Vol. 35, No. 3
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1991, p. 519-523
0066-4804/91/030519-05$02.00/0 Copyright © 1991, American Society for Microbiology
Mechanisms of Quinolone Resistance in a Clinical Isolate of Escherichia coli Highly Resistant to Fluoroquinolones but Susceptible to Nalidixic Acid N. MONIOT-VILLE,1 J. GUIBERT,2 N. MOREAU,3 J. F. ACAR,"2 E. COLLATZ,1 AND L. GUTMANN`.4* Laboratoire de Microbiologie Medicale, Universite Paris VI, 75270 Paris Cedex 06,1 Laboratoire de Microbiologie, H6pital Saint-Joseph, 75674 Paris Cedex 14,2 Centre National de la Recherche Scientifique, Centre d' Etudes et de
Recherches de Chimie Organique Appliquee, 94320 Thiais,3 and Laboratoire de Microbiologie, H6pital Broussais, 75014 Paris,4 France Received 17 July 1990/Accepted 5 January 1991
Two associated resistance mechanisms were found in a nalidixic acid-susceptible (4 ,ug/ml) but fluoroquinolone-resistant (8 to 16 ,ug/ml) strain of Escherichia coli Q2 selected under norfloxacin therapy. As compared with the susceptible E. coli Ql isolated before treatment, changes in outer membrane proteins and lipopolysaccharides in Q2 were associated with a 1.5- to 3-fold decrease in the uptake of fluoroquinolones but not nalidixic acid. A 50% inhibition of DNA synthesis in toluene-permeabilized cells of the resistant strain E. coli Q2 required up to 500-fold increased quantities of fluoroquinolones, whereas such inhibition was obtained in both E. coli Ql and Q2 with similar amounts of nalidixic acid. Selection from E. coli Ql on norfloxacin of one-step resistant mutants resembling E. coli Q2 was unsuccessful. From these results we infer that a decrease in outer membrane permeability, associated with a peculiar alteration of the DNA gyrase, was responsible for the unusual quinolone resistance phenotype of E. coli Q2.
Quinolones are synthetic antibiotics which can be grouped into the older compounds nalidixic acid, pipemidic acid, piromidic acid, and flumequine and the newer, fluorinated quinolones such as norfloxacin pefloxacin, ofloxacin, and ciprofloxacin. The latter have comparatively enlarged spectra of activity as well as better intrinsic activities. Many studies have been carried out with quinolone-resistant gramnegative bacilli. Two main mechanisms of resistance which may occur either alone or combined have been found (15): (i) modification of the DNA gyrase in either subunit A (8, 10, 15, 20) or B (16, 21), which can give rise to moderate or high levels of resistance, and (ii) decreased uptake of quinolones, which correlates with a decrease in the quantity of porins (5, 10, 13-15). Another not totally elucidated mechanism involving the marA gene may be responsible for low levels of resistance (7). It was the purpose of this study to describe the mechanism(s) which could explain the particular profiles of resistance to fluroroquinolones and susceptibility to nalidixic acid observed in a clinical isolate of Escherichia coli.
Hinton agar by using a Steers-type replicator device with ca. 104 CFU per spot and were read after 18 h of incubation at 37°C. MICs of novobiocin were determined as the quantities of antibiotic necessary to totally inhibit the growth of 100 CFU per plate after 18 h of incubation at 37°C (21). Selection of resistant mutants. Mutants resistant to norfloxacin were selected in vitro on plates containing a gradient of the antibiotic from 0 to 8 pug/ml. Extraction of OM proteins and LPS. Cell membranes were prepared from 200 ml of exponential-phase cultures at an optical density at 650 nm of 0.4 as previously described (19). Outer membrane (OM) proteins were obtained as follows: 100 jig of total-membrane protein was incubated for 45 min at 25°C in 70 RI of sodium phosphate buffer (50 mM, pH 7.0) containing 0.3% of N-laurylsarcosine N197 (ICN Biochemicals, Cleveland, Ohio) and centrifuged at 40,000 x g for 30 min at 15°C. The pellet containing the OM was resuspended in 60 RI of sample buffer and applied onto a sodium dodecyl sulfate (SDS)-containing 12% polyacrylamide gel as previously described (17). Lipopolysaccharide (LPS) was extracted from intact cells as previously described (12) and analyzed on a 15% polyacrylamide gel. Quinolone uptake and efflux. The uptake of quinolones was assayed by a modified method of Chapman and Georgopapadakou (4): mid-log-phase cells grown in 200 ml of antibiotic medium 3 (Difco) were harvested, washed once in sodium phosphate buffer (50 mM, pH 7), and resuspended in the same buffer at an optical density at 650 nm of 1. Samples (15 ml) were distributed into 50-ml Erlenmeyer flasks and incubated in a shaking water bath at 37°C. After 10 min, 10 ,ug of norfloxacin per ml, 20 jig of ciprofloxacin per ml, or 50 ,ug of pefloxacin, enoxacin, flumequine, or nalidixic acid per ml was added. Samples (1 ml) were taken at different time points, centrifuged, washed in 1 ml of phosphate buffer, suspended in 1 ml of glycine-HCl (0.1 M, pH 3), and left at 20°C for 75 min. The antibiotic in the supernatant was quantified by spectrofluorometry. For each time point, the
MATERIALS AND METHODS Bacterial strains and growth media. E. coli Ql, susceptible to all quinolones, was isolated from a lower urinary tract infection in a paraplegic patient. During treatment of this infection with norfloxacin, a pure culture of a quinoloneresistant strain, E. coli Q2, was selected after 7 days. Its resistance phenotype was stable in vitro. No reversion to quinolone susceptibility was observed after repeated regrowth in antibiotic-free medium. Strains were generally grown in Mueller-Hinton broth or on agar (Diagnostics Pasteur). Antibiotic medium 3 (Difco) or Luria-Bertani medium were also used in different assays. Susceptibility testing. MICs were determined on Mueller*
Corresponding author. 519
520
ANTIMICROB. AGENTS CHEMOTHER.
MONIOT-VILLE ET AL. TABLE 1. Susceptibility of E. coli Ql and E. coli Q2 to different quinolones MIC
Antibiotic
Nalidixic acid Flumequine Enoxacin Oxolinic acid Piromidic acid Norfloxacin Pefloxacin Pipemidic acid Ofloxacin Cinoxacin Rosoxacin Ciprofloxacin
(pug/ml) for:
E. coli Ql
2 0.5 0.5 0.25 16 0.12 0.12 2 0.12 4 0.5 0.015
Increase
E. coli Q2
4 2 8 16
>1,024 16 16 256 32 >1,024 >256 16
(fold)
2 4 16 64 >64 128 128 128 256 >256 >512
21.5
M~a 1 123
215-;t
FIG. 1. SDS-polyacrylamide gel electrophoresis of OM proteins (a) and LPS (b). (a) Lanes: 1, Mr standards (in thousands); 2, E. coli Q2; 3, E. coli Ql. (b) Lanes: 1, E. coli Q1; 2, E. coli Q2.
1,024
value obtained immediately after addition of the antibiotic substracted. The wavelengths of excitation and emission for the different compounds were, respectively, 246 and 354 nm for nalidixic acid, 245 and 365 nm for flumequine, 346 and 404 nm for enoxacin, 277 and 442 nm for pefloxacin, and 328 and 448 nm for ciprofloxacin. In each case, the quantity of antibiotic present in the supernatant was deduced from a reference curve. Inhibition of the active efflux (6) was measured during the uptake of norfloxacin, after the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (0.1 mM; Sigma), at the 15-min time point. Inhibition of DNA synthesis in toluene-permeabilized cells. DNA replication was assayed essentially as described by Badet et al. (2). Briefly, cells grown to mid-log phase in 10 ml of Luria-Bertani medium were suspended in 2 ml of a mixture containing 100 mM sodium phosphate (pH 7.4), 2 mM MgCl2, and 1% toluene and strongly vortexed for 2 min at room temperature. Ca. 108 toluene-treated cells were incubated in 175 ,ul of a mixture containing 35 mM Tris-HCl (pH 7.8); 2.8 mM MgCl2; 110 mM KCl; 0.7 mM 3-mercaptoethanol; 1.1 mM ATP; 22 F.M (each) dATP, dTTP, dCTP, and dGTP; 3H-thymidine (4.77 TBq/mmol); and different concentrations of antibiotics. After 60 min at 30°C, the reaction was stopped by addition of 2 ml of trichloroacetic acid (5%). After filtration of the cells through Whatman GF/C filters, three washes with 5 ml of ice-cold trichloroacetic acid, and one wash with 5 ml of ethanol, radioactivity was counted. was
RESULTS MICs of quinolones. E. coli Q2 was selected under norfloxacin treatment of a lower urinary tract infection caused by E. coli Ql. Neither E. coli Qi nor Q2 was phage typable. However, since both strains had the same tetracycline, chloramphenicol, and ampicillin resistance patterns and the same plasmid content (data not shown), and since the OM protein patterns (see below) appeared qualitatively identical, we considered strain Q2 as a possible derivative from Ql. The MICs of quinolones for E. coli Ql and Q2 are shown in Table 1. There was almost no increase in the MIC of nalidixic acid (2-fold) or flumequine (4-fold), while there were large increases (16- to 1,024-fold) in the MICs of the other quinolones tested, including several fluoroquinolones. The largest relative increases were observed for rosoxacin
and ciprofloxacin (Table 1). No difference in the susceptibility to novobiocin was found (data not shown). Selection of resistant strains. About 500 resistant clones, selected from E. coli Ql in the presence of different concentrations of norfloxacin, were tested to find a resistance pattern similar to that of E. coli Q2. Mutants with cross resistance to all quinolones and low-level resistance to norfloxacin (0.5 to 2 ,ug/ml) were selected at a frequency of ca. 10-', but none had the particular quinolone resistance phenotype of E. coli Q2. OM protein and LPS. Comparison of the OM protein patterns showed the disappearance of a major OM protein of ca. 40 kDa (OmpF), associated with an apparent compensatory increase of a ca. 38-kDa protein (OmpC) in E. coli Q2 (Fig. la). Analysis of the LPS patterns (Fig. lb) showed that in E. coli Q2, in contrast to Qi, only the core of the LPS was present. On the basis of these results, we supposed that decreased OM permeability could explain part of the resistance and therefore assayed the uptake of different quinolones. Uptake and inhibition of efflux. Quinolone uptake was measured by the fluorimetric assay previously described (4). For all of the quinolones tested, a rapid uptake was observed within 5 min, as noted by other workers (1, 9-11, 14). With the exception of nalidixic acid and with respect to strain Qi, all quinolones were taken up less rapidly initially by strain Q2, and total uptake was reduced 1.5- to 3-fold. Different concentrations of the individual antibiotics were used for this assay (Fig. 2). Concentrations of up to 50 jig of nalidixic acid, flumequine, pefloxacin, and enoxacin per ml were necessary to obtain an intracellular accumulation sufficient to be measured by the fluorimetric assay. Despite these high concentrations, we believe that the results are reliable, since the accumulation of quinolones is a nonsaturable process depending upon the external concentration of antibiotics (3, 4, 6). When 50 Fg of norfloxacin per ml was used instead of 10 ,ug/ml, similar (proportionally) results were obtained (data not shown). The inhibition of norfloxacin efflux by CCCP, added after 15 min, was also assayed. The effect of CCCP was more pronounced on the resistant strain, with a roughly threefold increase in uptake (Fig. 3). Similar observations were previously made with other isogenic susceptible and resistant strains (6, 14) and suggest that there is no impaired efflux in E. coli Q2. Inhibition of DNA synthesis. In order to avoid the permeability barrier, we used toluene-treated cells to examine the effect of the six quinolones studied in the uptake assay on DNA synthesis (Fig. 4). A clear correlation existed between the MIC ratios (Table 1) and the ratios of the quinolone
VOL. 35, 1991
NOVEL CROSS RESISTANCE TO QUINOLONES IN E. COLI
E L ,,
i'
114 i{ I -7'---7
c
-7-I-
1 10
5
606
- .~-
40
f20
1%
b 510 20 30
521
/d 5 10 20 30
--
f 510 20 30
Time (min) FIG. 2. Quinolone uptake in E. coli Ql ( ) and Q2 (---) at external concentrations of 10 ,ug of norfloxacin (d) per ml, 20 ,ug of ciprofloxacin (e) per ml, and 50 p.g of pefloxacin (c), enoxacin (f), flumequine (b), and nalidixic acid (a) per ml.
amounts necessary to inhibit DNA synthesis in E. coli Ql and Q2 (Fig. 4). While no difference was found between the concentrations of nalidixic acid needed to inhibit DNA synthesis in E. coli Ql and Q2, over 500 times more ciprofloxacin was required to inhibit DNA synthesis in E. coli Q2 to the same degree as in Ql. A good correlation between the concentrations inhibiting 50% of DNA synthesis and the MICs of the different compounds for E. coli Qi and Q2 was observed (Fig. 5).
DISCUSSION The quinolone resistance pattern of E. coli Q2 is peculiar. This strain is resistant to almost all quinolones, including the fluoroquinolones, but very susceptible to nalidixic acid and flumequine, with the MICs of the latter compounds increased by factors of only 2 and 4, respectively, compared with MICs in the susceptible strain Ql.
0
25_
-
U
CCCP
20_
E
0
L
15_
.
0
10-
0 co
//
o
0
/ 5
10 15 20 25 Time (min)
FIG. 3. Effect of CCCP (0.1 mM) on the uptake of norfloxacin (10 ,ug/ml) by E. coli Ql (0) and Q2 (0).
We have demonstrated that at least two mechanisms of resistance were operative in E. coli Q2: a decrease in OM permeability and a probable alteration of the DNA gyrase. The decrease in uptake of most of the quinolones into strain Q2 is compatible with the decrease in amount of the OmpF porin (1, 5, 9, 10, 13-15). However, there was no apparent decrease in the uptake of nalidixic acid. Since this quinolone is among the most hydrophobic (9), the absence of decreased uptake might be explained by the simultaneous LPS modification which could conceivably allow a better permeation via the hydrophobic route (non-porin pathway). Mutants of E. coli KEA16 with similar characteristics (OmpF-, modification of LPS) have been described by Hirai et al. (10). Under the same circumstances it is, however, not clear why there was some decrease in uptake of flumequine, a compound even more hydrophobic than nalidixic acid. We have observed an active efflux of norfloxacin in E. coli Q2, as demonstrated by its increased uptake in the presence of CCCP. We have, however, no information suggesting an increased efflux of flumequine relative to that of nalidixic acid, which could explain the relatively lower uptake of flumequine into strain Q2. The second mechanism of resistance is the decrease in the inhibition of DNA synthesis, which appears most likely to be due to a modification of the DNA gyrase. A priori, the unaltered susceptibility to novobiocin would argue against a modification of the B subunit. Curiously, nalidixic acid and flumequine, which showed the lowest increase in MICs as well as in the concentrations necessary to inhibit DNA synthesis in toluene-permeabilized cells, were the only compounds which have no cyclic subtituent at the C-7 position or a fused ring structure linking the C-6 and C-7 atoms (18, 20). To our knowledge, there has been no previous report on clinical isolates which present a high-level resistance to the newer quinolones while remaining susceptible to nalidixic acid. With respect to the association of two mechanisms of resistance, E. coli Q2 would resemble a clinical isolate of E. coli previously described (1) as well as in vitro-selected mutant KF111 (15). However, nothing is known about the
522
ANTIMICROB. AGENTS CHEMOTHER.
MONIOT-VILLE ET AL.
100-
0
0 4o._
0N
50-
CL o
u
*
'-02>~00_o
a
d
100-
r_
1._
GL)a 50-
C._
b
0
0
E 100-
-c-
I4-L, 50-
_ l ~ l___l_ ~ ~ ~ ~ ~ _l_ _l_ _l _l
\_ _o
0.01 0.1
1
s
\-°
10 1001000 0.01 0.1
°-
1
_
__l
__l
__
f
10 1001000
antibiotic concentration (pg/mi) FIG. 4. Synthesis of DNA in toluene-permeabilized E. coli Ql (0) and Q2 (0) cells in the presence of quinolones; 100% corresponded to ca. 3,000 to 4,500 cpm. (a) Nalidixic acid; (b) flumequine; (c) enoxacin; (d) norfloxacin; (e) pefloxacin; (f) ciprofloxacin.
LPS of these two strains, neither of which remained susceptible to nalidixic acid and flumequine. We suppose that the modification of the DNA gyrase is the major mechanism responsible for the high level of resistance, but we do not know yet whether one or several
1000_
ACKNOWLEDGMENTS We are grateful to A. Jaffd for helpful discussions. We thank C. Harcour for excellent secretarial assistance. This work was supported by a grant (CJF-89-01) from the Institut National de la Sante et de la Recherche Medicale.
PEF
NOR
100_
NALE NALO
10-
/
FLU
0) FLU 0-
1~
0
n..oI
otPEE
0.1 _
E/INOR
/-C(IP A) A1 i
0.01
0.1
1
10
mutations of the gyrase are required to yield this particular phenotype nor whether one or both gyrase subunits are affected. An argument for the alteration of the subunit A of the gyrase was obtained from a preliminary experiment in which the introduction of a plasmid harboring the wild-type gyrA gene into E. coli Q2 decreased the MICs of the fluoroquinolones 16- to 64-fold, which remained, however, 4- to 8-fold higher than the MICs observed for E. coli Ql. This difference in MICs could conceivably be explained by the permeability defect of the Q2 transformants.
100
MIC (pg/mi) FIG. 5. Correlation between MICs (Table 1) and concentrations inhibiting 50% of DNA synthesis (estimated from Fig. 4). El, E. coli Q1; *, E. coli Q2; CIP, ciprofloxacin; NOR, norfloxacin; PEF, pefloxacin; FLU, flumequine; NAL, nalidixic acid. Enoxacin was not included since 50% inhibition was not achieved for the resistant strain.
REFERENCES 1. Aoyama, H., K. Sato, T. Kato, K. Hirai, and S. Mitsuhashi. 1987. Norfloxacin resistance in a clinical isolate of Escherichia coli. Antimicrob. Agents Chemother. 31:1640-1641. 2. Badet, B., P. Hugues, M. Kohiyama, and P. Forterre. 1982. Inhibition of DNA replication in vitro by pefloxacin. FEBS Lett. 145:355-359. 3. Bedard, J., S. Wong, and L. E. Bryan. 1987. Accumulation of enoxacin by Escherichia coli and Bacillus subtilis. Antimicrob. Agents Chemother. 31:1348-1354. 4. Chapman, J. S., and N. H. Georgopapadakou. 1989. Fluorimetric assay for fleroxacin uptake by bacterial cells. Antimicrob. Agents Chemother. 33:27-29. 5. Chow, R. T., T. J. Dougherty, H. S. Fraimow, E. Y. Bellin, and M. H. Miller. 1988. Association between early inhibition of DNA synthesis and the MICs and MBCs of carboxyquinolone antimicrobial agents for wild-type and mutant [gyrA nfxB (ompF) acrA] Escherichia coli K-12. Antimicrob. Agents Chemother. 32:1113-1118. 6. Cohen, S. P., D. C. Hooper, J. S. Wolfson, K. S. Souza, L. M. McMurry, and S. B. Levy. 1988. Endogenous active efflux of norfloxacin in susceptible Escherichia coli. Antimicrob. Agents Chemother. 32:1187-1191. 7. Cohen, S. P., L. M. McMurry, D. C. Hooper, J. S. Wolfson, and S. B. Levy. 1989. Cross-resistance to fluoroquinolones in multiple-antibiotic-resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: decreased drug accumulation asso-
VOL. 35, 1991
8. 9.
10.
11.
12. 13. 14.
NOVEL CROSS RESISTANCE TO QUINOLONES IN E. COLI
ciated with membrane changes in addition to OmpF reduction. Antimicrob. Agents Chemother. 33:1318-1325. Hane, M. W., and T. H. Wood. 1969. Escherichia coli K-12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J. Bacteriol. 99:238-241. Hirai, K., H. Aoyama, T. Irikura, S. lyobe, and S. Mitsuhashi. 1986. Differences in susceptibility to quinolones of outer membane mutants of Salmonella typhimurium and Escherichia coli. Antimicrob. Agents Chemother. 29:535-538. Hirai, K., H. Aoyama, S. Suzue, T. Irikura, S. lyobe, and S. Mitsuhashi. 1986. Isolation and characterization of norfloxacinresistant mutants of Escherichia coli K-12. Antimicrob. Agents Chemother. 30:248-253. Hirai, K., S. Suzue, T. Irikura, S. lyobe, and S. Mitsuhashi. 1987. Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 31:582586. Hitchcock, P., and T. M. Brown. 1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154:269-277. Hooper, D. C., J. S. Wolfson, E. Y. Ng, and M. N. Swartz. 1987. Mechanisms of action of and resistance to ciprofloxacin. Am. J. Med. 82(Suppl. 4A):12-20. Hooper, D. C., J. S. Wolfson, K. S. Souza, E. Y. Ng, G. L. McHugh, and M. N. Swartz. 1989. Mechanism of quinolone
15.
16.
17. 18. 19.
20. 21.
523
resistance in Escherichia coli: characterization of nfxB and cfxB, two mutant resistance loci decreasing norfloxacin accumulation. Antimicrob. Agents Chemother. 33:283-290. Hooper, D. C., J. S. Wolfson, K. S. Souza, C. Tung, G. L. McHugh, and M. N. Swartz. 1986. Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli. Antimicrob. Agents Chemother. 29:639-644. Inoue, S., T. Ohue, J. Yamagishi, S. Nakamura, and M. Shimizu. 1978. Mode of incomplete cross-resistance among pipemidic, piromidic, and nalidixic acids. Antimicrob. Agents Chemother. 14:240-245. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 22:680-685. Smith, J. T. 1984. Awakening the slumbering potential of the 4-quinolone antibacterials. Pharmaceut. J. 233:299-305. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Escherichia coli K12. Eur. J. Biochem. 72:341-352. Wolfson, J. S., and D. C. Hooper. 1989. Fluoroquinolone antimicrobial agents. Clin. Microbiol. Rev. 2:378-424. Yamagishi, J.-I., Y. Furutani, S. Inoue, T. Ohue, S. Nakamura, and M. Shimizu. 1981. New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity. J. Bacteriol. 148:450-458.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1991, p. 519-523
0066-4804/91/030519-05$02.00/0 Copyright © 1991, American Society for Microbiology
Mechanisms of Quinolone Resistance in a Clinical Isolate of Escherichia coli Highly Resistant to Fluoroquinolones but Susceptible to Nalidixic Acid N. MONIOT-VILLE,1 J. GUIBERT,2 N. MOREAU,3 J. F. ACAR,"2 E. COLLATZ,1 AND L. GUTMANN`.4* Laboratoire de Microbiologie Medicale, Universite Paris VI, 75270 Paris Cedex 06,1 Laboratoire de Microbiologie, H6pital Saint-Joseph, 75674 Paris Cedex 14,2 Centre National de la Recherche Scientifique, Centre d' Etudes et de
Recherches de Chimie Organique Appliquee, 94320 Thiais,3 and Laboratoire de Microbiologie, H6pital Broussais, 75014 Paris,4 France Received 17 July 1990/Accepted 5 January 1991
Two associated resistance mechanisms were found in a nalidixic acid-susceptible (4 ,ug/ml) but fluoroquinolone-resistant (8 to 16 ,ug/ml) strain of Escherichia coli Q2 selected under norfloxacin therapy. As compared with the susceptible E. coli Ql isolated before treatment, changes in outer membrane proteins and lipopolysaccharides in Q2 were associated with a 1.5- to 3-fold decrease in the uptake of fluoroquinolones but not nalidixic acid. A 50% inhibition of DNA synthesis in toluene-permeabilized cells of the resistant strain E. coli Q2 required up to 500-fold increased quantities of fluoroquinolones, whereas such inhibition was obtained in both E. coli Ql and Q2 with similar amounts of nalidixic acid. Selection from E. coli Ql on norfloxacin of one-step resistant mutants resembling E. coli Q2 was unsuccessful. From these results we infer that a decrease in outer membrane permeability, associated with a peculiar alteration of the DNA gyrase, was responsible for the unusual quinolone resistance phenotype of E. coli Q2.
Quinolones are synthetic antibiotics which can be grouped into the older compounds nalidixic acid, pipemidic acid, piromidic acid, and flumequine and the newer, fluorinated quinolones such as norfloxacin pefloxacin, ofloxacin, and ciprofloxacin. The latter have comparatively enlarged spectra of activity as well as better intrinsic activities. Many studies have been carried out with quinolone-resistant gramnegative bacilli. Two main mechanisms of resistance which may occur either alone or combined have been found (15): (i) modification of the DNA gyrase in either subunit A (8, 10, 15, 20) or B (16, 21), which can give rise to moderate or high levels of resistance, and (ii) decreased uptake of quinolones, which correlates with a decrease in the quantity of porins (5, 10, 13-15). Another not totally elucidated mechanism involving the marA gene may be responsible for low levels of resistance (7). It was the purpose of this study to describe the mechanism(s) which could explain the particular profiles of resistance to fluroroquinolones and susceptibility to nalidixic acid observed in a clinical isolate of Escherichia coli.
Hinton agar by using a Steers-type replicator device with ca. 104 CFU per spot and were read after 18 h of incubation at 37°C. MICs of novobiocin were determined as the quantities of antibiotic necessary to totally inhibit the growth of 100 CFU per plate after 18 h of incubation at 37°C (21). Selection of resistant mutants. Mutants resistant to norfloxacin were selected in vitro on plates containing a gradient of the antibiotic from 0 to 8 pug/ml. Extraction of OM proteins and LPS. Cell membranes were prepared from 200 ml of exponential-phase cultures at an optical density at 650 nm of 0.4 as previously described (19). Outer membrane (OM) proteins were obtained as follows: 100 jig of total-membrane protein was incubated for 45 min at 25°C in 70 RI of sodium phosphate buffer (50 mM, pH 7.0) containing 0.3% of N-laurylsarcosine N197 (ICN Biochemicals, Cleveland, Ohio) and centrifuged at 40,000 x g for 30 min at 15°C. The pellet containing the OM was resuspended in 60 RI of sample buffer and applied onto a sodium dodecyl sulfate (SDS)-containing 12% polyacrylamide gel as previously described (17). Lipopolysaccharide (LPS) was extracted from intact cells as previously described (12) and analyzed on a 15% polyacrylamide gel. Quinolone uptake and efflux. The uptake of quinolones was assayed by a modified method of Chapman and Georgopapadakou (4): mid-log-phase cells grown in 200 ml of antibiotic medium 3 (Difco) were harvested, washed once in sodium phosphate buffer (50 mM, pH 7), and resuspended in the same buffer at an optical density at 650 nm of 1. Samples (15 ml) were distributed into 50-ml Erlenmeyer flasks and incubated in a shaking water bath at 37°C. After 10 min, 10 ,ug of norfloxacin per ml, 20 jig of ciprofloxacin per ml, or 50 ,ug of pefloxacin, enoxacin, flumequine, or nalidixic acid per ml was added. Samples (1 ml) were taken at different time points, centrifuged, washed in 1 ml of phosphate buffer, suspended in 1 ml of glycine-HCl (0.1 M, pH 3), and left at 20°C for 75 min. The antibiotic in the supernatant was quantified by spectrofluorometry. For each time point, the
MATERIALS AND METHODS Bacterial strains and growth media. E. coli Ql, susceptible to all quinolones, was isolated from a lower urinary tract infection in a paraplegic patient. During treatment of this infection with norfloxacin, a pure culture of a quinoloneresistant strain, E. coli Q2, was selected after 7 days. Its resistance phenotype was stable in vitro. No reversion to quinolone susceptibility was observed after repeated regrowth in antibiotic-free medium. Strains were generally grown in Mueller-Hinton broth or on agar (Diagnostics Pasteur). Antibiotic medium 3 (Difco) or Luria-Bertani medium were also used in different assays. Susceptibility testing. MICs were determined on Mueller*
Corresponding author. 519
520
ANTIMICROB. AGENTS CHEMOTHER.
MONIOT-VILLE ET AL. TABLE 1. Susceptibility of E. coli Ql and E. coli Q2 to different quinolones MIC
Antibiotic
Nalidixic acid Flumequine Enoxacin Oxolinic acid Piromidic acid Norfloxacin Pefloxacin Pipemidic acid Ofloxacin Cinoxacin Rosoxacin Ciprofloxacin
(pug/ml) for:
E. coli Ql
2 0.5 0.5 0.25 16 0.12 0.12 2 0.12 4 0.5 0.015
Increase
E. coli Q2
4 2 8 16
>1,024 16 16 256 32 >1,024 >256 16
(fold)
2 4 16 64 >64 128 128 128 256 >256 >512
21.5
M~a 1 123
215-;t
FIG. 1. SDS-polyacrylamide gel electrophoresis of OM proteins (a) and LPS (b). (a) Lanes: 1, Mr standards (in thousands); 2, E. coli Q2; 3, E. coli Ql. (b) Lanes: 1, E. coli Q1; 2, E. coli Q2.
1,024
value obtained immediately after addition of the antibiotic substracted. The wavelengths of excitation and emission for the different compounds were, respectively, 246 and 354 nm for nalidixic acid, 245 and 365 nm for flumequine, 346 and 404 nm for enoxacin, 277 and 442 nm for pefloxacin, and 328 and 448 nm for ciprofloxacin. In each case, the quantity of antibiotic present in the supernatant was deduced from a reference curve. Inhibition of the active efflux (6) was measured during the uptake of norfloxacin, after the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (0.1 mM; Sigma), at the 15-min time point. Inhibition of DNA synthesis in toluene-permeabilized cells. DNA replication was assayed essentially as described by Badet et al. (2). Briefly, cells grown to mid-log phase in 10 ml of Luria-Bertani medium were suspended in 2 ml of a mixture containing 100 mM sodium phosphate (pH 7.4), 2 mM MgCl2, and 1% toluene and strongly vortexed for 2 min at room temperature. Ca. 108 toluene-treated cells were incubated in 175 ,ul of a mixture containing 35 mM Tris-HCl (pH 7.8); 2.8 mM MgCl2; 110 mM KCl; 0.7 mM 3-mercaptoethanol; 1.1 mM ATP; 22 F.M (each) dATP, dTTP, dCTP, and dGTP; 3H-thymidine (4.77 TBq/mmol); and different concentrations of antibiotics. After 60 min at 30°C, the reaction was stopped by addition of 2 ml of trichloroacetic acid (5%). After filtration of the cells through Whatman GF/C filters, three washes with 5 ml of ice-cold trichloroacetic acid, and one wash with 5 ml of ethanol, radioactivity was counted. was
RESULTS MICs of quinolones. E. coli Q2 was selected under norfloxacin treatment of a lower urinary tract infection caused by E. coli Ql. Neither E. coli Qi nor Q2 was phage typable. However, since both strains had the same tetracycline, chloramphenicol, and ampicillin resistance patterns and the same plasmid content (data not shown), and since the OM protein patterns (see below) appeared qualitatively identical, we considered strain Q2 as a possible derivative from Ql. The MICs of quinolones for E. coli Ql and Q2 are shown in Table 1. There was almost no increase in the MIC of nalidixic acid (2-fold) or flumequine (4-fold), while there were large increases (16- to 1,024-fold) in the MICs of the other quinolones tested, including several fluoroquinolones. The largest relative increases were observed for rosoxacin
and ciprofloxacin (Table 1). No difference in the susceptibility to novobiocin was found (data not shown). Selection of resistant strains. About 500 resistant clones, selected from E. coli Ql in the presence of different concentrations of norfloxacin, were tested to find a resistance pattern similar to that of E. coli Q2. Mutants with cross resistance to all quinolones and low-level resistance to norfloxacin (0.5 to 2 ,ug/ml) were selected at a frequency of ca. 10-', but none had the particular quinolone resistance phenotype of E. coli Q2. OM protein and LPS. Comparison of the OM protein patterns showed the disappearance of a major OM protein of ca. 40 kDa (OmpF), associated with an apparent compensatory increase of a ca. 38-kDa protein (OmpC) in E. coli Q2 (Fig. la). Analysis of the LPS patterns (Fig. lb) showed that in E. coli Q2, in contrast to Qi, only the core of the LPS was present. On the basis of these results, we supposed that decreased OM permeability could explain part of the resistance and therefore assayed the uptake of different quinolones. Uptake and inhibition of efflux. Quinolone uptake was measured by the fluorimetric assay previously described (4). For all of the quinolones tested, a rapid uptake was observed within 5 min, as noted by other workers (1, 9-11, 14). With the exception of nalidixic acid and with respect to strain Qi, all quinolones were taken up less rapidly initially by strain Q2, and total uptake was reduced 1.5- to 3-fold. Different concentrations of the individual antibiotics were used for this assay (Fig. 2). Concentrations of up to 50 jig of nalidixic acid, flumequine, pefloxacin, and enoxacin per ml were necessary to obtain an intracellular accumulation sufficient to be measured by the fluorimetric assay. Despite these high concentrations, we believe that the results are reliable, since the accumulation of quinolones is a nonsaturable process depending upon the external concentration of antibiotics (3, 4, 6). When 50 Fg of norfloxacin per ml was used instead of 10 ,ug/ml, similar (proportionally) results were obtained (data not shown). The inhibition of norfloxacin efflux by CCCP, added after 15 min, was also assayed. The effect of CCCP was more pronounced on the resistant strain, with a roughly threefold increase in uptake (Fig. 3). Similar observations were previously made with other isogenic susceptible and resistant strains (6, 14) and suggest that there is no impaired efflux in E. coli Q2. Inhibition of DNA synthesis. In order to avoid the permeability barrier, we used toluene-treated cells to examine the effect of the six quinolones studied in the uptake assay on DNA synthesis (Fig. 4). A clear correlation existed between the MIC ratios (Table 1) and the ratios of the quinolone
VOL. 35, 1991
NOVEL CROSS RESISTANCE TO QUINOLONES IN E. COLI
E L ,,
i'
114 i{ I -7'---7
c
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1 10
5
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f20
1%
b 510 20 30
521
/d 5 10 20 30
--
f 510 20 30
Time (min) FIG. 2. Quinolone uptake in E. coli Ql ( ) and Q2 (---) at external concentrations of 10 ,ug of norfloxacin (d) per ml, 20 ,ug of ciprofloxacin (e) per ml, and 50 p.g of pefloxacin (c), enoxacin (f), flumequine (b), and nalidixic acid (a) per ml.
amounts necessary to inhibit DNA synthesis in E. coli Ql and Q2 (Fig. 4). While no difference was found between the concentrations of nalidixic acid needed to inhibit DNA synthesis in E. coli Ql and Q2, over 500 times more ciprofloxacin was required to inhibit DNA synthesis in E. coli Q2 to the same degree as in Ql. A good correlation between the concentrations inhibiting 50% of DNA synthesis and the MICs of the different compounds for E. coli Qi and Q2 was observed (Fig. 5).
DISCUSSION The quinolone resistance pattern of E. coli Q2 is peculiar. This strain is resistant to almost all quinolones, including the fluoroquinolones, but very susceptible to nalidixic acid and flumequine, with the MICs of the latter compounds increased by factors of only 2 and 4, respectively, compared with MICs in the susceptible strain Ql.
0
25_
-
U
CCCP
20_
E
0
L
15_
.
0
10-
0 co
//
o
0
/ 5
10 15 20 25 Time (min)
FIG. 3. Effect of CCCP (0.1 mM) on the uptake of norfloxacin (10 ,ug/ml) by E. coli Ql (0) and Q2 (0).
We have demonstrated that at least two mechanisms of resistance were operative in E. coli Q2: a decrease in OM permeability and a probable alteration of the DNA gyrase. The decrease in uptake of most of the quinolones into strain Q2 is compatible with the decrease in amount of the OmpF porin (1, 5, 9, 10, 13-15). However, there was no apparent decrease in the uptake of nalidixic acid. Since this quinolone is among the most hydrophobic (9), the absence of decreased uptake might be explained by the simultaneous LPS modification which could conceivably allow a better permeation via the hydrophobic route (non-porin pathway). Mutants of E. coli KEA16 with similar characteristics (OmpF-, modification of LPS) have been described by Hirai et al. (10). Under the same circumstances it is, however, not clear why there was some decrease in uptake of flumequine, a compound even more hydrophobic than nalidixic acid. We have observed an active efflux of norfloxacin in E. coli Q2, as demonstrated by its increased uptake in the presence of CCCP. We have, however, no information suggesting an increased efflux of flumequine relative to that of nalidixic acid, which could explain the relatively lower uptake of flumequine into strain Q2. The second mechanism of resistance is the decrease in the inhibition of DNA synthesis, which appears most likely to be due to a modification of the DNA gyrase. A priori, the unaltered susceptibility to novobiocin would argue against a modification of the B subunit. Curiously, nalidixic acid and flumequine, which showed the lowest increase in MICs as well as in the concentrations necessary to inhibit DNA synthesis in toluene-permeabilized cells, were the only compounds which have no cyclic subtituent at the C-7 position or a fused ring structure linking the C-6 and C-7 atoms (18, 20). To our knowledge, there has been no previous report on clinical isolates which present a high-level resistance to the newer quinolones while remaining susceptible to nalidixic acid. With respect to the association of two mechanisms of resistance, E. coli Q2 would resemble a clinical isolate of E. coli previously described (1) as well as in vitro-selected mutant KF111 (15). However, nothing is known about the
522
ANTIMICROB. AGENTS CHEMOTHER.
MONIOT-VILLE ET AL.
100-
0
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50-
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a
d
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_ l ~ l___l_ ~ ~ ~ ~ ~ _l_ _l_ _l _l
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1
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antibiotic concentration (pg/mi) FIG. 4. Synthesis of DNA in toluene-permeabilized E. coli Ql (0) and Q2 (0) cells in the presence of quinolones; 100% corresponded to ca. 3,000 to 4,500 cpm. (a) Nalidixic acid; (b) flumequine; (c) enoxacin; (d) norfloxacin; (e) pefloxacin; (f) ciprofloxacin.
LPS of these two strains, neither of which remained susceptible to nalidixic acid and flumequine. We suppose that the modification of the DNA gyrase is the major mechanism responsible for the high level of resistance, but we do not know yet whether one or several
1000_
ACKNOWLEDGMENTS We are grateful to A. Jaffd for helpful discussions. We thank C. Harcour for excellent secretarial assistance. This work was supported by a grant (CJF-89-01) from the Institut National de la Sante et de la Recherche Medicale.
PEF
NOR
100_
NALE NALO
10-
/
FLU
0) FLU 0-
1~
0
n..oI
otPEE
0.1 _
E/INOR
/-C(IP A) A1 i
0.01
0.1
1
10
mutations of the gyrase are required to yield this particular phenotype nor whether one or both gyrase subunits are affected. An argument for the alteration of the subunit A of the gyrase was obtained from a preliminary experiment in which the introduction of a plasmid harboring the wild-type gyrA gene into E. coli Q2 decreased the MICs of the fluoroquinolones 16- to 64-fold, which remained, however, 4- to 8-fold higher than the MICs observed for E. coli Ql. This difference in MICs could conceivably be explained by the permeability defect of the Q2 transformants.
100
MIC (pg/mi) FIG. 5. Correlation between MICs (Table 1) and concentrations inhibiting 50% of DNA synthesis (estimated from Fig. 4). El, E. coli Q1; *, E. coli Q2; CIP, ciprofloxacin; NOR, norfloxacin; PEF, pefloxacin; FLU, flumequine; NAL, nalidixic acid. Enoxacin was not included since 50% inhibition was not achieved for the resistant strain.
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NOVEL CROSS RESISTANCE TO QUINOLONES IN E. COLI
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