Alteration in the GyrA Subunit of DNA Gyrase and the ParC Subunit of DNA Topoisomerase IV in Quinolone-Resistant Clinical Isolates of Staphylococcus epidermidis

Abstract

We examined 22 clinical isolates of Staphylococcus epidermidis to analyze the association of alterations in GyrA and ParC with fluoroquinolone resistance. The simultaneous presence of GyrA and ParC alterations was associated with a high level of fluoroquinolone resistance in the clinical isolates of S. epidermidis.

Staphylococcus epidermidis is a resident on human skin that is now recognized as being an important opportunistic pathogen that may cause human infection. Recently, we have observed an increase in the number of urinary-tract-derived strains of S. epidermidis showing decreased susceptibilities to fluoroquinolones. Most studies on the mechanisms of quinolone resistance in bacterial species have focused on the alterations of two target enzymes of quinolones, DNA gyrase and DNA topoisomerase IV (2, 9). For this species, however, only alterations in the GyrA subunit of DNA gyrase have been analyzed (14). To extend the investigation of the association of target alterations with quinolone resistance in S. epidermidis, we examined clinical isolates for mutations in the regions of the gyrA and parC genes of this species, which are analogous to the quinolone resistance-determining region (QRDR) of the Escherichia coli gyrA gene (16).

The type strain of S. epidermidis, ATCC 14990, was purchased from the American Type Culture Collection. For this study, we selected 22 clinical isolates that were identified as S. epidermidis by the Vitek system with the Vitek GPI card (bioMérieux Vitek, Inc., Hazelwood, Mo.) and which showed a variety of susceptibilities to fluoroquinolones. They were isolated from 1993 through 1995 from Japanese patients with urinary tract infections complicated by functional or anatomical disorders of the urinary tract. The patients had not received antibiotic treatment when the isolates were recovered; however, sufficient information was not available regarding prior use of quinolones. We determined the susceptibilities of these isolates to norfloxacin, ofloxacin, and ciprofloxacin by the twofold agar dilution method (6). The MICs were defined as the lowest drug concentrations that completely inhibited visible growth of the inoculum after incubation for 18 h at 37°C (6).

The sequence of the parC gene of S. epidermidis has not been reported. To determine the region of the S. epidermidis parC gene which is analogous to the QRDR of the E. coli gyrA gene (16), we amplified a DNA fragment from the chromosomal DNA of the type strain by PCR with two primers named SA-PARC-A and SA-PARC-B. The primers were identical to nucleotide positions 2171 to 2190 and 2347 to 2367, respectively, of the Staphylococcus aureus grlA gene (3). The PCR product was sequenced as described previously (1). In brief, ligation of the PCR product to the vector, transformation into competent cells, and selection of colorless recombinant colonies were performed with the TA Cloning Kit (Invitrogen, San Diego, Calif.). Both strands of the plasmid DNA including the PCR product were sequenced with a nonradioactive cycle sequencing kit that included the M13 forward primer and the M13 reverse primer (Toyobo, Osaka, Japan).

To detect mutations in the gyrA gene region of S. epidermidis corresponding to the QRDR of the E. coli gyrA gene, DNA fragments were amplified from chromosomal DNAs of the type strain and the clinical isolates of S. epidermidis by PCR with the primers SE-GYRA-A and SE-GYRA-B. The primer sequences were identical to the nucleotide sequences of the S. epidermidis gyrA gene with nucleotide positions equivalent to positions 2353 to 2372 and 2453 to 2472, respectively, of the S. aureus gyrA gene (8). To examine the clinical isolates of S. epidermidis for mutations in the parC gene, DNA fragments were also amplified by PCR with primers SA-PARC-A and SA-PARC-B. All the amplified DNA fragments were sequenced as described above.

The primers SA-PARC-A and SA-PARC-B produced a 197-bp DNA fragment from the DNA of the S. epidermidis type strain. The 52-amino-acid sequence deduced from the nucleotide sequence of the amplified 156-bp DNA fragment between the primers showed 96% identity with the corresponding region of the S. aureus GrlA subunit (Fig. 1). In fluoroquinolone-resistant strains of S. aureus, amino acid changes have been commonly observed at Ser-80 and Glu-84 in GrlA (3, 4, 10, 15). The amino acid in ParC of the S. epidermidis type strain equivalent to Ser-80 of S. aureus GrlA was serine, but the amino acid equivalent to Glu-84 of GrlA was aspartate.

FIG. 1.

Comparisons of the nucleotide sequence of the amplified DNA fragment of S. epidermidis ATCC 14990 (SepparC) with the corresponding sequence of the S. aureus grlA gene (SaugrlA) and of the deduced amino acid sequence (SepParC) with the corresponding sequence of the S. aureus GrlA protein (SauGrlA). Primer sequences are excluded from the comparisons. The dashes in the lines for the SaugrlA and SauGrlA sequences indicate the nucleotides and amino acids which are identical to the nucleotides in SepparC and the amino acids in SepParC.

Of the 22 clinical isolates of S. epidermidis examined in this study, the 9 isolates for which the MICs of norfloxacin, ofloxacin, and ciprofloxacin were ≤12.5, ≤1.56, and ≤1.56 μg/ml, respectively, had no alterations in the determined regions of either GyrA or ParC (Table 1). Conversely, all 13 isolates for which MICs of norfloxacin, ofloxacin, and ciprofloxacin were ≥12.5, ≥1.56, and ≥1.56 μg/ml, respectively, had amino acid changes at Ser-84 and Glu-88 in GyrA and at Ser-80 and Glu-84 in ParC.

TABLE 1.

Alterations in GyrA and ParC in clinical isolates of S. epidermidis

Strain(s)	MIC (μg/ml)a			Amino acid (codon) at the position indicated
	NFLX	OFLX	CPFX	GyrA		ParC
				84	88	80	84
Type strain	0.78	0.39	0.2	Ser (TCT)	Glu (GAA)	Ser (TCT)	Asp (GAT)
25	0.39	0.39	0.39	—b	—	—	—
4, 19, 29, 35	0.78	0.39	0.2	—	—	—	—
11	0.78	0.2	0.2	—	—	—	—
1, 7	1.56	0.78	0.39	—	—	—	—
33	3.13	1.56	0.39	—	—	—	—
20	12.5	1.56	1.56	—	—	Phe (TTT)	—
14	25	3.13	1.56	Tyr (TAT)	—	—	Asn (AAT)
10	25	6.25	6.25	Phe (TTT)	—	Tyr (TAT)	—
28	25	12.5	6.25	Tyr (TAT)	—	Phe (TTT)	—
23	25	12.5	12.5	Tyr (TAT)	—	Phe (TTT)	—
30	25	12.5	12.5	Phe (TTT)	—	Tyr (TAT)	—
34	100	12.5	12.5	Phe (TTT)	—	Phe (TTT)	—
3	>100	25	12.5	Phe (TTT)	—	Tyr (TAT)	—
27	>100	25	100	Tyr (TAT)	—	Phe (TTT)	Ala (GCT)
38	>100	50	100	Phe (TTT)	—	Phe (TTT)	Asn (AAT)
9	>100	>100	100	Phe (TTT)	—	Phe (TTT)	Ala (GCT)
24	>100	>100	>100	Phe (TTT)	Lys (AAA)	Tyr (TAT)	Tyr (TAT)
21	>100	>100	>100	Phe (TTT)	Lys (AAA)	Tyr (TAT)	Asn (AAT)

^aNFLX, norfloxacin; OFLX, ofloxacin; CPFX, ciprofloxacin. 

^b—, identical to type strain. 

Of the 13 isolates with decreased susceptibilities to fluoroquinolones, one had a single amino acid change in ParC with no alteration in GyrA. The remaining 12 isolates had changes in both GyrA and ParC. The level of fluoroquinolone resistance in the single-alteration isolate was between that of isolates with alterations in neither GyrA nor ParC and that of the isolates with alterations in both GyrA and ParC. The increases in levels of fluoroquinolone resistance appeared to be associated with accumulation of amino acid changes in GyrA and ParC.

In this study, we did not examine the isolates for alterations in GyrB or ParE. We do not exclude the possibility that GyrB and/or ParE alterations could contribute to the decreased susceptibilities to fluoroquinolones of these isolates. In addition, recent studies on fluoroquinolone resistance in Streptococcus pneumoniae and S. aureus have reported that either DNA gyrase or topoisomerase IV can be the primary target of fluoroquinolones (5, 13). However, the findings obtained in the present study suggest that ParC alterations may be the primary ones associated with fluoroquinolone resistance in S. epidermidis as well as in other gram-positive bacterial species (4, 7, 10–12, 15). This study also implies that the simultaneous presence of alterations in GyrA and ParC may be responsible for the development of higher-level fluoroquinolone resistance in clinical isolates of S. epidermidis.

Nucleotide sequence accession number.

The partial sequence of the S. epidermidis parC gene reported here appears in the DDBJ, EMBL, and GenBank nucleotide sequence databases under the accession no. AB015166.