCR amplification mixture, consisting of
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 100 µM
deoxynucleoside triphosphates, 3 U of high-fidelity Taq DNA
polymerase, and 0.4 µM primers in a final volume of 50 µl. Samples
were denatured at 94°C for 10 min, followed by 25 amplification
cycles with the following parameters: 94°C for 20 s, 55°C for
20 s, and 72°C for 50 s. A final cycle of 72°C for 5 min
was used to fully extend amplicons.
PCR products were purified with a PCR purification kit
(Qiagen, Hilden, Germany). PCR-amplified DNA was sequenced by the
dye terminator method in both the forward and reverse directions. The
reaction was carried out with 50 ng of DNA and 0.1 µmol of primers,
by using a Ready Reaction Dye Terminator Cycle Sequencing Kit
(Perkin-Elmer) according to the manufacturer's instructions. The
products were resolved and automatically analyzed with a 310 DNA
sequencer (Perkin-Elmer).
Sequence data from codon 16 to codon 189 of the grlA gene,
from codon 386 to codon 497 of the grlB gene, from codon 70 to codon 121 of the gyrA gene, and from codon 413 to codon
483 of the gyrB gene were obtained for further analysis.
Wild-type sequences with no mutations were identified on the basis of
being identical to the published sequences of grlA and
grlB (19) and gyrA and gyrB
(8). Mutations in these genes were identified by comparison.
The mutations identified are summarized in Table
1. Within the grlA gene, 11 single or combination mutations were found in 84 isolates; within the
grlB gene, 9 single or combination mutations were located in
20 isolates; within the gyrA gene, 9 single or combination
mutations were found in 90 isolates; and within the gyrB
gene, 4 single or combination mutations were found in 46 isolates.
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of grlA,
grlB, gyrA, and gyrB Mutations in 116 Unrelated Isolates of Staphylococcus aureus and Effects
of Mutations on Ciprofloxacin MIC
ABSTRACT
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TABLE 1.
Mutations within the grlA, grlB,
gyrA, and gyrB genes in 116 clonally unrelated
clinical isolates of S. aureus from eight countries
The effect of the three amino acid changes within grlB (Table 2) is unclear, as the MICs of ciprofloxacin for several isolates without these mutations are lower (Table 2).
|
From the correlation of the characterized mutations with the resulting
MICs of ciprofloxacin (Table 2), it is clear that all the isolates
studied that do not have the grlA mutation Ser-80
Phe are ciprofloxacin susceptible.
All ciprofloxacin-resistant isolates for which MICs were 
4 µg/ml
had the grlA mutation Ser-80Phe in combination with
either a Ser-84Leu mutation or a Glu-88Lys mutation
within the gyrA gene.
In two isolates a Ser-80Phe mutation was combined with no mutations
in the gyrA gene, resulting in a ciprofloxacin MIC of 2 µg/ml, which, while elevated from a wild-type level, is still below
the breakpoint for resistance. These data support the finding that in
S. aureus, grlA mutations precede
gyrA mutations in the development of resistance to
ciprofloxacin (3). However, in contrast, two isolates, each
of which had a single mutation in the gyrA gene without a
corresponding Ser-80Phe mutation in grlA, were
associated with MICs of ciprofloxacin of 0.25 and 1 µg/ml.
Combinations of single point mutations within the gyrA gene
have been shown to be associated with higher ciprofloxacin MICs than
single point mutations (18). Similarly, two combinations of
single point mutations within grlA, a Glu-84Val or an
Ala-48Thr mutation in combination with a Ser-80Phe mutation, were
associated with relatively higher ciprofloxacin MICs (range, 64 to 256 µg/ml) than only a single Ser-80Phe mutation (range, 8 to 128 µg/ml) (Table 2).
However, other factors have some effect on ciprofloxacin resistance, as
evidenced, for example, by the fact that in the 47 isolates with a
single grlA mutation (Ser-80Phe) in combination with the
Ser-84Leu mutation in gyrA, 16-fold differences in
ciprofloxacin MICs occurred. This implicates additional resistance
mechanisms associated with elevated MICs of ciprofloxacin (1, 9,
14).
In summary, our data support previous findings and provide evidence that two mutations within the grlA gene (located at codons 80 and 84), as well as two mutations within the gyrA gene (located at codons 84 and 88), are clearly associated with the development of ciprofloxacin resistance. From 116 unrelated isolates we have found 9 combinations of amino acid changes within in GrlA, GrlB, and GyrA associated with resistance to ciprofloxacin. However, some mutations reported by previous workers (3, 6, 8, 17-19) as associated with ciprofloxacin resistance were not found among these isolates, suggesting that other, unknown mutations are likely to exist. The association of mutations within grlA at codon 48, as well as that of polymorphisms in grlB and gyrB, with increased ciprofloxacin MICs is not known. Sequence data from unrelated clones of S. aureus isolated from different countries show that some grlA and gyrA mutations are conserved in both MRSA and MSSA.
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FOOTNOTES |
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*
Corresponding author. Mailing address: Institute for
Medical Microbiology and Virology, Heinrich-Heine-Universität
Düsseldorf, Universitätsstra
e 1, Geb. 22.21, 40225 Düsseldorf, Germany. Phone: 49-2132-72040. Fax:
49-211-811-5323.
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