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Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii

Yoshinori Nishino, Takashi Deguchi, Mitsuru Yasuda, Takeshi Kawamura, Masahiro Nakano, Emiko Kanematsu, Shigehiko Ozeki, Yukimichi Kawada
DOI: http://dx.doi.org/10.1111/j.1574-6968.1997.tb12675.x 409-414 First published online: 1 September 1997

Abstract

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.

Keywords
  • Citrobacter freundii
  • DNA gyrase
  • Topoisomerase IV

1 Introduction

Citrobacter freundii is often isolated from patients with urinary tract infections. Fluoroquinolones, which have a good activity against C. freundii, have been effective in curing urinary tract infections with this pathogen [1]. However, we have found an increase in the number of C. freundii isolates with decreased susceptibilities to fluoroquinolones.

Recently, several mechanisms of quinolone resistance have been identified in some bacterial species [24]. In Gram-negative bacterial species, such as Escherichia coli, Neisseria gonorrhoeae, and Haemophilus influenzae, alterations in the GyrA subunit of DNA gyrase play a primary role in developing quinolone resistance [59]. In these species, alterations in the ParC subunit of DNA topoisomerase IV play a complementary role in increasing quinolone resistance [7, 912]. In C. freundii, however, the mechanisms of quinolone resistance have not been well studied. In the present study, we attempted to amplify the gyrA and parC genes of C. freundii, corresponding to the quinolone resistance-determining region (QRDR), and then we analyzed clinical strains of C. freundii isolated from patients with urinary tract infections for the association of alterations of GyrA and ParC with quinolone resistance.

2 Materials and methods

2.1 Bacterial strains

Type strain of C. freundii ATCC8090 was purchased from the American Type Culture Collection. Thirty-eight clinical strains of C. freundii used in this study were isolated from 1991 through 1994 from Japanese patients with urinary tract infection and maintained in our laboratory. To avoid testing multiple isolates from a single patient, C. freundii was isolated in only one urinary culture from each patient during the infection period and was used for this study. No patients were given any quinolones when they visited a clinic.

2.2 Determination of partial sequences of the C. freundii gyrA and parC genes

For partial sequencing of the regions of the gyrA and parC genes of C. freundii containing the region analogous to the QRDR of the E. coli gyrA gene, DNA fragments were amplified from the chromosomal DNA of type strain by polymerase chain reaction (PCR) with two sets of primers, EC-GYRA-A and EC-GYRA-B for gyrA, and EC-PARC-A and EC-PARC-B for parC, and the PCR products were sequenced. EC-GYRA-A (5′-CGCGTACTTTACGCCATGAACGTA-3′) and EC-GYRA-B (5′-CAGACGGATTTCCGTATAACGC-3′) were located within the consensus amino acids of the bacterial GyrA proteins and were identical to nucleotide positions 139 to 162 and 360 to 381 of the E. coli gyrA gene [5]. EC-PARC-A (5′-CTGAACGCCAGCGCGAAATT-3′) and EC-PARC-B (5′-GCGAAAGATTTGGGATCGTC-3′) were identical to nucleotide positions 185 to 204 and 353 to 372 of the E. coli parC gene [3]. DNA extraction, PCR amplification, and sequencing of the PCR products were performed as reported previously [8].

2.3 MIC testing

The susceptibilities of the strains to ciprofloxacin and norfloxacin were determined by the 2-fold agar dilution method. The strains were cultured overnight in Mueller-Hinton broth at 37°C, and using an inoculator (Microplanter; Sakura Seisakusho, Tokyo, Japan), an inoculum of 104 CFU per spot was applied to agar plates containing serial 2-fold dilutions of each drug. MICs were defined as the lowest concentrations of drug that completely inhibited visible growth of the inoculum after incubation for 18 h at 37°C.

2.4 Detection of mutations in the gyrA and parC genes

Thirty-eight clinical strains were examined for the presence of mutations in the gyrA and parC genes. For analysis of the mutations in the region corresponding to the QRDR of the E. coli gyrA and parC genes, DNA fragments were amplified using the following two sets of primers, EC-GYRA-A and EC-GYRA-B, and EC-PARC-A and EC-PARC-B, and sequencing of PCR products was performed by procedures similar to those previously reported [8, 10].

2.5 Case study of a fluoroquinolone treatment failure in urinary tract infection with emergence of a post-treatment isolate with enhanced resistance to fluoroquinolones

We examined clinical strains that were isolated from a case of fluoroquinolone treatment failure in urinary tract infection. A 65 year-old Japanese man with urethral stricture presented at the clinic with dysuria and urinary turbidity. A strain of C. freundii (named GU-CF08) was isolated from his urine (107 CFU/ml). He was treated with a novel fluoroquinolone, AM-1155 [13], 100 mg, twice daily for 7 days. When he returned to the clinic with continuing symptoms, a strain of C. freundii (named GU-CF09) was isolated again (107 CFU/ml). To assess whether the pre- and post-treatment isolates were isogenic, arbitrarily primed PCR analysis was performed [14], and biochemical characteristics were examined by using Enterotube II (Nippon Becton Dickinson Co., Tokyo, Japan). The two strains were tested for the MICs of ciprofloxacin, norfloxacin, AM-1155, piperacillin, cefazolin, cefotaxime, cefixime, aztreonam, imipenem, gentamicin, chloramphenicol, and tetracycline. They were also examined for the presence of mutations in the gyrA and parC genes. The analysis of the mutations and susceptibilities were performed in the manner identical to that described above.

2.6 Statistical analysis

Statistical analysis was conducted using the Wilcoxon rank sum test. All statistical comparisons were two-tailed and were performed with the significance set at P<0.05.

2.7 Nucleotide sequence accession number

The partial sequence of the C. freundii gyrA and parC gene reported here appears in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the accession numbers AB003913 and AB003914, respectively.

3 Results and discussion

3.1 Amplification of the C. freundii gyrA and parC genes

The primers EC-GYRA-A and EC-GYRA-B amplified a DNA fragment of the expected 243 bp from the chromosomal DNA of type strain of C. freundii. The PCR product was sequenced, and the nucleotide sequence and amino acid sequence of the C. freundii gyrA gene and GyrA protein are shown in Fig. 1. The determined nucleotide sequence of a 197 bp DNA fragment excluding the primers showed 87.3% similarity with the corresponding region of the gyrA gene of E. coli. The deduced 64 amino acid sequence showed 98.5% identity with the GyrA protein of E. coli and exhibited 60 to 82% identities with the corresponding regions of other bacterial GyrA proteins [8, 15, 16]. Conversely, the primers EC-PARC-A and EC-PARC-B amplified a DNA fragment of the expected 188 bp from the chromosomal DNA of type strain of C. freundii. The 148 bp DNA fragment excluding the primers exhibited 93.3% identity with the corresponding regions of the parC gene of E. coli. The deduced 49 amino acid sequence was identical to that of the ParC protein of E. coli (Fig. 2) and exhibited 51 to 69% identities with the regions of the Neisseria gonorrhoeae ParC and Staphylococcus aureus GrlA proteins, respectively [7, 17]. From these findings, we concluded that the determined sequences were partial sequences of the C. freundii gyrA and parC genes.

Figure 1

Comparisons of particular regions of the nucleotide sequence of the DNA fragment amplified from the chromosomal DNA of C. freundii ATCC 8090 (CfgyrA) with the equivalent region of E. coli (EcogyrA) and of the deduced amino acid sequence (CfGyrA) with the equivalent regions of E. coli (EcoGyrA). Portions of the primers are excluded from the sequence. Dashes on the lines of EcogyrA and EcoGyrA indicate nucleotides and amino acids identical to nucleotides in CfgyrA and amino acids in CfGyrA, respectively.

Figure 2

Comparisons of particular regions of the nucleotide sequence of the DNA fragment amplified from the chromosomal DNA of C. freundii ATCC 8090 (CfparC) with the equivalent region of E. coli (EcoparC) and of the deduced amino acid sequence (CfParC) with the equivalent regions of E. coli (EcoParC). Portions of the primers are excluded from the sequence. Dashes on the lines of EcoparC and EcoParC indicate nucleotides and amino acids identical to nucleotides in CfparC and amino acids in CfParC, respectively.

3.2 Detection of mutations in the gyrA and parC genes amplified from clinical isolates of C. freundii

The association of mutations in gyrA and parC genes with susceptibilities to fluoroquinolones was determined in 38 urinary tract-derived clinical strains of C. freundii. In 17 isolates with both ciprofloxacin and norfloxacin MICs of 1.56 mg/l, the nucleotide sequences in the regions of the gyrA and parC genes analyzed in this study contained no mutations resulting in amino acid changes in GyrA and ParC proteins (Table 1). Seven strains with a ciprofloxacin MIC of 6.25 mg/l and norfloxacin MICs of 12.5 mg/l to 25 mg/l had single mutations in the gyrA gene alone, resulting in an amino acid change of Thr-83→Ile. Six strains with ciprofloxacin MICs of 12.5 mg/l to 25 mg/l and norfloxacin MICs of 25 mg/l to 50 mg/l had single mutations in the gyrA and parC genes. Eight strains with ciprofloxacin MICs of 50 mg/l to 200 mg/l and norfloxacin MICs of 100 mg/l to 200 mg/l had double mutations in the gyrA gene and single mutations in the parC gene. All the mutations observed in codon 83 of the gyrA gene were C-to-T substitutions, generating an amino acid change of Thr-83→Ile. The mutations in codon 87 were G-to-A, G-to-C, and A-to-C substitutions, resulting in amino acid changes of Asp-87→Asn, Asp-87→Tyr, and Asp-87→Val, respectively. All the mutations in the codons of the parC gene corresponding to Ser-80 and Glu-84 of the E. coli ParC protein were a G-to-T substitutions, generating Ser-80→Ile. All the mutations in the codon corresponding to Glu-84 of the E. coli ParC protein were G-to-A substitutions, resulting in Glu-84→Lys. These alterations observed in GyrA and ParC of C. freundii were analogous to those that were frequently found to be responsible for fluoroquinolone resistance in E. coli and other bacterial species [6, 7, 12, 1517]. In this study, we found no strains having alterations in ParC without the simultaneous presence of alterations in GyrA.

View this table:
1

Alterations in GyrA and ParC and susceptibilities to ciprofloxacin and norfloxacin in clinical isolates of C. freundii

MIC (mg/l)Amino acid change
NFLXaCPFXaGyrAParC
Strain83878084
Type strain≤0.025≤0.025Thr(ACC)Asp(GAC)Ser(AGC)Glu(GAA)
01,11,660.10.05b
12,580.20.05
60,690.20.1
04,150.780.39
03,070.780.78
10,19,65,671.560.78
59,681.561.56
71,72,7312.56.25Ile(ATC)
05,06,23,70256.25Ile(ATC)
022512.5Ile(ATC)Lys(AAA)
362512.5Ile(ATC)Ile(ATC)
34,352525Ile(ATC)Lys(AAA)
16,295025Ile(ATC)Ile(ATC)
1810050Ile(ATC)Tyr(TAC)Ile(ATC)
31,48,5110050Ile(ATC)Tyr(TAC)Lys(AAA)
55100100Ile(ATC)Tyr(TAC)Ile(ATC)
13,49,50200200Ile(ATC)Val(GTC)Ile(ATC)
  • aNFLX, norfloxacin; CPFX, ciprofloxacin.

  • b–, identical to type strain.

The strains having a single or double amino acid change in GyrA exhibited significantly higher-level resistance to ciprofloxacin and norfloxacin than those without alterations in either GyrA or ParC (P<0.01). The six strains with single amino acid changes in GyrA and ParC were significantly more resistant to ciprofloxacin and norfloxacin than the seven strains with a single amino acid change in GyrA alone (ciprofloxacin, P<0.05; norfloxacin, P<0.01). The eight strains with a double amino acid change in GyrA and a single amino acid change in ParC were significantly more resistant to ciprofloxacin and norfloxacin than the six strains with single amino acid changes in GyrA and ParC (P<0.01).

3.3 Case study of a fluoroquinolone treatment failure in urinary tract infection caused by a quinolone-resistant C. freundii strain

The electrophoresis profile of the DNAs amplified by AP-PCR from the post-treatment isolate GU-CF09 was identical to that from the pre-treatment isolate GU-CF08. The biochemical characteristics of the pre- and post-treatment isolates also were identical to each other. These analyses suggested that the pre- and post-treatment isolates were isogenic. For these strains, Table 2 shows the antimicrobial susceptibilities and amino acid changes in GyrA and ParC. The isolate GU-CF08 exhibited 4-fold higher MICs of fluoroquinolones than GU-CF09, but showed MICs of the other agents identical to those of GU-CF08. In the isolate GU-CF08, the threonine at position 83 was changed into an isoleucine in the GyrA, and the serine at position 80 was substituted with an isoleucine in ParC. In the isolate GU-CF09, an alteration of Asp-87→Asn in GyrA was observed in addition to the amino acid changes in GyrA and ParC identical to those found in the isolate GU-CF08. The accumulation of amino acid changes in GyrA appeared to contribute to a specific increase in fluoroquinolone resistance in the isolate GU-CF09.

View this table:
2

Antimicrobial susceptibility profiles and alterations in GyrA and ParC of pre- and post-treatment C. freundii strains isolated from a case of fluoroquinolone treatment failure in urinary tract infection

MICs and alterationsPre-treatment isolate GU-CF08Post-treatment isolate GU-CF09
MIC (mg/l) of:
Norfloxacin25100
Ciprofloxacin12.550
Ofloxacin2550
AM-11556.2525
Piperacillin6.256.25
Cefazolin3.133.13
Cefotaxime0.780.78
Cefixime3.133.13
Aztreonam1.561.56
Imipenem0.20.2
Gentamicin0.390.39
Chloramphenicol0.780.78
Tetracycline3.133.13
Alterations in:
GyrAThr-83→IleThr-83→Ile
Asp-87→Asn
ParCSer-80→IleSer-80→Ile

In this study, we determined the association of mutations in gyrA and parC genes with susceptibilities to fluoroquinolones, and reported a case study of a fluoroquinolone treatment failure in an urinary tract infection, accompanied with emergence of a post-treatment isolate with enhanced resistance to fluoroquinolones. Although we have not proved the alterations in GyrA and ParC actually cause the resistance phenotype, the findings of this study do suggest, in C. freundii as well as in E. coli and N. gonorrhoeae, that DNA gyrase is a primary target of quinolones, that only a single amino acid change at Thr-83 in GyrA is sufficient to generate high-level resistance to ciprofloxacin and norfloxacin, and that the accumulation of amino acid changes in GyrA with the simultaneous presence of alterations in ParC contributes to increment in ciprofloxacin and norfloxacin resistance [810]. To our knowledge, this is the first report to identify the mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of C. freundii. This study should provide useful information for understanding the molecular mechanisms of fluoroquinolone resistance in C. freundii.

Acknowledgements

The authors thank Ms. Kyoko Hirata for technical assistance and laboratory analysis.

References

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