ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1990, p. 1889-1894

0066-4804/90/101889-06$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 34, No. 10

Broad-Host-Range Gyrase A Gene Probe N. J. ROBILLARD Pharmaceutical Division, Miles Inc., West Haven, Connecticut 06516 Received 19 March 1990/Accepted 16 July 1990

The Escherichia coli gyrase A gene was cloned in the broad-host-range cosmid vector pLA2917. The resulting plasmid, pNJR3-2, conferred quinolone susceptibility on a gyrA mutant of E. coli. To analyze the expression of this E. coli gene in Pseudomonas aeruginosa, pNJR3-2 or pLA2917 was mobilized via conjugation into P. aeruginosa PAO2 and several well-characterized quinolone-resistant mutants of this strain. The vector pLA2917 did not significantly affect the quinolone susceptibilities of any of the P. aeruginosa strains. However, pNJR3-2 conferred wild-type quinolone susceptibility on P. aeruginosa cfxA (gyrA) mutants and intermediate quinolone susceptibility on cfxA-cfxB double mutants of P. aeruginosa. The quinolone susceptibility of P. aeruginosa PAO2 gyrA + was unaffected by pNJR3-2. Also, pNJR3-2 had no significant effect on P. aeruginosa cfxB (permeability) mutants. These results demonstrate that the DNA gyrase A gene from E. coli is expressed in P. aeruginosa and confers dominant susceptibility on gyrA mutants. Thus, pNJR3-2 can be used to detect the quinolone resistance mutations that occur in the gyrase A gene of this organism. pNJR3-2 also appears to discriminate between mutations in gyrA and mutations which alter permeability. This gyrase A probe was used successfully in the analysis of quinolone resistance in clinical isolates of P. aeruginosa.

The newer 4-quinolones are highly effective as therapeutic

study series, Special considerations in the development of 4-quinolone antibacterial agents [N. J. Robillard, B. L. Masecar, B. Brescia, and R. A. Celesk, University of York, York, England, and Springer-Verlag London, Ltd., 1989].)

agents because of their extreme potencies and broad spectra of activity. These agents are indicated for the treatment of

infections caused by gram-positive or gram-negative bacteria, including Pseudomonas aeruginosa. The bactericidal activities of 4-quinolones have been attributed to the inhibition of DNA gyrase (9, 14, 15, 24), a type II topoisomerase which is unique to bacteria. DNA gyrase, which is essential for the replication of bacterial DNA, is involved in DNA repair, transcription, and recombination (8). The gyrase holoenzyme is composed of two dimeric subunits. The A subunit, which is susceptible to quinolone inhibition, mediates breakage and reunion of double-stranded DNA (9, 29), while the B subunit, which is susceptible to novobiocin and coumermycin, is the site of ATP hydrolysis (28). The holoenzyme, using energy supplied by ATP, converts relaxed DNA to the supercoiled form (5). Analyses of quinolone-resistant mutants of P. aeruginosa by this laboratory (3, 24) and others (13, 15, 23) have identified three classes of mutation which reduce susceptibilities to 4-quinolones. cfxA (gyrA) mutations alter the A subunit of DNA gyrase and result in an 8- to 16-fold reduction in susceptibility to 4-quinolones. Two other classes of mutation appear to affect quinolone permeation. cfxB (3, 24) mutations reduce quinolone susceptibility by four- to eightfold and also reduce susceptibility to novobiocin, carbenicillin, chloramphenicol, and tetracycline. The nfxB mutation (13), which is distinct from cfxB, decreases quinolone susceptibility by 4- to 16-fold, has no effect on tetracycline or chloramphenicol susceptibility, and increases susceptibility to P-lactams and aminoglycosides. To further understand the role of gyrase A mutations and their relative frequencies in quinolone-resistant clinical isolates, I investigated the expression of the Escherichia coli wild-type gyrase A gene in P. aeruginosa. This led to the development of a broad-host-range gyrase A gene probe which can detect gyrase A mutations in P. aeruginosa, E. coli, and other gram-negative bacteria. This probe discriminates between gyrA mutations and other quinolone resistance mutations. (A part of this study was presented during the advanced

MATERIALS AND METHODS Media. ML broth has been described previously (19). For ML agar plates, 1.5% agar (Difco Laboratories, Detroit, Mich.) was added. Cation-supplemented Mueller-Hinton broth (Difco) was used for antimicrobial susceptibility testing, which was performed by standard methods (21). Antibiotics (when added for plasmid selection) were used at the following final concentrations: 100 ,ug/ml, ampicillin; 50 ,ug/ml, kanamycin; 10 ,ug/ml, tetracycline for E. coli; and 200 ,ug/ml, tetracycline for P. aeruginosa. Tetracycline (2 ,ug/ml for E. coli and 200 ,ug/ml for P. aeruginosa) also was used for plasmid maintenance during MIC determinations. Bacterial strains and plasmids. The bacterial strains and plasmids used or constructed in this study are listed in Table 1. Plasmid pSLS447 was kindly provided by Stephen Swanberg and James Wang, Harvard University, Cambridge, Mass. P. aeruginosa MP001 (quinolone susceptible) was recovered from a patient with a complicated infection prior to ciprofloxacin therapy. P. aeruginosa MP002 and MP003 were recovered from the same patient approximately 3 weeks after initiation of parenteral ciprofloxacin therapy. The isolates were verified as one strain by John Ogle (University of Colorado School of Medicine, Denver) using an epidemiological DNA probe (22). MP002 and MP003 were quinolone-resistant gyrase A mutants which also lacked the long-chain O-polysaccharide component of lipopolysaccharide (LPS) (17). P. aeruginosa MP004 and MP005 were uncharacterized clinical isolates. Antibiotics. The following antibiotics were obtained from the indicated sources: ciprofloxacin (Miles Inc., Pharmaceutical Div., West Haven, Conn.); nalidixic acid, ampicillin, tetracycline, kanamycin, and chloramphenicol (Sigma Chemical Co., St. Louis, Mo.); norfloxacin (Merck Sharp & Dohme, Rahway, N.J.); ofloxacin (Ortho Diagnostics, Inc., Raritan, N.J.); and enoxacin (Warner-Lambert Co., Ann Arbor, Mich.). 1889

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TABLE 1. Bacterial strains and plasmids used in this study Strain or

Genotype or

plasmid

phenotype

E. coli DH2

DH1

S17-lb

recAl relAl endAl thi-l hsdRJ7 supE44 gyrA96 recAl relAl endAl thi1 hsdRJ7 supE44 Pro- Res- Mod' Tpr Smr

Reference or source

Bachmann collectiona Bachmann collection

Simon et al. (26)

P. aeruginosa

PAO2 PA04701 PA04702 PA04703 PA04704 PA04741 PA04742 PA04700 PA04739 PA04740 MP001 MP002 MP003 MP004 MP005 Plasmids pSLS447 pLA2917

pNJR3-2

ser-3 cfxA2 ser-3 cfxA3 ser-3 cfxA4 ser-3 cfxAS ser-3 cfxB4 ser-3 cfxBS ser-3 cfxAI cfxBI ser-3 cfxA6 cfxB2 ser-3 cfxA7 cfxB3 ser-3

Holloway collectionc This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory This laboratory Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate

Gyrase A clone (pBR322) Cloning vector Kmr Tcr Gyrase A clone (pLA2917)

Swanberg and Wang (30) Allen and Hanson (1) This study

a E. coli Genetic Stock Center, Yale University School of Medicine, New Haven, Conn. b S17-1 is a mobilizing E. coli strain which carries the transfer genes of the broad-host-range incompatibility group P-type plasmid RP4 integrated in its chromosome. This strain can transfer any plasmid containing a P-type Mob site to any gram-negative bacterium. c Bruce Holloway, Department of Genetics, Monash University, Clayton, Victoria, Australia.

Isolation of DNA gyrase subunits. The DNA gyrase A and B subunits of E. coli were kindly provided by Linus Shen (Abbott Laboratories, Abbott Park, Ill.). The DNA gyrase A and B subunits of P. aeruginosa were isolated as described previously (24, 27), with the following modifications. Gyrase A was eluted from a novobiocin-Sepharose column with a 0.05 to 1.0 M KCl gradient in buffer B (1 mM EDTA, 1 mM dithiothreitol 10% [wt/vol] glycerol, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). Bound gyrase B was washed extensively with 3 M KCI in buffer B and then eluted with 5 M urea in buffer B. DNA supercoiling assay and ICss determination. Relaxed plasmid pBR322 substrate DNA was prepared by treatment with calf thymus topoisomerase I (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) by the protocol recommended by the manufacturer. DNA supercoiling assays were performed as described previously (18). One unit of gyrase was defined as that amount of enzyme which catalyzed one-half maximal supercoiling in 30 min at 37°C in the standard gyrase reaction containing 0.4 ,ug of DNA. Approximately 6 U of gyrases A and B was used per reaction. The 50% inhibitory concentration (IC50) was defined as the

concentration of antibiotic that inhibited 50% of the supercoiling activity of gyrase in a standard gyrase reaction. Antibiotic was added to each reaction before the addition of gyrase. A control reaction without drug was included in each assay. The IC50 was determined by visual inspection of the most supercoiled band in ethidium bromide-stained 1.0% agarose gels by comparing each reaction to the drug-free control. Construction of a broad-host-range gyrase A gene probe. pSLS447 contains the wild-type gyrase A gene of E. coli on a 7-kilobase (kb) BamHI fragment inserted in the BamHI site of pBR322 (30). pSLS447 was digested with BamHI and ligated to BglII-digested pLA2917, a 21-kb broad-host-range cosmid vector (1) which was treated with calf intestinal phosphatase. The ligation mixture was transformed into E. coli DH1 with selection for tetracycline resistance. Kanamycin-susceptible (insertion into the BglII site of pLA2917), ampicillin-susceptible (absence of pBR322 sequences) clones were tested for nalidixic acid-sensitizing activity (the wild-type gyrase A gene confers nalidixic acid susceptibility on DH1 gyrA). One of the recombinant plasmids (pNJR3-2, 28 kb) which conferred nalidixic acid susceptibility was analyzed by restriction analysis to confirm the presence of the gyrase A gene. pNJR3-2 was transformed into E. coli S17-1. Analysis of gyrase expression in E. coli. E. coli DH2 (wild-type gyrase A allele) and E. coli DH1 (gyrA mutant) were transformed with pLA2917 (vector control) or pNJR3-2 (gyrase A probe). The MICs of ciprofloxacin, norfloxacin, enoxacin, and ofloxacin were determined for each strain in the presence or absence of each plasmid. The MICs for strains containing plasmid were determined (in duplicate) in the presence of 2 ,ug of tetracycline per ml. Analysis of gyrase expression in P. aeruginosa. pLA2917 and pNJR3-2 were introduced into P. aeruginosa strains by conjugation from E. coli S17-1 (26), which contains chromosomal tra genes that are capable of mobilizing pLA2917 or pNJR3-2. For matings, E. coli S17-1 donor strains containing pLA2917 or pNJR3-2 and P. aeruginosa recipient strains were grown in ML broth overnight at 32°C with aeration. Donor strains were grown in the presence of 5 jig of tetracycline per ml. One-half milliliter of donor culture was added to 0.5 ml of recipient culture in a microcentrifuge tube and centrifuged. The mating mixture was suspended in 50 lil of 0.15 M NaCl and plated onto ML agar. Mating and expression were allowed to occur during a 5-h incubation at 35°C. The mating mixture was harvested in 3 ml of 0.15 M NaCl and plated onto ML agar containing 200 ,ug of tetracycline per ml. For P. aeruginosa MP004 and MP005 recipients, 400 ,ug of tetracycline per ml was used in Pseudomonas isolation agar (Difco). Plasmid-containing E. coli donor strains could not grow in the presence of these tetracycline concentrations, while P. aeruginosa recipient strains that inherited pLA2917 or pNJR3-2 could. Transconjugants were purified on selection media. The MICs of several quinolones were determined for each strain in the presence or absence of each plasmid. The MICs for strains containing plasmid were performed (in duplicate) in the presence of 200 jig of tetracycline per ml. RESULTS DNA gyrase assays. As a preliminary to gyrase A gene expression studies, DNA gyrase enzymes from E. coli and P. aeruginosa were compared by using in vitro supercoiling inhibition assays. The sensitivity of each holoenzyme to

GYRASE A GENE PROBE

VOL. 34, 1990

r Sx

e

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d~~~rexe

supercld

T4 Ligase

I1

FIG. 1. DNA supercoiling of relaxed pBR322 plasmid DNA by reconstituted E. coli and P. aeruginosa DNA gyrase subunits A and B. Lanes: A, no enzyme control; B, P. aeruginosa gyrase A; C, P. aeruginosa gyrase B; D, P. aeruginosa gyrases A and B; E, E. coli gyrase A; F, E. coli gyrase B; G, E. coli gyrases A and B; H, P. aeruginosa gyrase A and E. coli gyrase B; I, E. coli gyrase A and P. aeruginosa gyrase B; J, Micrococcus luteus gyrase control.

ciprofloxacin and nalidixic acid was determined. E. coli DNA gyrase was 50% inhibited by 0.5 ,ug of ciprofloxacin per ml or 50 ,ug of nalidixic acid per ml. P. aeruginosa DNA gyrase was 50% inhibited by 1.0 ,ug of ciprofloxacin per ml or 50 p.g of nalidixic acid per ml. To determine whether the P. aeruginosa gyrase A or B subunit could be complemented by the E. coli gyrase B or A subunit, heterologous mixed subunits were assayed for supercoiling. As shown in Fig. 1 (lanes H and I), the gyrase A or B subunit from P. aeruginosa can supercoil relaxed pBR322 DNA in the presence of the complementing gyrase subunit from E. coli H560. The heterologous gyrase combinations also were assayed for inhibition of supercoiling by ciprofloxacin and nalidixic acid. The A subunit of E. coli complemented by the B subunit of P. aeruginosa was inhibited by 0.5 ,g of ciprofloxacin per ml, while the A subunit of P. aeruginosa complemented by the B subunit of E. coli was inhibited by 1.0 ,ug of ciprofloxacin per ml. The IC50 of nalidixic acid was the same for both subunit combinations (50 ,ug/ml). These results indicate that the DNA gyrase A subunits of E. coli and P. aeruginosa complement the heterologous B subunits and that the quinolone sensitiivities of E. coli and P. aeruginosa DNA gyrases are similar. Analysis of E. coli gyrase A gene expression. In E. coli gyrase A merodiploids, the wild-type gyrase A gene is dominant over quinolone-resistant gyrA alleles (12, 20). This implies that when both the resistant and sensitive gyrase A proteins are present in the cell, some function of gyrase will be quinolone sensitive. The results of the heterologous in vitro gyrase assays demonstrate that the A subunit of E. coli is capable of replacing the A subunit of P. aeruginosa. Furthermore, this hybrid enzyme exhibits a level of quinolone sensitivity similar to that of the P. aeruginosa holoenzyme. Based on these results, one might predict that the E. coli gyrase A subunit is capable of replacing the P. aeruginosa gyrase A subunit in vivo (provided that the gene is expressed) and that this would not significantly alter the quinolone susceptibility of wild-type P. aeruginosa. One might also predict that the wild-type E. coli gyrase A gene would confer quinolone susceptibility on a quinolone-resistant P. aeruginosa gyrase A mutant in vivo, but the quinolone susceptibility of this heterodiploid would only de-

FIG. 2. Construction of pNJR3-2. The gyrA-containing 7-kb BamHI fragment from pSLS447 was cloned into the BglII site of the broad-host-range plasmid pLA2917 (21 kb) to give pNJR3-2 (28 kb). B, S, P, H, and Bg denote cleavage sites recognized by BamHI, Sall, PstI, HindIII, and BglII, respectively. B/Bg indicates the fusion of BamHI sites to BglII sites in pNJR3-2. Tc, Ap, and Km denote genes encoding resistance to tetracycline, ampicillin, and kanamycin, respectively. CIP denotes calf intestinal phosphatase.

crease to the P. aeruginosa wild-type level and not to that of E. coli. To determine whether these predictions were correct, the wild-type gyrase A gene of E. coli was cloned into the broad-host-range plasmid vector pLA2917 for expression studies in P. aeruginosa (Fig. 2; see also Materials and Methods). The recombinant broad-host-range gyrase A plasmid was designated pNJR3-2. The presence of the gyrase A gene in pNJR3-2 was confirmed by demonstrating the ability of pNJR3-2 to confer nalidixic acid susceptibility on the gyrA mutant E. coli DH1 (see below). In addition, the restriction map of pNJR3-2 was consistent with the published nucleotide sequence of the gyrA gene (Fig. 2) (30). The dominant susceptibility trait of the wild-type gyrase A gene also was used to monitor its expression. To confirm gyrase A expression in E. coli, pLA2917 or pNJR3-2 was introduced into the wild-type gyrase A strain DH2 as well as the gyrA mutant DH1. The MICs of selected 4-quinolones for these strains are presented in Table 2. The vector pLA2917 had no significant effect on the quinolone susceptibility of either strain DH2 or the gyrA mutant DH1. pNJR3-2 had no effect on DH2, but conferred quinolone susceptibility on the gyrA mutant DH1, confirming that quinolone susceptibility is dominant in this gyrA merodip-

loid. For analysis in P. aeruginosa, pLA2917 and pNJR3-2 were introduced via conjugal mating into the gyrase A-positive strain PAO2 as well as several well-characterized gyrA mutants of this strain (24). Since these mutants were isolated by selection with ciprofloxacin, they have cfxA designations. The 4-quinolone MICs for these strains are presented in Table 3. The vector pLA2917 had no effect on PAO2 or the

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TABLE 2. Effect of E. coli gyrase A gene on gyrA+ and gyrA E. coli strains MIC (>g/ml) Strain and plasmid

Ciprofloxacin

Norfloxacin

Enoxacin

Ofloxacin

DH2 (no plasmid) DH2(pLA2917)a DH2(pNJR3-2)b

c0.008 c0.008 c0.008

'0.015 '0.015 '0.015

0.03 0.03 0.03

'0.015 '0.015 '0.015

DH1 (no plasmid) DH1(pLA2917) DH1(pNJR3-2)

0.03 0.03 c0.008

0.125 0.25 0.03

0.5 1.0 0.06

0.125 0.125 '0.015

a Vector control plasmid. b Gyrase A gene probe.

gyrA mutants. As predicted, pNJR3-2 also had no effect on PA02. However, this result also would be expected if the E. coli gyrase A gene was not expressed in P. aeruginosa. In each of four P. aeruginosa gyrA mutants, pNJR3-2 conferred wild-type susceptibility to the 4-quinolones that were tested, thus demonstrating expression of the E. coli gyrase A gene in P. aeruginosa. As with E. coli, quinolone susceptibility was dominant in these heterodiploids. That pNJR3-2 induced a level of 4-quinolone susceptibility in these gyrA mutants, which is characteristic of wild-type P. aeruginosa, and had no effect on PA02 demonstrates the usefulness of this probe in detecting gyrA mutations in P. aeruginosa. These results corroborate the results of the in vitro gyrase assays, suggesting that DNA gyrase from P. aeruginosa is similar to that of E. coli with respect to quinolone susceptibility. The ability of pNJR3-2 to discriminate between gyrA and permeability mutations in P. aeruginosa was tested by monitoring the effect of pLA2917 and pNJR3-2 on the quinolone susceptibility of cfxB and cfxA cfxB double mutants of P. aeruginosa. The quinolone susceptibilities of these heterodiploids are presented in Table 4. In the case of the cfxB mutants, pLA2917 had no effect, while pNJR3-2 in some cases had a slight effect, increasing the quinolone susceptibility by twofold. This is in contrast to the eightfold TABLE 3. Expression of the E. coli gyrase A gene in gyrA+ and gyrA P. aeruginosa strains MIC

Strain and plasmid

(jg/ml)

Ciprofloxacin Norfloxacin Enoxacin Ofloxacin

PAO2 (no plasmid) PAO2(pLA2917) PAO2(pNJR3-2)

0.25 0.25 0.25

1.0 1.0 1.0

1.0 2.0 2.0

2.0 2.0 2.0

PA04701 (no plasmid) PA04701(pLA2917) PA04701(pNJR3-2)

2.0

8.0 8.0 1.0

8.0 8.0 2.0

8.0 8.0 1.0

PA04702 (no plasmid) PA04702(pLA2917) PA04702(pNJR3-2)

4.0 4.0 0.25

8.0

16.0 16.0 2.0

16.0 16.0 2.0

PA04703 (no plasmid) PA04703(pLA2917) PA04703(pNJR3-2)

4.0 2.0 0.25

8.0

2.0

16.0 16.0 2.0

16.0 16.0 2.0

PA04704 (no plasmid) PA04704(pLA2917) PA04704(pNJR3-2)

4.0 4.0 0.25

8.0 8.0 2.0

16.0 16.0 2.0

32.0 16.0 2.0

2.0 0.25

8.0 2.0

8.0

TABLE 4. Expression of the E. coli gyrase A gene in cfxB and cfxA cfxB double mutants of P. aeruginosa MIC (pg/ml) Strain and plasmid Ciprofloxacin Norfloxacin Enoxacin Ofloxacin

cfxB mutants PA04741 (no plasmid) PA04741(pLA2917) PA04741(pNJR3-2)

1.0 1.0 0.5

4.0 4.0 2.0

4.0 4.0 4.0

8.0 8.0 4.0

PA04742 (no plasmid) PA04742(pLA2917) PA04742(pNJR3-2)

1.0 1.0 0.5

4.0 4.0 4.0

4.0 8.0 4.0

8.0 8.0 4.0

cfxA cfxB double mutants PA04700 (no plasmid) PA04700(pLA2917) PA04700(pNJR3-2)

16.0 1.0

32.0 16.0 4.0

32.0 32.0 4.0

>32.0 32.0 4.0

PA04739 (no plasmid) PA04739(pLA2917) PA04739(pNJR3-2)

16.0 16.0 1.0

>32.0 32.0 8.0

>32.0 >32.0 8.0

>32.0 >32.0 8.0

PA04740 (no plasmid) PA04740(pLA2917) PA04740(pNJR3-2)

16.0 16.0

32.0 32.0 8.0

>32.0 32.0 8.0

>32.0 >32.0 8.0

8.0

1.0

increase in the quinolone susceptibility of gyrA mutants by pNJR3-2. For cfxA cfxB double mutants, pLA2917 either had no effect or increased the quinolone susceptibility by twofold, while pNJR3-2 increased the quinolone susceptibility of these mutants to that of cfxB single mutants. These results suggest that pNJR3-2 can distinguish between gyrA (cfxA) and cfxB mutants of P. aeruginosa PA02. Probing clinical isolates of P. aeruginosa. P. aeruginosa MPOO1 (quinolone susceptible) and MPO02 and MPOO3, two quinolone-resistant gyrase A, LPS mutants (see Materials and Methods), were probed with pNJR3-2 to determine whether the quinolone resistance that occurred during therapy was due solely to the gyrase A mutation. The quinolone susceptibilities of these heterodiploids are presented in Table 5. pLA2917 had no effect on the pre- and posttherapy strains. pNJR3-2 slightly sensitized the pretherapy strain to quinolones (twofold, except for enoxacin, for which there was no change in susceptibility) and induced intermediate susceptibility in the posttherapy isolates. The susceptibilities of the posttherapy isolates decreased four- to eightfold in the presence of the probe plasmid. However, the MICs for these heterodiploids were still four- to eightfold higher than that for the pretherapy isolate. These results may be indicative of a gyrase A mutation coupled with a non-gyrase A resistance mechanism (possibly related to the LPS alteration observed in strains MP002 and MP003). Two other quinolone-resistant clinical isolates of P. aeruginosa (MP004 and MP005), which were obtained from different sources, were also probed. The results, which are included in Table 5, indicate that pNJR3-2 conferred susceptibility on MP004 but not MPOO5, indicating that MP004 was a gyrA mutant. MP005 most likely possessed gyrase A-independent resistance, although dominant gyrase A-mediated quinolone resistance, gyrase B-mediated quinolone resistance, or poor expression of the E. coli gyrA gene in this strain cannot be ruled out. Nor can these possibilities be ruled out for the additional quinolone resistance expressed by MP002 and MPOO3.

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TABLE 5. Probing quinolone-resistant P. aeruginosa clinical strains MIC (Lg/ml)

Strain and plasmid

Ciprofloxacin Norfloxacin Enoxacin Ofloxacin

Pretherapy strains MP001 (no plasmid)

MP001(pLA2917) MP001(pNJR3-2)

0.5 0.5 0.25

2.0 2.0 1.0

2.0 2.0 2.0

4.0 4.0 2.0

Posttherapy strains MP002 (no plasmid) MP002(pLA2917) MP002(pNJR3-2)

>16.0 >16.0 4.0

>32.0 >32.0 8.0

>32.0 >32.0 16.0

>32.0 >32.0 16.0

MP003 (no plasmid) MP003(pLA2917) MP003(pNJR3-2)

16.0 16.0 2.0

32.0 >32.0 8.0

>32.0 >32.0 16.0

>32.0 >32.0 16.0

16.0 8.0 0.5

32.0 16.0 4.0

>32.0 32.0 4.0

>32.0 32.0 4.0

8.0 4.0 4.0

32.0 16.0 16.0

16.0

16.0

32.0 16.0 16.0

Other clinical strains MP004 (no plasmid)

MP004(pLA2917) MP004(pNJR3-2) MP005 (no plasmid) MP005(pLA2917) MP005(pNJR3-2)

16.0

DISCUSSION DNA gyrase A-mediated quinolone resistance can be detected by four methods. (i) The most definitive approach is to clone and sequence the gyrase A gene (7, 31). (ii) Another method involves the isolation of DNA gyrase coupled with an in vitro assay for either quinolone-mediated inhibition of DNA supercoiling or quinolone-induced DNA doublestranded cleavage (15, 24, 25, 29). Both of these methods, while providing definitive results, are laborious and timeconsuming. (iii) Fisher et al. (7a) have recently developed a restriction fragment length polymorphism method to detect the loss of a Hinfl restriction endonuclease cleavage site at nucleotide 244 in the E. coli gyrase A gene. This polymorphism is caused by a high-level quinolone-resistance mutation at nucleotide 248. Southern blot analysis of quinoloneresistant clinical strains of E. coli showed that eight of nine strains tested lacked the Hinfl site, thereby demonstrating the usefulness of this method for the analysis of clinical strains of E. coli. (iv) gyrA mutations in clinical strains of E. coli and P. aeruginosa also have been detected by using the dominant susceptibility trait of the wild-type gyrase A gene (20, 32). In each case a recombinant plasmid harboring the wild-type E. coli gyrA allele was introduced via transformation into quinolone-resistant clinical strains. Several gyrA mutants were identified by this method, although in some cases the strains could not be transformed. Results of this investigation confirm the usefulness of the E. coli wild-type gyrA allele in probing quinolone resistance in P. aeruginosa. In this study a mobilizable vector encoding tetracycline resistance was used. The results of heterologous supercoiling assays showed that the reason this technique works is that the E. coli and P. aeruginosa gyrase A and B subunits are able to interact functionally and that the A subunits from these organisms have similar quinolone susceptibilities. Analyses of P. aeruginosa cfxA, cfxB, and cfxA cfxB heterodiploids harboring pNJR3-2 demonstrated expression of the E. coli gyrase A gene in P. aeruginosa and specificity of this probe for gyrA mutations.

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Initial testing of this probe in the clinical setting was conducted on five P. aeruginosa clinical isolates. Strain MP004 was identified as a gyrA mutant, while strain MP005 appeared to possess a gyrA-independent mechanism of quinolone resistance. These strains were not characterized further. The other isolates were recovered from the same patient prior to (strain MP001) and following (strains MP002 and MP003) the initiation of intravenous ciprofloxacin therapy. All three isolates were of the same strain. Strains MP002 and MP003 were shown previously to possess quinolone-resistant DNA gyrase A subunits and LPS alterations (17). The intermediate susceptibility conferred by pNJR3-2 on the posttherapy isolates MP002 and MP003 is indicative of gyrase A-mediated quinolone resistance coupled with a non-gyrase A resistance mechanism. These results are similar to those obtained by probing P. aeruginosa PAO2 cfxA cfxB double mutants with pNJR3-2. While a gyrase A (cfxA) mutation is harbored by MP002 and MPOO3, these mutants are phenotypically distinct from cfxB mutants which possess a novel outer membrane protein, exhibit pleiotropic drug resistance, and accumulate less ciprofloxacin in the presence of carbonyl cyanide m-chlorophenylhydrazone (3, 17). However, the loss of long-chain LPS moieties from MP002 and MP003 correlates with resistance since the pretherapy isolate, MPOO1, contained intact LPS. Although ciprofloxacin accumulation studies produced ambiguous results (17), the inherent limitations of the technique used did not preclude a role for the LPS loss in reduced permeability or altered drug binding in these mutants. LPS alterations have been reported for other quinolone-resistant mutants of P. aeruginosa which exhibited minor decreases in drug accumulation (4, 16). A decrease in the length of LPS side chains in P. aeruginosa also has been associated with decreased P-lactam permeability (10, 11) or, alternatively, with hypersusceptibility to P-lactams, gentamicin, and hydrophobic agents

(2).

Other possible explanations for the partial dominance exhibited by MP002 and MPO03 gyrase A heterodiploids (and the resistance expressed by MP005) include primary or secondary gyrase A mutations which confer some degree of dominant quinolone resistance, gyrase B-mediated quinolone resistance, or poor expression of the E. coli gyrA gene in these ogranisms. These possibilities have not been ruled out.

The pNJR3-2 gyrase A probe has recently been used successfully in the analysis of quinolone-resistant isolates of Serratia marcescens (unpublished data) and should also prove useful in the analysis of several other gram-negative organisms. Assuming that the E. coli gyrase A subunit interacts functionally with the gyrase B subunit of gramnegative bacteria other than P. aeruginosa and E. coli and that the gyrase A gene is expressed in these organisms, the usefulness of pNJR3-2 will be limited only by the host range of pLA2917. Since pLA2917 contains the vegetative replication origin of the broad-host-range incompatibility group P plasmid RK2 (1, 6), pNJR3-2 can be introduced into a wide range of gram-negative organisms. In addition to possessing the RK2 broad-host-range replicon, pNJR3-2 has the ability to be mobilized conjugally. Thus, pNJR3-2 can be used with bacterial strains which transform poorly. ACKNOWLEDGMENTS I thank Debra Tonetti and Robert Miller for helpful discussions on DNA gyrase genetics and Barbara Thurberg for determining the MICs presented in this paper. I also thank Barbara Masecar, Janet Herrington, and Denise Rasimas for excellent technical support.

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