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

FEMS Microbiology Letters 154 (1997) 409^414

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

Yoshinori Nishino, Takashi Deguchi, Mitsuru Yasuda, Takeshi Kawamura, Masahiro Nakano, Emiko Kanematsu, Shigehiko Ozeki, Yukimichi Kawada * Department of Urology, Gifu University School of Medicine, Gifu 500, Japan

Received 3 June 1997; revised 23 July 1997; accepted 28 July 1997

Abstract

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. 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. freundii

Keywords : Citrobacter freundii

; DNA gyrase; Topoisomerase IV

1. Introduction

Neisseria gonorrhoeae, and Haemo, alterations in the GyrA subunit of DNA gyrase play a primary role in developing quinolone resistance [5^9]. In these species, alterations in the ParC subunit of DNA topoisomerase IV play a complementary role in increasing quinolone resistance [7,9^12]. 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. Escherichia coli

,

philus in£uenzae

Citrobacter freundii is often isolated from patients with urinary tract infections. Fluoroquinolones, which have a good activity against C. freundii, have been e¡ective 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 £uoroquinolones. Recently, several mechanisms of quinolone resistance have been identi¢ed in some bacterial species [2^4]. In Gram-negative bacterial species, such as

* Corresponding author. Tel: +81 (58) 265 1241; Fax: +8 (58) 265 9009.

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 3 6 1 - 3

FEMSLE 7789 25-10-97

Y. Nishino et al. / FEMS Microbiology Letters 154 (1997) 409^414

410

Japan), an inoculum of 10

2. Materials and methods

4

CFU per spot was ap-

plied to agar plates containing serial 2-fold dilutions

2.1. Bacterial strains

of each drug. MICs were de¢ned as the lowest concentrations of drug that completely inhibited visible

C. freundii

Type strain of

ATCC8090 was pur-

chased from the American Type Culture Collection.

C. freundii

Thirty-eight clinical strains of

growth of the inoculum after incubation for 18 h at 37³C.

used in this

study were isolated from 1991 through 1994 from Japanese patients with urinary tract infection and

2.4. Detection of mutations in the gyrA and parC genes

maintained in our laboratory. To avoid testing multiple isolates from a single patient,

C. freundii

was

Thirty-eight clinical strains were examined for the

gyrA

and

parC

isolated in only one urinary culture from each pa-

presence of mutations in the

tient during the infection period and was used for

For analysis of the mutations in the region corre-

genes.

this study. No patients were given any quinolones

sponding to the QRDR of the

when they visited a clinic.

parC

E. coli gyrA

and

genes, DNA fragments were ampli¢ed using

the following two sets of primers, EC-GYRA-A

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

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 re-

For partial sequencing of the regions of the and

parC

genes of

C. freundii

gyrA

analogous to the QRDR of the

E. coli gyrA

gene,

DNA fragments were ampli¢ed 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 EC-PARC-B for

parC,

gyrA,

ported [8,10].

containing the region

2.5. Case study of a £uoroquinolone treatment failure in urinary tract infection with emergence of a post-treatment isolate with enhanced resistance to £uoroquinolones

and EC-PARC-A and

and the PCR products were

We examined clinical strains that were isolated

sequenced. EC-GYRA-A (5P-CGCGTACTTTACG-

from a case of £uoroquinolone treatment failure in

CCATGAACGTA-3P) and EC-GYRA-B (5P-CAG-

urinary tract infection. A 65 year-old Japanese man

ACGGATTTCCGTATAACGC-3P)

with urethral stricture presented at the clinic with

were

located

C. freundii

within the consensus amino acids of the bacterial

dysuria and urinary turbidity. A strain of

GyrA proteins and were identical to nucleotide posi-

7 (named GU-CF08) was isolated from his urine (10

E. coli gyrA

CFU/ml). He was treated with a novel £uoroquino-

gene [5]. EC-PARC-A (5P-CTGAACGCCAGCGC-

lone, AM-1155 [13], 100 mg, twice daily for 7 days.

GAAATT-3P) and EC-PARC-B (5P-GCGAAAGA-

When

TTTGGGATCGTC-3P) were identical to nucleotide

symptoms,

tions 139 to 162 and 360 to 381 of the

positions 185 to 204 and 353 to 372 of the

parC

he

returned a

to

strain

of

the

clinic

with

C. freundii

continuing

(named

E. coli

GU7 CF09) was isolated again (10 CFU/ml). To assess

gene [3]. DNA extraction, PCR ampli¢cation,

whether the pre- and post-treatment isolates were

and sequencing of the PCR products were performed

isogenic, arbitrarily primed PCR analysis was per-

as reported previously [8].

formed [14], and biochemical characteristics were examined

2.3. MIC testing

by

Dickinson

using Co.,

Enterotube

Tokyo,

II

Japan).

(Nippon

Becton

The

strains

two

were tested for the MICs of cipro£oxacin, nor£oxaThe susceptibilities of the strains to cipro£oxacin

cin, AM-1155, piperacillin, cefazolin, cefotaxime, ce-

and nor£oxacin were determined by the 2-fold agar

¢xime, aztreonam, imipenem, gentamicin, chloram-

dilution method. The strains were cultured overnight

phenicol,

in Mueller-Hinton broth at 37³C, and using an in-

examined for the presence of mutations in the

oculator (Microplanter ; Sakura Seisakusho, Tokyo,

and

parC

FEMSLE 7789 25-10-97

and

tetracycline.

They

were

also

gyrA

genes. The analysis of the mutations and

Y. Nishino et al. / FEMS Microbiology Letters 154 (1997) 409^414

411

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 signi¢cance set at P 6 0.05. 2.7. Nucleotide sequence accession number

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

Fig. 2. Comparisons of particular regions of the nucleotide sequence of the DNA fragment ampli¢ed 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. Results and discussion

3.1. Ampli¢cation of the C. freundii gyrA and parC genes

Fig. 1. Comparisons of particular regions of the nucleotide sequence of the DNA fragment ampli¢ed 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.

The primers EC-GYRA-A and EC-GYRA-B ampli¢ed 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 ampli¢ed 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

FEMSLE 7789 25-10-97

412

Y. Nishino et al. / FEMS Microbiology Letters 154 (1997) 409^414

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 ¢ndings, we concluded that the determined sequences were partial sequences of the C. freundii gyrA and parC genes. 3.2. Detection of mutations in the gyrA and parC genes ampli¢ed from clinical isolates of C. freundii

The association of mutations in gyrA and parC genes with susceptibilities to £uoroquinolones was determined in 38 urinary tract-derived clinical strains of C. freundii. In 17 isolates with both cipro£oxacin and nor£oxacin 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 cipro£oxacin MIC of 6.25 mg/l and nor£oxacin 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-83CIle. Six strains with cipro£oxacin MICs of 12.5 mg/l to 25 mg/l and nor£oxacin MICs of 25 mg/ l to 50 mg/l had single mutations in the gyrA and parC genes. Eight strains with cipro£oxacin MICs of 50 mg/l to 200 mg/l and nor£oxacin 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-83CIle. The mutations in codon 87 were G-to-A, G-to-C, and A-to-C substitutions, resulting in amino acid changes of Asp-87CAsn, Asp-87CTyr, and Asp-87CVal, 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 Ser80CIle. All the mutations in the codon corresponding to Glu-84 of the E. coli ParC protein were G-toA substitutions, resulting in Glu-84CLys. These alterations observed in GyrA and ParC of C. freundii were analogous to those that were frequently found to be responsible for £uoroquinolone resistance in E. coli and other bacterial species [6,7,12,15^17]. In this study, we found no strains having alterations in

Table 1 Alterations in GyrA and ParC and susceptibilities to cipro£oxacin and nor£oxacin in clinical isolates of C. freundii MIC (mg/l) Amino acid change a a NFLX CPFX GyrA ParC Strain 83 87 80 Type strain 90.025 90.025 Thr(ACC) Asp(GAC) Ser(AGC) ^ ^ 01,11,66 0.1 0.05 ^b 12,58 0.2 0.05 ^ ^ ^ 60,69 0.2 0.1 ^ ^ ^ 04,15 0.78 0.39 ^ ^ ^ 03,07 0.78 0.78 ^ ^ ^ 10,19,65,67 1.56 0.78 ^ ^ ^ 59,68 1.56 1.56 ^ ^ ^ 71,72,73 12.5 6.25 Ile(ATC) ^ ^ 05,06,23,70 25 6.25 Ile(ATC) ^ ^ 02 25 12.5 Ile(ATC) ^ ^ 36 25 12.5 Ile(ATC) ^ Ile(ATC) 34,35 25 25 Ile(ATC) ^ ^ 16,29 50 25 Ile(ATC) ^ Ile(ATC) 18 100 50 Ile(ATC) Tyr(TAC) Ile(ATC) 31,48,51 100 50 Ile(ATC) Tyr(TAC) ^ 55 100 100 Ile(ATC) Tyr(TAC) Ile(ATC) 13,49,50 200 200 Ile(ATC) Val(GTC) Ile(ATC) a NFLX, nor£oxacin; CPFX, cipro£oxacin. b ^, identical to type strain.

FEMSLE 7789 25-10-97

84 Glu(GAA) ^ ^ ^ ^ ^ ^ ^ ^ ^ Lys(AAA) ^ Lys(AAA) ^ ^ Lys(AAA) ^ ^

Y. Nishino et al. / FEMS Microbiology Letters 154 (1997) 409^414

413

Table 2 Antimicrobial susceptibility pro¢les and alterations in GyrA and ParC of pre- and post-treatment

C. freundii

strains isolated from a case

of £uoroquinolone treatment failure in urinary tract infection MICs and alterations

Pre-treatment isolate GU-CF08

Post-treatment isolate GU-CF09

Nor£oxacin

25

100

Cipro£oxacin

12.5

50

O£oxacin

25

50

MIC (mg/l) of :

AM-1155

6.25

Piperacillin

6.25

25 6.25

Cefazolin

3.13

3.13

Cefotaxime

0.78

0.78

Ce¢xime

3.13

3.13

Aztreonam

1.56

1.56

Imipenem

0.2

0.2

Gentamicin

0.39

0.39

Chloramphenicol

0.78

0.78

Tetracycline

3.13

3.13

Alterations in : GyrA

C C

Thr-83

ParC

Ser-80

Ile

C C C

Thr-83

Asp-87

Ile

Ser-80

Ile Asn

Ile

ParC without the simultaneous presence of altera-

tical to each other. These analyses suggested that the

tions in GyrA.

pre- and post-treatment isolates were isogenic. For

The strains having a single or double amino acid

these strains, Table 2 shows the antimicrobial sus-

change in GyrA exhibited signi¢cantly higher-level

ceptibilities and amino acid changes in GyrA and

resistance

ParC. The isolate GU-CF08 exhibited 4-fold higher

to

cipro£oxacin

and

nor£oxacin

than

those without alterations in either GyrA or ParC

MICs

(P

0.01). The six strains with single amino acid

showed MICs of the other agents identical to those

changes in GyrA and ParC were signi¢cantly more

of GU-CF08. In the isolate GU-CF08, the threonine

resistant to cipro£oxacin and nor£oxacin than the

at position 83 was changed into an isoleucine in the

seven strains with a single amino acid change in

GyrA, and the serine at position 80 was substituted

6

GyrA

P

6

alone

(cipro£oxacin,

P

6

0.05 ;

nor£oxacin,

0.01). The eight strains with a double amino

of

£uoroquinolones

than

GU-CF09,

but

C

with an isoleucine in ParC. In the isolate GU-CF09, an alteration of Asp-87

Asn in GyrA was observed

acid change in GyrA and a single amino acid change

in addition to the amino acid changes in GyrA and

in ParC were signi¢cantly more resistant to cipro-

ParC identical to those found in the isolate GU-

£oxacin and nor£oxacin than the six strains with

CF08. The accumulation of amino acid changes in

single

GyrA appeared to contribute to a speci¢c increase in

(P

6

amino

acid

changes

in

GyrA

and

ParC

0.01).

£uoroquinolone resistance in the isolate GU-CF09. In this study, we determined the association of

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

mutations in

gyrA

and

parC

genes with susceptibil-

ities to £uoroquinolones, and reported a case study of a £uoroquinolone treatment failure in an urinary tract infection, accompanied with emergence of a

The electrophoresis pro¢le of the DNAs ampli¢ed

post-treatment isolate with enhanced resistance to

by AP-PCR from the post-treatment isolate GU-

£uoroquinolones. Although we have not proved the

CF09 was identical to that from the pre-treatment

alterations in GyrA and ParC actually cause the re-

isolate GU-CF08. The biochemical characteristics of

sistance phenotype, the ¢ndings of this study do sug-

the pre- and post-treatment isolates also were iden-

gest, in

FEMSLE 7789 25-10-97

C. freundii

as well as in

E. coli

and

N. go-

Y. Nishino et al. / FEMS Microbiology Letters 154 (1997) 409^414

414

norrhoeae,

that DNA gyrase is a primary target of

quinolones, that only a single amino acid change at Thr-83 in GyrA is su¤cient to generate high-level resistance

to

cipro£oxacin

and

nor£oxacin,

and

gyrA and parC in £uoroquinolone-resistant isolates.

regions of

Mol. Microbiol. 14, 371^380. [8] Deguchi, T., Yasuda, M., Asano, M., Tada, K., Iwata, H., Komeda, H., Ezaki, T., Saito, I. and Kawada, Y. (1995) DNA gyrase mutations in quinolone-resistant clinical isolates of

that the accumulation of amino acid changes in

Neisseria gonorrhoeae.

GyrA with the simultaneous presence of alterations

561^563.

in ParC contributes to increment in cipro£oxacin and nor£oxacin resistance [8^10]. To our knowledge, this is the ¢rst report to identify the mutations in the

gyrA

parC

and

genes associated with £uoroquino-

lone resistance in clinical isolates of

C. freundii.

This study should provide useful information for understanding the molecular mechanisms of £uoroquinolone resistance in

C. freundii.

Antimicrob. Agents Chemother. 39,

[9] Georgiou, M., Munoz, R., Roman, F., Canton, R., GomezLus, R., Campos, J. and De la Campa, A.G. (1996) Cipro£oxacin-resistant

Hemophilus in£uenzae

strains possess muta-

tions in analogous positions GyrA and ParC. Antimicrob. Agents Chemother. 40, 1741^1744. [10] Deguchi, T., Yasuda, M., Nakano, M., Ozeki, S., Ezaki, T., Saito, I. and Kawada, Y. (1996) Quinolone-resistant

gonorrhoeae ;

Neisseria

Correlation of alterations in the GyrA subunit

of DNA gyrase and the ParC subunit of topoisomerase IV with antimicrobial susceptibility pro¢les. Antimicrob. Agents Chemother. 40, 1020^1023. [11] Vila, J., Ruiz, J., Goni, P. and Jimenez de Anta, M.T. (1996)

Acknowledgments

parC in quinolone-resistant clinical Escherichia coli. Antimicrob. Agents Chemother.

Detection of mutations in isolates of

40, 491^493.

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

[12] Kumagai, Y., Kato, J-I., Hoshino, K., Akasaka, T., Sato, K. and Ikeda, H. (1996) Quinolone-resistant mutants of

chia coli

DNA topoisomerase IV

parC

Escheri-

gene. Antimicrob.

Agents Chemother. 40, 710^714.

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