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Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa
International Journal of Antimicrobial Agents 21 (2003) 409 /413 www.ischemo.org
Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa P.G. Higgins a,*, A.C. Fluit c, D. Milatovic c, J. Verhoef c, F.-J. Schmitz b,c a
b
Institute of Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelstrasse 19-21, 50935 Cologne, Germany Institute for Medical Microbiology and Virology, Universita¨tsklinikum Du¨sseldorf, Universita¨tsstraße 1, Geb. 22.21, 40225 Dusseldorf, Germany c Eijkman /Winkler Institute for Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands Received 15 July 2002; accepted 29 August 2002
Abstract The target enzymes GyrA and ParC and two efflux pump regulatory genes mex R and nfx B were analysed to determine changes associated with fluoroquinolone resistance in Pseudomonas aeruginosa . Both low- and high-level ciprofloxacin resistance was associated with a Thr-83Ile substitution in GyrA. A ParC Ser-80Leu substitution was found in highly resistant isolates in tandem with the Thr-83Ile substitution in GyrA. Mutations in the efflux regulatory genes were associated with resistance only when in tandem with a mutation in GyrA or ParC. These data show that the main mechanism of fluoroquinolone resistance in P. aeruginosa is mediated primarily through mutations in GyrA, and that mutations in ParC and the efflux regulatory genes are secondary. # 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Pseudomonas aeruginosa ; Fluoroquinolone; GyrA; ParC; Efflux
1. Introduction Pseudomonas aeruginosa is a major threat within the hospital environment and is one of the most common Gram-negative microbes isolated from patients. It is responsible for a number of opportunist infections, including pneumonias, septicaemias, wound infections and urinary tract infections. It is also a major cause of morbidity in cystic fibrosis sufferers. Innately resistant to many antibiotics, treatment options are limited to a few drugs such as the ureidopenicillins, the carbapenems, ceftazidime and ciprofloxacin. Unfortunately, resistance to these drugs is increasing and the selection of resistant organisms during fluoroquinolone therapy, particularly multi-drug resistant phenotypes, have been well documented [1 /4]. Although fluoroquinolone development has produced more potent drugs, for example moxifloxacin, gatifloxacin and trovafloxacin, these do not exhibit enhanced activity against P. aeruginosa .
* Corresponding author. Tel.: /49-221-478-3064; fax: /49-221478-3067. E-mail address: [email protected] (P.G. Higgins).
Fluoroquinolone resistance in P. aeruginosa is mediated primarily through mutations in the target enzymes GyrA and ParC, although there are reports on the involvement of GyrB mutations contributing to reduced sensitivity [1]. A secondary resistance mechanism, active drug efflux, is characterised not only by reduced sensitivity to the fluoroquinolones but also its effect on a broad spectrum of unrelated antibiotics [1,5,6]. Fluoroquinolones, as well as non-fluoroquinolones are pumped out of the cell, reducing intracellular drug concentration to below inhibitory levels leading to the emergence of the multi drug resistant phenotype. This is brought about by increased expression of the efflux pumps MexAB /OprM and MexCD /OprJ through mutations in their regulatory genes mex R and nfx B, respectively [3,5]. Two other efflux pumps are known to reduce fluoroquinolone sensitivity in P. aeruginosa , MexEF and MexXY although their regulators are less well characterised. As multi-drug resistance rates are increasing with P. aeruginosa , new therapeutic options are in demand. In the present study we sought to compare the potency of the newer fluoroquinolones, sitafloxacin and clinafloxacin with ciprofloxacin against 58 imipenem-resistant P.
0924-8579/03/$30 # 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/S0924-8579(03)00009-8
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P.G. Higgins et al. / International Journal of Antimicrobial Agents 21 (2003) 409 /413
aeruginosa isolates. Their gyr A and par C genes were amplified to identify target-site mutations. The efflux regulatory genes mex R and nfx B were also sequenced.
2. Materials and methods 2.1. Bacterial strains A total of 58 unrelated P. aeruginosa strains used in this study were clinical isolates from European hospitals as part of the SENTRY antimicrobial surveillance programme. These were isolated in 1998/1999. The isolates were typed using ribotyping and pulsed field gel electrophoresis [7]. 2.2. Antimicrobial agents and susceptibility testing Susceptibility of the isolates to sitafloxacin, ciprofloxacin, trovafloxacin, levofloxacin, clinafloxacin, gatifloxacin, moxifloxacin and imipenem were performed by broth microdilution according to the NCCLS guidelines [8]. 2.3. PCR amplification and DNA sequencing The PCR amplification of the QRDR of gyr A, par C, mex R and nfxB genes was carried out using the primers and conditions previously described [5]. Amplified PCR products were visualised by electrophoresis in agarose gels containing 0.5 mg/l ethidium bromide.
3. Results In this study we examined by sequencing the QRDR of gyr A and par C as well as two efflux pump regulatory genes, mex R and nfx B, of 58 imipenem-resistant P. aeruginosa . Mutational changes and MIC data are summarised in Table 1. Results of ribotyping and PFGE did not show clustering of isolates indicating the mutations recorded were not due to clonal spread. Irrespective of mutational changes, sitafloxacin and clinafloxacin were the most potent of the drugs tested. Trovafloxacin, gatifloxacin and moxifloxacin were the least potent. Ciprofloxacin resistance was found in 88% of the isolates. Six strains showed no alterations to the amino acid sequences of the genes investigated, however one of these isolates was resistant to the fluoroquinolones tested, with a ciprofloxacin MIC of 4 mg/l. A single NfxB Ala-124Glu substitution was fluoroquinolone susceptible. One other isolate did not carry a GyrA or ParC mutation and had a ciprofloxacin of 4 mg/l and gatifloxacin and moxifloxacin MIC of 16 mg/l. Isolates with a single GyrA Thr-83Ile substitution had cipro-
floxacin MICs of 2 /16 mg/l. This was the most common GyrA mutation, found in 42 of 48 isolates with a ciprofloxacin MIC E/4 mg/l. Mutations in at least one of the efflux regulatory genes were found in 35 isolates. A Ser-80Phe substitution in ParC was found in 24 isolates, all of them ciprofloxacin-resistant and was associated with the Thr-83Ile mutation in GyrA in all but one isolate. In MexR, an Asn-53Asp was the most frequent substitution, found in nine isolates, all of which were ciprofloxacin-resistant. An Asn-53Tyr in MexR combined with a novel Ala-136Gly substitution in GyrA did not confer resistance. In NfxB, an Ala-124Glu substitution was found in 15 isolates, 14 of which were ciprofloxacin-resistant. With one exception, these efflux regulatory-gene substitutions were associated with the Thr-83Ile GyrA mutation. A single Ala-124Glu strain was ciprofloxacin susceptible. Eleven isolates had at least one mutation in both regulatory genes and these all had ciprofloxacin MICs E/4 mg/l. There were no nucleotide deletions or additions within these genes. Low-level ciprofloxacin resistance (4 /8 mg/l) was associated with a Thr-83Ile mutation in GyrA. A single Asp-87Gly GyrA mutation was found in one isolate that had a ciprofloxacin MIC of 8 mg/l. However, a single Asp-87Asn recorded a ciprofloxacin MIC of 1 mg/l. Two isolates had a mutation in ParC without a GyrA mutation. Both of these isolates also carried a mutation in one of the efflux regulatory genes; a His107-Pro in MexR combined with a Ser-80Phe substitution in ParC gave a ciprofloxacin MIC /16 mg/l while a double Arg21His/Asp-56Gly in NfxB with a Lys-73Val in ParC had a lower ciprofloxacin MIC of 4 mg/l. High-level ciprofloxacin resistance (E/16 mg/l) was associated with a Thre-83Ile substitution in GyrA and a Ser-80Phe/Leu/Trp substitution in ParC. These mutations were also found in combination with other GyrA/ ParC and MexR/NfxB mutations. Three isolates had a single Thr-83Ile GyrA mutation and recorded a ciprofloxacin MIC of 16 mg/l, suggesting the contribution of other factors. Sitafloxacin and clinafloxacin MICs E/8 mg/l were found in 11 isolates. Each of these isolates carried the Thr-83Ile GyrA mutation and eight of these strains also carried a Ser-80Leu/Trp substitution in ParC. Those in this group without a ParC mutation carried substitutions in MexR and NfxB. The most resistant isolate did not have a mutation in ParC but a double GyrA mutation (Thr-83Ile and Asp-87Asn) combined with an Asn-53Asp in MexR and Ala124Glu in NfxB.
4. Discussion These data confirm that the primary mechanism of ciprofloxacin resistance in P. aeruginosa is mediated
P.G. Higgins et al. / International Journal of Antimicrobial Agents 21 (2003) 409 /413
411
Table 1 MICs of P. aeruginosa isolates and amino acid substitutions sorted by low /high ciprofloxacin MIC N a Amino acid substitutions GyrA c
MIC (mg/l)b
ParC
MexR
NfxB
SITX
CIPX
CLIX
LEVX
TRFX
GATX
MOXX
6 1 1 1
wt wt Ala-136Gly Asp-87Asn
wt wt wt wt
wt wt Asn-53Tyr wt
wt Ala-124Glu wt wt
B/0.06 /2 0.25 0.5 1
B/0.06 /4 0.25 1 1
B/0.06 /2 0.12 0.25 1
0.25 /8 0.5 2 2
0.12 /8 0.5 2 2
0.25 /8 1 4 4
0.5 /16 2 8 8
1
wt
wt
Cys-30Arg Gly-77Ala Asn-79Ser Gln-106Arg
Met-1Ile
2
Arg-114Cys
1
8
4
16
16
1
Thr-83Ile
wt
wt
Ala-38Gly
2
4
2
8
8
8
16
1
Thr-83Ile Ile-155Val
wt
wt
wt
2
4
2
8
8
8
16
1
Thr-83Ile
Ser-80Leu
Asn-86Ile
wt
2
4
2
8
16
16
16
1
wt
Lys-73Val
wt
Arg-21His Asp-56Gly
2
4
2
8
8
16
/16
1 1 1 6 2 1
Thr-83Ile Thr-83Ile Asp-87Gly Thr-83Ile Thr-83Ile Thr-83Ile
wt wt wt wt wt Leu-71Phe
Ser-88Cys Arg-63His wt wt Asn-53Asp Glu-48Lys
wt wt wt wt wt Ala-124Glu
2 2 2 1 /4 2 /4 2
4 4 8 2 /16 8 /16 8
2 2 2 1 /4 2 2
8 16 8 4 / /16 16 /16
8 16 8 4 / /16 16 /16
8 8 16 4 / /16 16 /16
16 16 /16 8 / /16 /16 /16
1
Thr-83Ile
Ser-80Leu
Asn-53Asp
Ala-124Glu Arg-161Pro
2
8
2
16
16
16
16
1
Asn-60Lys Pro-62Ala Thr-83Ile
wt
Asn-53Asp
wt
2
8
2
16
/16
16
/16
1 1
Thr-83Ile Thr-83Ile
Gly-78Cys Ser-80Leu
wt Asn-53Asp
Ala-124Glu Glu-111Lys
2 2
16 16
2 2
16 /16
/16 /16
16 /16
/16 /16
1
Ser-53Arg Thr-83Ile Gln-94His
Glu-84Lys
wt
wt
2
16
2
16
/16
16
/16
1
Thr-83Ile
Ser-80Trp
wt
Arg-21His Asp-56Gly Ala-124Glu
2
/16
2
16
/16
/16
/16
1
Thr-83Ile
Leu-71Phe
wt
Ala-124Glu
2
/16
2
/16
/16
/16
/16
1
Thr-83Ile Ala-93Gly
Ser-80Leu
wt
wt
2
/16
4
/16
/16
/16
/16
1 2 1
Thr-83Ile Thr-83Ile wt
Ser-80Leu Ser80Leu Ser-80Leu
wt wt His-107Pro
Glu-8Lys wt wt
4 4 4
/16 /16 /16
2 2-4 4
/16 /16 /16
/16 /16 /16
/16 /16 /16
/16 /16 /16
1
Thr-83Ile Tyr-100Cys
Ser-80Leu
Arg-21Gly Ser-26Gly
Ala-124Glu
4
/16
4
/16
/16
/16
1
Lys-61Arg Thr-83Ile Ala-136Gly
Gly-72Cys Ser-80Leu
wt
wt
4
/16
4
/16
/16
/16
/16
1 1 1
Thr-83Ile Thr-83Ile Thr-83Ile
Asp-79Asn Ser80-Trp Ser63-Leu
Asn-79Gly wt Asn-53Asp
wt Ala124-Glu Ala124-Glu
4 4 4
/16 /16 /16
4 4 4
/16 /16 /16
/16 /16 /16
/16 /16 /16
/16 /16 /16
1
Thr-83Ile Asn-86Ile
Ser-80Leu
Asn-53Asp
Gln64-His
4
/16
4
/16
/16
/16
/16
1
Thr-83Ile Ala-93Gly
Ser-80Leu
wt
Ala124-Glu
4
/16
4
/16
/16
/16
/16
1
Thr-83Ile
wt
Met-10Arg
wt
8
16
8
/16
/16
/16
/16
P.G. Higgins et al. / International Journal of Antimicrobial Agents 21 (2003) 409 /413
412 Table 1 (Continued ) N a Amino acid substitutions
MIC (mg/l)b
GyrA
ParC
MexR
NfxB
SITX
CIPX
CLIX
LEVX
TRFX
GATX
MOXX
1 1
Thr-83Ile Thr-83Ile
Ser-80Leu wt
Ala-103Thr Asn-53Tyr
wt wt
8 8
16 16
8 8
/16 /16
/16 /16
/16 /16
/16 /16
1
Ser-53Gly Thr-83Ile Ala-131Asp Ala-136Val
Ser-80Leu
wt
Ala-124Glu Ala-141Gly
4
/16
8
/16
/16
/16
/16
2 3
Thr-83Ile Thr-83Ile
Ser80-Trp Ser-80Leu
wt wt
wt Ala-124Glu
4 /8 4 /8
/16 /16
4 /8 4 /8
/16 /16
/16 /16
/16 /16
/16 /16
1
Thr-83Ile
Gly-72Arg Ser-80Leu
wt
Ser-36Gly Gln-52His Glu-75Gln
8
/16
4
/16
/16
/16
/16
1
Thr-83Ile Asp87-Tyr
Ser-80Leu
Ser-88Cys Gln-106His
wt
8
/16
8
/16
/16
/16
/16
1
Thr-83Ile Asp-87His
Ser-80Leu
wt
wt
8
/16
16
/16
/16
/16
/16
1
Thr-83Ile Asp-87Asn
wt
Asn-53Asp
Ala-124Glu
16
/16
16
/16
/16
/16
/16
Arg-78Ile
a
N , number of strains. SITX, sitafloxacin; CIPX, ciprofloxacin; CLIX, clinafloxacin; LEVX, levofloxacin; TRFX, trovafloxacin; GATX, gatifloxacin; MOXX, moxifloxacin. c wt, wild-type. b
through target site alterations, specifically a Thr-83Ile substitution in GyrA. Sitafloxacin and clinafloxacin MICs were elevated in strains with this as a sole substitution but only one isolate had an MIC /2 mg/ l for both drugs. A Ser-80Leu ParC substitution was found in high-level ciprofloxacin-resistant isolates. The MexR Asn-53Asp and NfxB Ala-124Glu substitutions were associated with ciprofloxacin-resistance only when expressed in tandem with the Thr-83Ile GyrA alteration. The mechanism of imipenem resistance was not investigated in this study, however upregulation of MexAB / OprM does not result in raised imipenem MICs [9]. Sitafloxacin and clinafloxacin MICs were the least affected by the mutational changes. Their greater potency over ciprofloxacin suggests that they may be of therapeutic value against low-level fluoroquinoloneresistance P. aeruginosa. However no breakpoints have been established for these drugs to date and their in vivo efficacy is unknown. Our data suggest the involvement of other resistance factors. The evidence for this comes in part from MIC differentials with isolates not harbouring mutations in any of the genes investigated. Their ciprofloxacin MICs ranged from /0.06 to 4 mg/l. Further evidence comes from single GyrA Thr-83Ile mutants with a ciprofloxacin MIC range of 2 /16 mg/l, a phenotype previously reported by Jalal and Wretlind [3]. These other factors can include mutations in GyrB [4,10], ParE and the intergenic region between mex R and the mex AB /opr M
operon which has previously been shown to harbour mutations that affect the regulation of mex R [1]. The efflux pump MexXY has also been shown to be involved in fluoroquinolone resistance and upregulation of MexEF-OprN may affect both fluoroquinolones and imipenem [9,11 /13]. Okazaki and Hirai report resistance mutations within NfxB are found in a putative DNA binding domain between amino acid residues 26 and 42 [14]. While we found two resistant isolates with mutations within this region (Ser-36Gly and Ala-38Gly), they both carried the GyrA Thr-83Ile mutation. Given that the GyrA mutation on its own can cause reduced sensitivity to the fluoroquinolones, the contribution of the NfxB mutations is speculative. The crystal structure of the E. coli efflux pump regulator MarR has recently been resolved [15]. MexR is part of the MarR family and they share 31% amino acid identity. The DNA-binding domain is thought to be found between amino acid residues 55 and 100 in MarR. This region shows the greatest homology between the two proteins. The crystal structure suggests that DNA-binding is centred between amino residues 70 /90. The majority of isolates in the present study with mutations in MexR were unaffected in this area. We found that an Asn-53Asp substitution was the most common substitution in this regulatory protein. However, as with the NfxB mutations, they were always
P.G. Higgins et al. / International Journal of Antimicrobial Agents 21 (2003) 409 /413
found in combination with other mutations, most notably Thr-83Ile in GyrA. The role of the amino acid substitutions we found within the regulators MexR and NfxB require further investigation. In particular, the role of MexR Asn53Asp and NfxB Ala-124Glu. We did not test for increased expression of the outer membrane components of the efflux pumps (OprM and OprJ) and therefore do not know if the substitutions lead to increased expression. However, these data suggest that target-site mutations, particulary in the GyrA Thr-83 codon, are the primary cause of reduced fluoroquinolone sensitivity in P. aeruginosa .
References [1] Le Thomas I, Couetdic G, Clermont O, Brahimi N, Plesiat P, Bingen E. In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy. J Antimicrob Chemother 2001;48:553 /5. [2] Nakano M, Yasuda M, Yokoi S, Takahashi Y, Ishihara S, Deguchi T. In vivo selection of Pseudomonas aeruginosa with decreased susceptibilities to fluoroquinolones during fluoroquinolone treatment of urinary tract infection. Urology 2001;58:125 /8. [3] Jalal S, Ciofu O, Hoiby N, Gotoh N, Wretlind B. Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother 2000;44:710 /2. [4] Ziha-Zarifi I, Llanes C, Kohler T, Pechere JC, Plesiat P. In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA /MexB / OprM. Antimicrob Agents Chemother 1999;43:287 /91. [5] Jalal S, Wretlind B. Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa . Microb Drug Resist 1998;4:257 /61.
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[6] Evans K, Adewoye L, Poole K. MexR repressor of the mex AB / opr M multidrug efflux operon of Pseudomonas aeruginosa : Identification of MexR binding sites in the mex A /mex R intergenic region. J Bacteriol 2001;183:807 /12. [7] Brisse S, Milatovic S, Fluit AC, et al. Molecular surveillance of European quinolone-resistant clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. using automated ribotyping. J Clin Microbiol 2000;38:3636 /45. [8] National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A5, 5th ed. Wayne, PA, NCCLS 2000. [9] Livermore DM. Of Pseudomonas, porins, pumps and carbapenems. J Antimicrob Chemother 2001;47:247 /50. [10] Akasaka T, Tanaka M, Yamaguchi A, Sato K. Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrob Agents Chemother 2001;45:2263 /8. [11] Mine T, Morita Y, Kataoka A, Mizushima T, Tsuchiya T. Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa . Antimicrob Agents Chemother 1999;43:415 /7. [12] Kohler T, Micheahamzehpour M, Plesiat P, Kahr AL, Pechere JC. Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa . Antimicrob Agents Chemother 1997;41:2540 /3. [13] Fukuda H, Hosaka M, Iyobe S, Gotoh N, Nishino T, Hirai K. nfxc -type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa . Antimicrob Agents Chemother 1995;39:790 /2. [14] Okazaki T, Hirai K. Cloning and nucleotide-sequence of the Pseudomonas aeruginosa nfx B gene, conferring resistance to new quinolones. FEMS Microbiol Lett 1992;97:197 /202. [15] Alekshun MN, Levy SB, Mealy TR, Seaton BA, Head JF. The crystal structure of MarR, a regulator of multiple antibiotic resistance, at 2.3 angstrom resolution. Nat Struct Biol 2001;8:710 /4.
Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa P.G. Higgins a,*, A.C. Fluit c, D. Milatovic c, J. Verhoef c, F.-J. Schmitz b,c a
b
Institute of Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelstrasse 19-21, 50935 Cologne, Germany Institute for Medical Microbiology and Virology, Universita¨tsklinikum Du¨sseldorf, Universita¨tsstraße 1, Geb. 22.21, 40225 Dusseldorf, Germany c Eijkman /Winkler Institute for Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands Received 15 July 2002; accepted 29 August 2002
Abstract The target enzymes GyrA and ParC and two efflux pump regulatory genes mex R and nfx B were analysed to determine changes associated with fluoroquinolone resistance in Pseudomonas aeruginosa . Both low- and high-level ciprofloxacin resistance was associated with a Thr-83Ile substitution in GyrA. A ParC Ser-80Leu substitution was found in highly resistant isolates in tandem with the Thr-83Ile substitution in GyrA. Mutations in the efflux regulatory genes were associated with resistance only when in tandem with a mutation in GyrA or ParC. These data show that the main mechanism of fluoroquinolone resistance in P. aeruginosa is mediated primarily through mutations in GyrA, and that mutations in ParC and the efflux regulatory genes are secondary. # 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Pseudomonas aeruginosa ; Fluoroquinolone; GyrA; ParC; Efflux
1. Introduction Pseudomonas aeruginosa is a major threat within the hospital environment and is one of the most common Gram-negative microbes isolated from patients. It is responsible for a number of opportunist infections, including pneumonias, septicaemias, wound infections and urinary tract infections. It is also a major cause of morbidity in cystic fibrosis sufferers. Innately resistant to many antibiotics, treatment options are limited to a few drugs such as the ureidopenicillins, the carbapenems, ceftazidime and ciprofloxacin. Unfortunately, resistance to these drugs is increasing and the selection of resistant organisms during fluoroquinolone therapy, particularly multi-drug resistant phenotypes, have been well documented [1 /4]. Although fluoroquinolone development has produced more potent drugs, for example moxifloxacin, gatifloxacin and trovafloxacin, these do not exhibit enhanced activity against P. aeruginosa .
* Corresponding author. Tel.: /49-221-478-3064; fax: /49-221478-3067. E-mail address: [email protected] (P.G. Higgins).
Fluoroquinolone resistance in P. aeruginosa is mediated primarily through mutations in the target enzymes GyrA and ParC, although there are reports on the involvement of GyrB mutations contributing to reduced sensitivity [1]. A secondary resistance mechanism, active drug efflux, is characterised not only by reduced sensitivity to the fluoroquinolones but also its effect on a broad spectrum of unrelated antibiotics [1,5,6]. Fluoroquinolones, as well as non-fluoroquinolones are pumped out of the cell, reducing intracellular drug concentration to below inhibitory levels leading to the emergence of the multi drug resistant phenotype. This is brought about by increased expression of the efflux pumps MexAB /OprM and MexCD /OprJ through mutations in their regulatory genes mex R and nfx B, respectively [3,5]. Two other efflux pumps are known to reduce fluoroquinolone sensitivity in P. aeruginosa , MexEF and MexXY although their regulators are less well characterised. As multi-drug resistance rates are increasing with P. aeruginosa , new therapeutic options are in demand. In the present study we sought to compare the potency of the newer fluoroquinolones, sitafloxacin and clinafloxacin with ciprofloxacin against 58 imipenem-resistant P.
0924-8579/03/$30 # 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/S0924-8579(03)00009-8
410
P.G. Higgins et al. / International Journal of Antimicrobial Agents 21 (2003) 409 /413
aeruginosa isolates. Their gyr A and par C genes were amplified to identify target-site mutations. The efflux regulatory genes mex R and nfx B were also sequenced.
2. Materials and methods 2.1. Bacterial strains A total of 58 unrelated P. aeruginosa strains used in this study were clinical isolates from European hospitals as part of the SENTRY antimicrobial surveillance programme. These were isolated in 1998/1999. The isolates were typed using ribotyping and pulsed field gel electrophoresis [7]. 2.2. Antimicrobial agents and susceptibility testing Susceptibility of the isolates to sitafloxacin, ciprofloxacin, trovafloxacin, levofloxacin, clinafloxacin, gatifloxacin, moxifloxacin and imipenem were performed by broth microdilution according to the NCCLS guidelines [8]. 2.3. PCR amplification and DNA sequencing The PCR amplification of the QRDR of gyr A, par C, mex R and nfxB genes was carried out using the primers and conditions previously described [5]. Amplified PCR products were visualised by electrophoresis in agarose gels containing 0.5 mg/l ethidium bromide.
3. Results In this study we examined by sequencing the QRDR of gyr A and par C as well as two efflux pump regulatory genes, mex R and nfx B, of 58 imipenem-resistant P. aeruginosa . Mutational changes and MIC data are summarised in Table 1. Results of ribotyping and PFGE did not show clustering of isolates indicating the mutations recorded were not due to clonal spread. Irrespective of mutational changes, sitafloxacin and clinafloxacin were the most potent of the drugs tested. Trovafloxacin, gatifloxacin and moxifloxacin were the least potent. Ciprofloxacin resistance was found in 88% of the isolates. Six strains showed no alterations to the amino acid sequences of the genes investigated, however one of these isolates was resistant to the fluoroquinolones tested, with a ciprofloxacin MIC of 4 mg/l. A single NfxB Ala-124Glu substitution was fluoroquinolone susceptible. One other isolate did not carry a GyrA or ParC mutation and had a ciprofloxacin of 4 mg/l and gatifloxacin and moxifloxacin MIC of 16 mg/l. Isolates with a single GyrA Thr-83Ile substitution had cipro-
floxacin MICs of 2 /16 mg/l. This was the most common GyrA mutation, found in 42 of 48 isolates with a ciprofloxacin MIC E/4 mg/l. Mutations in at least one of the efflux regulatory genes were found in 35 isolates. A Ser-80Phe substitution in ParC was found in 24 isolates, all of them ciprofloxacin-resistant and was associated with the Thr-83Ile mutation in GyrA in all but one isolate. In MexR, an Asn-53Asp was the most frequent substitution, found in nine isolates, all of which were ciprofloxacin-resistant. An Asn-53Tyr in MexR combined with a novel Ala-136Gly substitution in GyrA did not confer resistance. In NfxB, an Ala-124Glu substitution was found in 15 isolates, 14 of which were ciprofloxacin-resistant. With one exception, these efflux regulatory-gene substitutions were associated with the Thr-83Ile GyrA mutation. A single Ala-124Glu strain was ciprofloxacin susceptible. Eleven isolates had at least one mutation in both regulatory genes and these all had ciprofloxacin MICs E/4 mg/l. There were no nucleotide deletions or additions within these genes. Low-level ciprofloxacin resistance (4 /8 mg/l) was associated with a Thr-83Ile mutation in GyrA. A single Asp-87Gly GyrA mutation was found in one isolate that had a ciprofloxacin MIC of 8 mg/l. However, a single Asp-87Asn recorded a ciprofloxacin MIC of 1 mg/l. Two isolates had a mutation in ParC without a GyrA mutation. Both of these isolates also carried a mutation in one of the efflux regulatory genes; a His107-Pro in MexR combined with a Ser-80Phe substitution in ParC gave a ciprofloxacin MIC /16 mg/l while a double Arg21His/Asp-56Gly in NfxB with a Lys-73Val in ParC had a lower ciprofloxacin MIC of 4 mg/l. High-level ciprofloxacin resistance (E/16 mg/l) was associated with a Thre-83Ile substitution in GyrA and a Ser-80Phe/Leu/Trp substitution in ParC. These mutations were also found in combination with other GyrA/ ParC and MexR/NfxB mutations. Three isolates had a single Thr-83Ile GyrA mutation and recorded a ciprofloxacin MIC of 16 mg/l, suggesting the contribution of other factors. Sitafloxacin and clinafloxacin MICs E/8 mg/l were found in 11 isolates. Each of these isolates carried the Thr-83Ile GyrA mutation and eight of these strains also carried a Ser-80Leu/Trp substitution in ParC. Those in this group without a ParC mutation carried substitutions in MexR and NfxB. The most resistant isolate did not have a mutation in ParC but a double GyrA mutation (Thr-83Ile and Asp-87Asn) combined with an Asn-53Asp in MexR and Ala124Glu in NfxB.
4. Discussion These data confirm that the primary mechanism of ciprofloxacin resistance in P. aeruginosa is mediated
P.G. Higgins et al. / International Journal of Antimicrobial Agents 21 (2003) 409 /413
411
Table 1 MICs of P. aeruginosa isolates and amino acid substitutions sorted by low /high ciprofloxacin MIC N a Amino acid substitutions GyrA c
MIC (mg/l)b
ParC
MexR
NfxB
SITX
CIPX
CLIX
LEVX
TRFX
GATX
MOXX
6 1 1 1
wt wt Ala-136Gly Asp-87Asn
wt wt wt wt
wt wt Asn-53Tyr wt
wt Ala-124Glu wt wt
B/0.06 /2 0.25 0.5 1
B/0.06 /4 0.25 1 1
B/0.06 /2 0.12 0.25 1
0.25 /8 0.5 2 2
0.12 /8 0.5 2 2
0.25 /8 1 4 4
0.5 /16 2 8 8
1
wt
wt
Cys-30Arg Gly-77Ala Asn-79Ser Gln-106Arg
Met-1Ile
2
Arg-114Cys
1
8
4
16
16
1
Thr-83Ile
wt
wt
Ala-38Gly
2
4
2
8
8
8
16
1
Thr-83Ile Ile-155Val
wt
wt
wt
2
4
2
8
8
8
16
1
Thr-83Ile
Ser-80Leu
Asn-86Ile
wt
2
4
2
8
16
16
16
1
wt
Lys-73Val
wt
Arg-21His Asp-56Gly
2
4
2
8
8
16
/16
1 1 1 6 2 1
Thr-83Ile Thr-83Ile Asp-87Gly Thr-83Ile Thr-83Ile Thr-83Ile
wt wt wt wt wt Leu-71Phe
Ser-88Cys Arg-63His wt wt Asn-53Asp Glu-48Lys
wt wt wt wt wt Ala-124Glu
2 2 2 1 /4 2 /4 2
4 4 8 2 /16 8 /16 8
2 2 2 1 /4 2 2
8 16 8 4 / /16 16 /16
8 16 8 4 / /16 16 /16
8 8 16 4 / /16 16 /16
16 16 /16 8 / /16 /16 /16
1
Thr-83Ile
Ser-80Leu
Asn-53Asp
Ala-124Glu Arg-161Pro
2
8
2
16
16
16
16
1
Asn-60Lys Pro-62Ala Thr-83Ile
wt
Asn-53Asp
wt
2
8
2
16
/16
16
/16
1 1
Thr-83Ile Thr-83Ile
Gly-78Cys Ser-80Leu
wt Asn-53Asp
Ala-124Glu Glu-111Lys
2 2
16 16
2 2
16 /16
/16 /16
16 /16
/16 /16
1
Ser-53Arg Thr-83Ile Gln-94His
Glu-84Lys
wt
wt
2
16
2
16
/16
16
/16
1
Thr-83Ile
Ser-80Trp
wt
Arg-21His Asp-56Gly Ala-124Glu
2
/16
2
16
/16
/16
/16
1
Thr-83Ile
Leu-71Phe
wt
Ala-124Glu
2
/16
2
/16
/16
/16
/16
1
Thr-83Ile Ala-93Gly
Ser-80Leu
wt
wt
2
/16
4
/16
/16
/16
/16
1 2 1
Thr-83Ile Thr-83Ile wt
Ser-80Leu Ser80Leu Ser-80Leu
wt wt His-107Pro
Glu-8Lys wt wt
4 4 4
/16 /16 /16
2 2-4 4
/16 /16 /16
/16 /16 /16
/16 /16 /16
/16 /16 /16
1
Thr-83Ile Tyr-100Cys
Ser-80Leu
Arg-21Gly Ser-26Gly
Ala-124Glu
4
/16
4
/16
/16
/16
1
Lys-61Arg Thr-83Ile Ala-136Gly
Gly-72Cys Ser-80Leu
wt
wt
4
/16
4
/16
/16
/16
/16
1 1 1
Thr-83Ile Thr-83Ile Thr-83Ile
Asp-79Asn Ser80-Trp Ser63-Leu
Asn-79Gly wt Asn-53Asp
wt Ala124-Glu Ala124-Glu
4 4 4
/16 /16 /16
4 4 4
/16 /16 /16
/16 /16 /16
/16 /16 /16
/16 /16 /16
1
Thr-83Ile Asn-86Ile
Ser-80Leu
Asn-53Asp
Gln64-His
4
/16
4
/16
/16
/16
/16
1
Thr-83Ile Ala-93Gly
Ser-80Leu
wt
Ala124-Glu
4
/16
4
/16
/16
/16
/16
1
Thr-83Ile
wt
Met-10Arg
wt
8
16
8
/16
/16
/16
/16
P.G. Higgins et al. / International Journal of Antimicrobial Agents 21 (2003) 409 /413
412 Table 1 (Continued ) N a Amino acid substitutions
MIC (mg/l)b
GyrA
ParC
MexR
NfxB
SITX
CIPX
CLIX
LEVX
TRFX
GATX
MOXX
1 1
Thr-83Ile Thr-83Ile
Ser-80Leu wt
Ala-103Thr Asn-53Tyr
wt wt
8 8
16 16
8 8
/16 /16
/16 /16
/16 /16
/16 /16
1
Ser-53Gly Thr-83Ile Ala-131Asp Ala-136Val
Ser-80Leu
wt
Ala-124Glu Ala-141Gly
4
/16
8
/16
/16
/16
/16
2 3
Thr-83Ile Thr-83Ile
Ser80-Trp Ser-80Leu
wt wt
wt Ala-124Glu
4 /8 4 /8
/16 /16
4 /8 4 /8
/16 /16
/16 /16
/16 /16
/16 /16
1
Thr-83Ile
Gly-72Arg Ser-80Leu
wt
Ser-36Gly Gln-52His Glu-75Gln
8
/16
4
/16
/16
/16
/16
1
Thr-83Ile Asp87-Tyr
Ser-80Leu
Ser-88Cys Gln-106His
wt
8
/16
8
/16
/16
/16
/16
1
Thr-83Ile Asp-87His
Ser-80Leu
wt
wt
8
/16
16
/16
/16
/16
/16
1
Thr-83Ile Asp-87Asn
wt
Asn-53Asp
Ala-124Glu
16
/16
16
/16
/16
/16
/16
Arg-78Ile
a
N , number of strains. SITX, sitafloxacin; CIPX, ciprofloxacin; CLIX, clinafloxacin; LEVX, levofloxacin; TRFX, trovafloxacin; GATX, gatifloxacin; MOXX, moxifloxacin. c wt, wild-type. b
through target site alterations, specifically a Thr-83Ile substitution in GyrA. Sitafloxacin and clinafloxacin MICs were elevated in strains with this as a sole substitution but only one isolate had an MIC /2 mg/ l for both drugs. A Ser-80Leu ParC substitution was found in high-level ciprofloxacin-resistant isolates. The MexR Asn-53Asp and NfxB Ala-124Glu substitutions were associated with ciprofloxacin-resistance only when expressed in tandem with the Thr-83Ile GyrA alteration. The mechanism of imipenem resistance was not investigated in this study, however upregulation of MexAB / OprM does not result in raised imipenem MICs [9]. Sitafloxacin and clinafloxacin MICs were the least affected by the mutational changes. Their greater potency over ciprofloxacin suggests that they may be of therapeutic value against low-level fluoroquinoloneresistance P. aeruginosa. However no breakpoints have been established for these drugs to date and their in vivo efficacy is unknown. Our data suggest the involvement of other resistance factors. The evidence for this comes in part from MIC differentials with isolates not harbouring mutations in any of the genes investigated. Their ciprofloxacin MICs ranged from /0.06 to 4 mg/l. Further evidence comes from single GyrA Thr-83Ile mutants with a ciprofloxacin MIC range of 2 /16 mg/l, a phenotype previously reported by Jalal and Wretlind [3]. These other factors can include mutations in GyrB [4,10], ParE and the intergenic region between mex R and the mex AB /opr M
operon which has previously been shown to harbour mutations that affect the regulation of mex R [1]. The efflux pump MexXY has also been shown to be involved in fluoroquinolone resistance and upregulation of MexEF-OprN may affect both fluoroquinolones and imipenem [9,11 /13]. Okazaki and Hirai report resistance mutations within NfxB are found in a putative DNA binding domain between amino acid residues 26 and 42 [14]. While we found two resistant isolates with mutations within this region (Ser-36Gly and Ala-38Gly), they both carried the GyrA Thr-83Ile mutation. Given that the GyrA mutation on its own can cause reduced sensitivity to the fluoroquinolones, the contribution of the NfxB mutations is speculative. The crystal structure of the E. coli efflux pump regulator MarR has recently been resolved [15]. MexR is part of the MarR family and they share 31% amino acid identity. The DNA-binding domain is thought to be found between amino acid residues 55 and 100 in MarR. This region shows the greatest homology between the two proteins. The crystal structure suggests that DNA-binding is centred between amino residues 70 /90. The majority of isolates in the present study with mutations in MexR were unaffected in this area. We found that an Asn-53Asp substitution was the most common substitution in this regulatory protein. However, as with the NfxB mutations, they were always
P.G. Higgins et al. / International Journal of Antimicrobial Agents 21 (2003) 409 /413
found in combination with other mutations, most notably Thr-83Ile in GyrA. The role of the amino acid substitutions we found within the regulators MexR and NfxB require further investigation. In particular, the role of MexR Asn53Asp and NfxB Ala-124Glu. We did not test for increased expression of the outer membrane components of the efflux pumps (OprM and OprJ) and therefore do not know if the substitutions lead to increased expression. However, these data suggest that target-site mutations, particulary in the GyrA Thr-83 codon, are the primary cause of reduced fluoroquinolone sensitivity in P. aeruginosa .
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