ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1990,
p.
2142-2147
Vol. 34, No. 11
0066-4804/90/112142-06$02.00/0 Copyright ) 1990, American Society for Microbiology
Development of Quinolone-Imipenem Cross Resistance in Pseudomonas aeruginosa during Exposure to Ciprofloxacin GUNILLA RADBERG,* LENNART E. NILSSON, AND STEFAN SVENSSON Department of Clinical Bacteriology, University Hospital, S-581 85 Linkoping, Sweden Received 27 March 1990/Accepted 31 August 1990
Selection and regrowth of ciprofloxacin-resistant variants, which were present in low frequencies in the initial inoculum, were seen when large inocula of Pseudomonas aeruginosa were incubated with ciprofloxacin. These variants showed cross resistance to other quinolones. In 8 of 13 strains tested, ciprofloxacin selected imipenem-resistant variants in a similar way to imipenem. The opposite phenomenon of ciprofloxacinimipenem cross resistance after exposure to imipenem was not detected. None of the ciprofloxacin-resistant variants showed cross resistance to aztreonam, piperacillin, or tobramycin. These findings indicate that widespread and uncritical use of ciprofloxacin gives a potential risk of development of resistance in P. aeruginosa not only to quinolones but also to another unrelated useful agent, imipenem. In vitro evaluation of this phenomenon in isolates from patients with P. aeruginosa infections may be justified, since strains differ in development of quinolone-imipenem cross resistance after ciprofloxacin exposure.
MIC determination. Serial twofold dilutions from freshly prepared stock solutions of active drug were made in Mueller-Hinton broth (Gibco Limited, Renfrewshire, Scotland) supplemented with 25 mg of Mg2+ per liter and 50 mg of Ca2+ per liter. Samples of these dilutions (0.5 ml) were added to test tubes. Overnight cultures of the bacterial strains were diluted to approximately 105 or 107 CFU/ml, and 0.5-ml samples of these cultures were added to the tubes. Visible growth was recorded after 24 h of incubation at 37°C. The lowest concentration in which no visible growth could be detected was designated the MIC. Disk susceptibility tests. Disk susceptibility tests were performed with the method of Eriksson and Sherris (3a) on Mueller-Hinton agar (Gibco). Disks contained 30 ,ug of aztreonam, 10 ,ug of ciprofloxacin, 10 jig of imipenem, 10 p,g of norfloxacin, 10 pug of ofloxacin, 30 ,ug of piperacillin, and 30 ,ug of tobramycin (AB Biodisk, Solna, Sweden). Bacterial population analysis. Large inocula (107 CFU/ml) were incubated in supplemented Mueller-Hinton broth (Gibco) with and without ciprofloxacin and imipenem in a total volume of 1 ml for 24 h at 37°C. After thorough mixing, 0.1-ml samples from cultures without antibiotic and from cultures containing 1/2 the MIC of ciprofloxacin or 1/2 the MIC of imipenem were diluted serially in 0.9 ml of physiological saline. Drops (100 ,ul) from each dilution were placed on Mueller-Hinton agar (Gibco) containing different concentrations of ciprofloxacin (0.0625 to 16 ,ug/ml) or imipenem (0.25 to 64 ,ug/ml). The drops were allowed to dry at room temperature, and the plates were incubated overnight at 37°C. The colonies were then counted, and the number of viable bacteria was calculated. Stability of resistant variants. The resistant variants selected and enriched after exposure to 1/2 the MIC of ciprofloxacin in broth were colony purified on agar plates containing ciprofloxacin or imipenem. To test the stability of these colony-purified resistant variants, they were passaged 10 times in Mueller-Hinton broth (Gibco) for 24 h at 37°C. The susceptibilities to ciprofloxacin, imipenem, norfloxacin, and ofloxacin were tested with disk diffusion, as described above, after each passage.
The development of resistance to new antimicrobial agents as well as cross resistance to related and unrelated drugs is seldom thoroughly evaluated when new drugs are introduced. Recently there have been several reports of emergence of resistance to ciprofloxacin in both clinical and laboratory-derived strains of Pseudomonas aeruginosa (1-7, 10-12). Cross resistance to other quinolones (1, 2, 4-7, 11, 12) as well as to chemically unrelated antibiotics has also been described previously (5, 7, 10). The mechanism of resistance to ciprofloxacin appears to involve either an alteration in the target, subunit A protein of DNA gyrase (4, 11) or an alteration in drug permeation through the outer membrane of the cell (3-5, 12). In this study, the frequency and the selective growth of variants resistant to ciprofloxacin in clinical isolates and in an ATCC strain of P. aeruginosa were studied. Furthermore, the occurrence of cross resistance between ciprofloxacin and other quinolones, beta-lactam antibiotics, and aminoglycosides was studied. MATERIALS AND METHODS Bacterial strains. A total of 12 clinical isolates of P. aeruginosa and P. aeruginosa ATCC 27853 were used. Ten strains were blood isolates (LU I, LU II, LU IV, LU V, LU VII, LU VIII, LU IX, LU X, LU XI, and LU XII), and two strains were sputum isolates (LU IIIA and LU IIIB) from a patient with cystic fibrosis. Antimicrobial agents. Stock solutions of active drug (1,000 ,ug/ml) were prepared from ciprofloxacin (827 ,g/mg; Bayer, Leverkusen, Federal Republic of Germany), which was dissolved in 0.1 M NaOH, and from imipenem (964 pug/mg; Merck Sharp & Dohme Research Laboratories, Rahway, N.J.), which was dissolved in 0.01 M phosphate buffer (pH 7.0). Aqueous solutions of active drug (1,000 ,ug/ml) were prepared from aztreonam (964 ,g/mg; Squibb Institute for Medical Research, Princeton, N.J.), piperacillin (1,010 ,ug/ mg; Lederle Inc., Carolina, P.R.), and tobramycin (942 ,ug/mg; Eli Lilly and Co., Indianapolis, Ind.). *
Corresponding author. 2142
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TABLE 1. MICs of ciprofloxacin and imipenem for 13 strains of P. aeruginosa with small (105 CFU/ml) and large (107 CFU/ml) inoculum MIC (jLg/ml) P. aeruginosa Ciprofloxacin Imipenem _____________ ________ strain 105 CFU/ml 107 CFU/ml 10i CFU/ml 107 CFU/ml
ATCC 27852 LU I LU II LU IIIA LU IIIB LU IV LU V LU VII LU VIII LU IX LU X LU XI LU XII
1 0.25 0.5 1 1 4 4 0.25 0.25 0.25 0.5 0.25 0.25
1 1 1 1 2 8 4 1 1 1 1 0.5 1
2 2 1 2 4 4 8 1 2 1 2 1 1
8 16 8 8 4 8 16 4 8 8 8 2 4
RESULTS MICs. The MICs of ciprofloxacin and imipenem for 12 clinical isolates of P. aeruginosa and P. aeruginosa ATCC 27853 with large inocula (107 CFU/ml) and with small inocula (105 CFU/ml) are shown in Table 1. Resistance patterns of unexposed and ciprofloxacin- or imipenem-exposed bacteria. The susceptibilities of the P. aeruginosa strains to aztreonam, ciprofloxacin, imipenem, piperacillin, and tobramycin were determined by the disk diffusion method (Table 2). Large inocula (107 CFU/ml) of cultures grown without antibiotic and those exposed to 1/2 the MIC of ciprofloxacin or 1/2 the MIC of imipenem for 24 h were tested (Table 2). The mean inhibition zone given by a ciprofloxacin (10-,ug) disk for the 13 strains tested was 9 mm smaller for cells
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grown in the presence of 1/2 the MIC of ciprofloxacin than for cells grown without antibiotic (Table 2). For 8 of the strains exposed to 1/2 the MIC of ciprofloxacin, the mean inhibition zone given by an imipenem (10-,ug) disk was 11 mm smaller than for unexposed bacteria (Table 2). For the other five strains tested, there were no differences in inhibition zone diameter given by an imipenem (10-,ug) disk for the unexposed bacteria and for the bacteria exposed to 1/2 the MIC of ciprofloxacin (Table 2). The inhibition zone diameter given by an imipenem (10,ug) disk for all bacterial strains grown in the presence of 1/2 the MIC of imipenem was less than half the diameter of that produced when the bacterial strains were grown without antibiotic, while the zone diameter given by a ciprofloxacin (10-,ug) disk was similar for bacteria exposed to 1/2 the MIC of imipenem and for unexposed bacteria (Table 2). The inhibition zone diameters given by aztreonam, piperacillin, and tobramycin disks were the same for the bacteria from unexposed cultures and for those grown in cultures containing 1/2 the MIC of ciprofloxacin or 1/2 the MIC of imipenem (Table 2). The inhibition zone diameters given by 10-,ug disks of norfloxacin and ofloxacin decreased to the same extent as the zone given by a 10-,ug disk of ciprofloxacin, when cultures exposed to 1/2 the MIC of ciprofloxacin were compared with the corresponding unexposed cultures (see Fig. 3a). These results were reproduced for each strain, and the individual strain differences in each case were concordant with the results given for the differences between the means of the two groups. The margins of the zones of inhibition for the bacteria from the cultures exposed to drug were as distinct and uniform as the zones of inhibition of bacteria from the unexposed cultures. MIC determination of ciprofloxacin, imipenem, aztreonam, piperacillin, and tobramycin for six strains of P. aeruginosa was performed. Large inocula (107 CFU/ml) of
TABLE 2. Susceptibility of 13 strains of P. aeruginosa to ciprofloxacin, imipenem, aztreonam, piperacillin, and tobramycin in cultures incubated without antibiotic, with ciprofloxacin, and with imipenem Zone of inhibition (mm)
Strains and antibiotic disk
Culture exposed to 1/2 the MIC of the following drug:
Unexposed culture
Ciprofloxacin Range
Mean + SD
Range
Imipenem
Mean ± SD
Range
Mean ± SD
8 strains of P. aeruginosa showing ciprofloxacinimipenem cross resistance
Ciprofloxacin Imipenem
29.0-35.5
Aztreonam
21.5-29.0 6.0-30.5
Piperacillin Tobramycin
6.0-29.0 25.5-28.0
32.5 26.2 18.8 18.8 26.9
± 2.1 ± 2.7 ± 10.9
30.8 26.9 27.7 26.6 26.0
± 3.6
± 10.8 ± 0.8
20.0-26.5 11.5-21.0
6.0-34.0 6.0-32.0 25.5-32.0
23.4 15.0 21.4 21.0 29.1
± 2.4 ± 3.2 ± 11.7
± 11.9 ± 2.1
27.0-35.5 10.0-15.5 6.0-30.5 6.0-29.5 24.5-29.0
30.6 11.9 18.8 18.9 26.6
± 3.8 ± 2.1 ± 10.3 ± 10.5 ± 1.6
26.5-36.5 8.5-16.5 25.0-33.0
30.7 11.5 27.3 24.7 25.2
± ± ± ±
5 strains of P. aeruginosa showing no cross resistance
Ciprofloxacin Imipenem
26.0-36.0
Aztreonam Piperacillin Tobramycin
24.5-33.0
24.0-28.5 25.0-29.0 24.5-28.0
+ 2.2 ± 3.3 ± 1.6 ± 1.5
10.5-29.0 21.5-31.5
28.0-33.0 26.5-29.0 26.0-31.5
22.0 27.6 30.4 27.8 28.5
± 7.5 ± ± ± ±
4.1 2.5 1.1 2.2
23.0-26.5 23.5-28.0
± 3.7
3 3| 3.2 1.2 1.8
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. MICs of ciprofloxacin, imipenem, aztreonam, piperacillin, and tobramycin for six strains of P. aeruginosa with small inocula (105 CFU/ml) in cultures incubated without antibiotic or with ciprofloxacin MICa (,ug/ml)
Strains Ciprofloxacin
Imipenem
Aztreonam
Piperacillin
Tobramycin
3 strains of P. aeruginosa showing cross resistance LU I LU VIII LU XI
0.25/4 0.25/2 0.25/4
2/32 2/32 1/32
>32/>32 >32/>32 4/4
>32/>32 >32/>32 4/4
2/2 4/4 1/1
3 strains of P. aeruginosa showing no cross resistance LU IV LU V LU IX
4/16 2/8 0.25/4
4/4 8/8 1/1
4/4 16/16 4/2
8/8 >321>32 4/2
1/1 2/2 2/4
a
The values before the shills show the MICs for unexposed cultures, and the values after the shills show the
these strains were grown without antibiotic and with 1/2 the MIC of ciprofloxacin for 24 h. The results of MIC determination of these bacteria with a small inoculum (105 CFU/ml) are shown in Table 3. The MICs of ciprofloxacin were 4 to 16 times higher for cells grown in the presence of 1/2 the MIC of ciprofloxacin than for cells grown without antibiotic for all strains tested (Table 3). Three of the strains (P. aeruginosa LU I, LU VIII, and LU XI) showed MICs for imipenem which increased 16- to 32-fold after exposure to ciprofloxacin, while three strains
MICs for ciprofloxacin-exposed cultures.
(P. aeruginosa LU IV, LU V, and LU IX) did not show an increase in imipenem MIC (Table 3). The MICs of aztreonam, piperacillin, and tobramycin were almost the same (one dilution step difference) for the bacteria from unexposed cultures and for those grown in 1/2 the MIC of ciprofloxacin. These results (Table 3) are in full agreement with the results obtained with disk diffusion (Table 2). Population analysis. Six strains were chosen for population analysis. Three of the strains (P. aeruginosa LU I, LU VIII, and LU XI) showed ciprofloxacin-imipenem cross resistance
(a)
(b)
0
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-2
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-7.
-7. 1/8 1/4
1/2
1
2
4
8
16
1/8'1/4 1/2
1
2
4
8
16
Concentration of imipenem in agarplates expressed as multiples of MIC FIG. 1. Frequencies of variants resistant to different multiples of MIC of imipenem in broth cultures of strains of P. aeruginosa incubated without antibiotic (0), with imipenem (1/2 the MIC) (A), and with ciprofloxacin (1/2 the MIC) (-) for 24 h. (a) Mean frequency + standard deviation of three strains (P. aeruginosa LU I, LU VIII, and LU XI) showing cross resistance. (b) Mean frequency ± standard deviation for three strains (P. aeruginosa LU IV, LU V, and LU IX) showing no cross resistance.
QUINOLONE-IMIPENEM CROSS RESISTANCE IN PSEUDOMONAS AERUGINOSA
VOL. 34, 1990
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(b)
(a) 0*
4 -1
-1-
-2
-2-
-3
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-4
-4
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-7.
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Concentration
of
4
8
16
1/8
1/4
2
1/2
ciprofloxacin in agarplates expressed
as
4
8
16
multiples of MIC
FIG. 2. Frequencies of variants resistant to different multiples of MIC of ciprofloxacin in broth cultures of strains of P. aeruginosa incubated without antibiotic (0), with imipenem (1/2 the MIC) (A), and with ciprofloxacin (1/2 the MIC) (A) for 24 h. (a) Mean frequency + standard deviation for three strains (P. aeruginosa LU I, LU VIII, and LU XI) showing cross resistance. (b) Mean frequency + standard deviation for three strains (P. aeruginosa LU IV, LU V, and LU IX) showing no cross resistance.
(Fig. la) and three strains (P. aeruginosa LU IV, LU V, and LU IX) did not (Fig. lb) after exposure of large inocula (107 CFU/ml) to 1/2 the MIC of ciprofloxacin in broth, when studied by disk diffusion (Table 2) and MIC determination (Table 3). The frequency of variants resistant to different concentrations of ciprofloxacin or imipenem was calculated by dividing the number of colonies on plates with a particular concentration of ciprofloxacin or imipenem by the number of colonies on plates without antibiotic. Results shown in Fig. la are based on pooled population analysis data of three strains showing cross resistance (P. aeruginosa LU I, LU VIII, and LU XI). The frequency of bacteria resistant to the MIC was about 10' in the unexposed cultures. After exposure of large inocula (107 CFU/ml) to 1/2 the MIC of imipenem or 1/2 the MIC of ciprofloxacin, the frequency curve of variants resistant to imipenem was shifted toward higher concentrations of the drug. The frequencies of bacteria resistant to 4, 8, and 16 times the MIC of imipenem were about 10-1, 10-3, and 10-6, respectively, after exposure to both imipenem and ciprofloxacin. The pooled population analysis data of three strains (P. aeruginosa LU IV, LU V, and LU IX) showing no cross resistance is shown in Fig. lb. The frequency of bacteria resistant to MIC was about 10' in the unexposed cultures. After exposure to 1/2 the MIC of imipenem, the frequency curve was shifted toward higher imipenem concentrations. The frequencies of bacteria resistant to 2, 4, and 8 times the MIC of imipenem were 10-2 to 10-1, 10-4 to 10-3, and 10-5 to 10-4, respectively, while the frequency curve after expo-
sure to 1/2 the MIC of ciprofloxacin was similar to the frequency curve for the unexposed cultures (Fig. lb). The phenomenon of development of ciprofloxacin resistance after exposure to imipenem was not detected in any of these tested strains (Fig. lb and 2b). Stability of resistance. The resistant variants selected and enriched after exposure to 1/2 the MIC of ciprofloxacin in broth were colony purified on agar plates containing ciprofloxacin and imipenem. These colony-purified resistant variants were passaged in antibiotic-free broth for 10 passages. The resistant variants of six strains selected and enriched in 1/2 the MIC of ciprofloxacin and purified on ciprofloxacin agar plates were still, after 10 passages, less susceptible to ciprofloxacin, norfloxacin, and ofloxacin than unexposed bacteria (Fig. 3). The stability of the ciprofloxacin-imipenem cross-resistant variants was studied in three strains (Fig. 4). Bacteria exposed to 1/2 the MIC of ciprofloxacin in broth, which were purified on ciprofloxacin agar plates, were still less susceptible to imipenem than unexposed bacteria after 10 passages (Fig. 4). However, the difference in zone diameter between ciprofloxacin-exposed and unexposed bacteria had decreased during the 10 passages. The ciprofloxacin-imipenem cross-resistant bacteria from the 1/2-MIC ciprofloxacin broth culture were more stable during the 10 passages if they were purified on imipenem agar plates before the passages in antibiotic-free broth (Fig. 4). A proper determination of the growth rates was not made, but the resistant variants caused about the same turbidity as unexposed bacteria during the passage in antibiotic-free
2146
RADBERG
ANTIMICROB. AGENTS CHEMOTHER.
ET AL. 40-
40
(a)
(b)
Imipenem
Imipenem disc
(a)
E
30-
30-
E E
E
C
I
C
0
0
20-
20c
c 0
0
N
N
10-
0
L--j
m
Ciprofloxacin disc
[aALi
I
Nort loxacin disc
Ofloxacin disc
40 (b)
30E
E
1:-
disc
FIG. 4. Mean inhibition zone diameters given by 10-,ug disks of imipenem for broth cultures of three strains of P. aeruginosa incubated without antibiotic (E) and with ciprofloxacin (1/2 the MIC) after purification of resistant variants on ciprofloxacin agar ). Ciprofloxacinplates (DEmI) and on imipenem agar plates ( imipenem cross resistance before passages in antibiotic-free broth (a) and after 10 passages in antibiotic-free broth (b).
0
.Q20C
0
0
N
10A
Ciprofloxacin disc
Norfloxacin disc
Ofloxacin disc
FIG. 3. Mean inhibition zone + standard deviation given by disks of ciprofloxacin, norfloxacin, and ofloxacin for broth cultures of six strains of P. aeruginosa incubated without antibiotic (El) and with ciprofloxacin (1/2 the MIC) after purification of resistant variants on ciprofloxacin agar plates (Em). Quinolone cross resistance before passages in antibiotic-free broth (a) and after 10 passages in antibiotic-free broth (b). 10-lg
broth. The growth rate of the resistant variants seems to be comparable with the growth of the wild-type cells.
DISCUSSION This study shows that ciprofloxacin-resistant variants were selected in large inocula (107 CFU/ml) of P. aeruginosa by subinhibitory concentrations of ciprofloxacin (1/2 the MIC). This has been described in earlier reports (1, 2, 4-7, 10-12). The frequencies of variants resistant to ciprofloxacin in P. aeruginosa at different multiples of the MIC were similar to the frequencies of variants resistant to aminoglycosides (9) and to imipenem (8; also this study). In all P. aeruginosa strains tested, the selected resistant variants showed cross resistance to quinolones, which is in agreement with earlier reports (1, 2, 4-7, 11, 12). Development of resistance to ciprofloxacin in P. aeruginosa after therapy with enoxacin has been described previously (10). These posttherapy isolates showed cross resistance between quinolones and ureidopenicillins (carben-
icillin, mezlocillin, and piperacillin) but no resistance to imipenem (10). In another study, ciprofloxacin-resistant variants of P. aeruginosa showed cross resistance to aminoglycosides and moxalactam (5). The quinolone-resistant variants selected in our study did not show such a cross resistance to piperacillin, tobramycin, or aztreonam. In another study, posttherapy isolates showed resistance to quinolones after ciprofloxacin therapy and no cross resistance to aminoglycosides, moxalactam, and ureidopenicillins (piperacillin and ticarcillin) (3). Similar results were obtained with P. aeruginosa in a study in which resistance to quinolones was obtained after selection with quinolones, but no resistance to aminoglycosides or 3-lactam antibiotics was demonstrated (12). In our study, ciprofloxacin selected and enriched imipenem-resistant variants in a similar way to imipenem in 8 of 13 strains of P. aeruginosa tested. The opposite phenomenon of ciprofloxacin-imipenem cross resistance after exposure to imipenem was not detected. The development of ciprofloxacin-imipenem cross resistance after exposure to ciprofloxacin has been shown earlier in an experimental model of P. aeruginosa infection in mice (7). The variants selected in that study (7) did not show cross resistance to aztreonam, piperacillin, or aminoglycosides, which is in agreement with the results obtained in our study. The mechanism of resistance to ciprofloxacin has been evaluated in several studies (3, 5, 7, 10-12). Most researchers describe two major mechanisms of resistance: changes in the target subunit A of DNA gyrase (4, 11) and alteration in permeability of the bacteria for the quinolones (3-5, 12). When dealing with two chemically different antibiotics, such as quinolones and imipenem, the most likely explanation for decreased susceptibility would be changed drug permeation. To test the stability of quinolone resistance and quinoloneimipenem cross resistance, the bacteria were passaged in antibiotic-free broth. The quinolone-resistant variants were still, after 10 passages, less susceptible to ciprofloxacin, norfloxacin, and ofloxacin than the bacteria from unexposed cultures. The ciprofloxacin-imipenem cross-resistant vari-
VOL. 34, 1990
QUINOLONE-IMIPENEM CROSS RESISTANCE IN PSEUDOMONAS AERUGINOSA
ants purified on ciprofloxacin agar plates were less stable, but after 10 passages these variants were still more resistant than the unexposed bacteria. However, the ciprofloxacinimipenem cross-resistant variants were more stable if they were purified on imipenem agar plates before passages in antibiotic-free broth. This phenomenon can be explained by the facts that the cross-resistant population purified on ciprofloxacin agar plates is not homogeneous and contains a smaller part of the original more susceptible population and that the remaining susceptible bacteria will outgrow the resistant ones during the incubation in antibiotic-free broth. In conclusion, this study indicates that widespread and uncritical use of ciprofloxacin gives a potential risk of development of resistance in P. aeruginosa not only to quinolones but also to another unrelated useful agent, imipenem. Furthermore, the differences between strains in development of resistance to imipenem after exposure to ciprofloxacin might justify in vitro evaluation of this phenomenon in isolates from patients with P. aeruginosa infections.
LITERATURE CITED 1. Barry, A. L., and R. N. Jones. 1984. Cross-resistance among cinoxacin, ciprofloxacin, DJ-6783, enoxacin, nalidixic acid, norfloxacin, and oxolinic acid after in vitro selection of resistant populations. Antimicrob. Agents Chemother. 25:775-777. 2. Culiman, W., M. Stieglitz, B. Baars, and W. Opferkuch. 1985. Comparative evaluation of recently developed quinolone compounds-with a note on the frequency of resistant mutants. Chemotherapy (Basel) 31:19-28. 3. Daikos, G. L., V. T. Lolans, and G. G. Jackson. 1988. Alterations in outer membrane proteins of Pseudomonas aeruginosa associated with selective resistance to quinolones. Antimicrob.
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Agents Chemother. 32:785-787. 3a.Eriksson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. B Suppl. 217:1-90. 4. Kaatz, G. W., and S. M. Seo. 1988. Mechanism of ciprofloxacin resistance in Pseudomonas aeruginosa. J. Infect. Dis. 158:537541. 5. Legakis, N. J., L. S. Tzouvelekis, A. Makris, and H. Kotsifaki. 1989. Outer membrane alterations in multiresistant mutants of Pseudomonas aeruginosa selected by ciprofloxacin. Antimicrob. Agents Chemother. 33:124-127. 6. Limb, D. I., D. J. W. Dabbs, and R. C. Spencer. 1987. In-vitro selection of bacteria resistant to the 4-quinolone agents. J. Antimicrob. Chemother. 19:65-71. 7. Michea-Hamzehpour, R. Auckenthaler, P. Regamey, and J.-C. Pechere. 1987. Resistance occurring after fluoroquinolone therapy of experimental Pseudomonas aeruginosa peritonitis. Antimicrob. Agents Chemother. 31:1803-1808. 8. Nilsson, L., M. Nilsson, and J. Jendle. 1988. Subpopulations of variants resistant to imipenem in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 22:643-649. 9. Nilsson, L., L. Soren, and G. R&dberg. 1987. Frequencies of variants resistant to different aminoglycosides in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 20:255-259. 10. Piddock, L. J. V., W. J. A. Wjnands, and R. Wise. 1987. Quinolone/ureidopenicillin cross-resistance. Lancet ii:907. 11. Robillard, N. J., and A. L. Scarpa. 1988. Genetic and physiological characterization of ciprofloxacin resistance in Pseudomonas aeruginosa PAO. Antimicrob. Agents Chemother. 32: 535-539. 12. Sanders, C. C., W. E. Sanders, Jr., R. V. Goering, and V. Werner. 1984. Selection of multiple antibiotic resistance by quinolones, P-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes. Antimicrob. Agents Chemother. 26:797-801.
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Vol. 34, No. 11
0066-4804/90/112142-06$02.00/0 Copyright ) 1990, American Society for Microbiology
Development of Quinolone-Imipenem Cross Resistance in Pseudomonas aeruginosa during Exposure to Ciprofloxacin GUNILLA RADBERG,* LENNART E. NILSSON, AND STEFAN SVENSSON Department of Clinical Bacteriology, University Hospital, S-581 85 Linkoping, Sweden Received 27 March 1990/Accepted 31 August 1990
Selection and regrowth of ciprofloxacin-resistant variants, which were present in low frequencies in the initial inoculum, were seen when large inocula of Pseudomonas aeruginosa were incubated with ciprofloxacin. These variants showed cross resistance to other quinolones. In 8 of 13 strains tested, ciprofloxacin selected imipenem-resistant variants in a similar way to imipenem. The opposite phenomenon of ciprofloxacinimipenem cross resistance after exposure to imipenem was not detected. None of the ciprofloxacin-resistant variants showed cross resistance to aztreonam, piperacillin, or tobramycin. These findings indicate that widespread and uncritical use of ciprofloxacin gives a potential risk of development of resistance in P. aeruginosa not only to quinolones but also to another unrelated useful agent, imipenem. In vitro evaluation of this phenomenon in isolates from patients with P. aeruginosa infections may be justified, since strains differ in development of quinolone-imipenem cross resistance after ciprofloxacin exposure.
MIC determination. Serial twofold dilutions from freshly prepared stock solutions of active drug were made in Mueller-Hinton broth (Gibco Limited, Renfrewshire, Scotland) supplemented with 25 mg of Mg2+ per liter and 50 mg of Ca2+ per liter. Samples of these dilutions (0.5 ml) were added to test tubes. Overnight cultures of the bacterial strains were diluted to approximately 105 or 107 CFU/ml, and 0.5-ml samples of these cultures were added to the tubes. Visible growth was recorded after 24 h of incubation at 37°C. The lowest concentration in which no visible growth could be detected was designated the MIC. Disk susceptibility tests. Disk susceptibility tests were performed with the method of Eriksson and Sherris (3a) on Mueller-Hinton agar (Gibco). Disks contained 30 ,ug of aztreonam, 10 ,ug of ciprofloxacin, 10 jig of imipenem, 10 p,g of norfloxacin, 10 pug of ofloxacin, 30 ,ug of piperacillin, and 30 ,ug of tobramycin (AB Biodisk, Solna, Sweden). Bacterial population analysis. Large inocula (107 CFU/ml) were incubated in supplemented Mueller-Hinton broth (Gibco) with and without ciprofloxacin and imipenem in a total volume of 1 ml for 24 h at 37°C. After thorough mixing, 0.1-ml samples from cultures without antibiotic and from cultures containing 1/2 the MIC of ciprofloxacin or 1/2 the MIC of imipenem were diluted serially in 0.9 ml of physiological saline. Drops (100 ,ul) from each dilution were placed on Mueller-Hinton agar (Gibco) containing different concentrations of ciprofloxacin (0.0625 to 16 ,ug/ml) or imipenem (0.25 to 64 ,ug/ml). The drops were allowed to dry at room temperature, and the plates were incubated overnight at 37°C. The colonies were then counted, and the number of viable bacteria was calculated. Stability of resistant variants. The resistant variants selected and enriched after exposure to 1/2 the MIC of ciprofloxacin in broth were colony purified on agar plates containing ciprofloxacin or imipenem. To test the stability of these colony-purified resistant variants, they were passaged 10 times in Mueller-Hinton broth (Gibco) for 24 h at 37°C. The susceptibilities to ciprofloxacin, imipenem, norfloxacin, and ofloxacin were tested with disk diffusion, as described above, after each passage.
The development of resistance to new antimicrobial agents as well as cross resistance to related and unrelated drugs is seldom thoroughly evaluated when new drugs are introduced. Recently there have been several reports of emergence of resistance to ciprofloxacin in both clinical and laboratory-derived strains of Pseudomonas aeruginosa (1-7, 10-12). Cross resistance to other quinolones (1, 2, 4-7, 11, 12) as well as to chemically unrelated antibiotics has also been described previously (5, 7, 10). The mechanism of resistance to ciprofloxacin appears to involve either an alteration in the target, subunit A protein of DNA gyrase (4, 11) or an alteration in drug permeation through the outer membrane of the cell (3-5, 12). In this study, the frequency and the selective growth of variants resistant to ciprofloxacin in clinical isolates and in an ATCC strain of P. aeruginosa were studied. Furthermore, the occurrence of cross resistance between ciprofloxacin and other quinolones, beta-lactam antibiotics, and aminoglycosides was studied. MATERIALS AND METHODS Bacterial strains. A total of 12 clinical isolates of P. aeruginosa and P. aeruginosa ATCC 27853 were used. Ten strains were blood isolates (LU I, LU II, LU IV, LU V, LU VII, LU VIII, LU IX, LU X, LU XI, and LU XII), and two strains were sputum isolates (LU IIIA and LU IIIB) from a patient with cystic fibrosis. Antimicrobial agents. Stock solutions of active drug (1,000 ,ug/ml) were prepared from ciprofloxacin (827 ,g/mg; Bayer, Leverkusen, Federal Republic of Germany), which was dissolved in 0.1 M NaOH, and from imipenem (964 pug/mg; Merck Sharp & Dohme Research Laboratories, Rahway, N.J.), which was dissolved in 0.01 M phosphate buffer (pH 7.0). Aqueous solutions of active drug (1,000 ,ug/ml) were prepared from aztreonam (964 ,g/mg; Squibb Institute for Medical Research, Princeton, N.J.), piperacillin (1,010 ,ug/ mg; Lederle Inc., Carolina, P.R.), and tobramycin (942 ,ug/mg; Eli Lilly and Co., Indianapolis, Ind.). *
Corresponding author. 2142
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TABLE 1. MICs of ciprofloxacin and imipenem for 13 strains of P. aeruginosa with small (105 CFU/ml) and large (107 CFU/ml) inoculum MIC (jLg/ml) P. aeruginosa Ciprofloxacin Imipenem _____________ ________ strain 105 CFU/ml 107 CFU/ml 10i CFU/ml 107 CFU/ml
ATCC 27852 LU I LU II LU IIIA LU IIIB LU IV LU V LU VII LU VIII LU IX LU X LU XI LU XII
1 0.25 0.5 1 1 4 4 0.25 0.25 0.25 0.5 0.25 0.25
1 1 1 1 2 8 4 1 1 1 1 0.5 1
2 2 1 2 4 4 8 1 2 1 2 1 1
8 16 8 8 4 8 16 4 8 8 8 2 4
RESULTS MICs. The MICs of ciprofloxacin and imipenem for 12 clinical isolates of P. aeruginosa and P. aeruginosa ATCC 27853 with large inocula (107 CFU/ml) and with small inocula (105 CFU/ml) are shown in Table 1. Resistance patterns of unexposed and ciprofloxacin- or imipenem-exposed bacteria. The susceptibilities of the P. aeruginosa strains to aztreonam, ciprofloxacin, imipenem, piperacillin, and tobramycin were determined by the disk diffusion method (Table 2). Large inocula (107 CFU/ml) of cultures grown without antibiotic and those exposed to 1/2 the MIC of ciprofloxacin or 1/2 the MIC of imipenem for 24 h were tested (Table 2). The mean inhibition zone given by a ciprofloxacin (10-,ug) disk for the 13 strains tested was 9 mm smaller for cells
2143
grown in the presence of 1/2 the MIC of ciprofloxacin than for cells grown without antibiotic (Table 2). For 8 of the strains exposed to 1/2 the MIC of ciprofloxacin, the mean inhibition zone given by an imipenem (10-,ug) disk was 11 mm smaller than for unexposed bacteria (Table 2). For the other five strains tested, there were no differences in inhibition zone diameter given by an imipenem (10-,ug) disk for the unexposed bacteria and for the bacteria exposed to 1/2 the MIC of ciprofloxacin (Table 2). The inhibition zone diameter given by an imipenem (10,ug) disk for all bacterial strains grown in the presence of 1/2 the MIC of imipenem was less than half the diameter of that produced when the bacterial strains were grown without antibiotic, while the zone diameter given by a ciprofloxacin (10-,ug) disk was similar for bacteria exposed to 1/2 the MIC of imipenem and for unexposed bacteria (Table 2). The inhibition zone diameters given by aztreonam, piperacillin, and tobramycin disks were the same for the bacteria from unexposed cultures and for those grown in cultures containing 1/2 the MIC of ciprofloxacin or 1/2 the MIC of imipenem (Table 2). The inhibition zone diameters given by 10-,ug disks of norfloxacin and ofloxacin decreased to the same extent as the zone given by a 10-,ug disk of ciprofloxacin, when cultures exposed to 1/2 the MIC of ciprofloxacin were compared with the corresponding unexposed cultures (see Fig. 3a). These results were reproduced for each strain, and the individual strain differences in each case were concordant with the results given for the differences between the means of the two groups. The margins of the zones of inhibition for the bacteria from the cultures exposed to drug were as distinct and uniform as the zones of inhibition of bacteria from the unexposed cultures. MIC determination of ciprofloxacin, imipenem, aztreonam, piperacillin, and tobramycin for six strains of P. aeruginosa was performed. Large inocula (107 CFU/ml) of
TABLE 2. Susceptibility of 13 strains of P. aeruginosa to ciprofloxacin, imipenem, aztreonam, piperacillin, and tobramycin in cultures incubated without antibiotic, with ciprofloxacin, and with imipenem Zone of inhibition (mm)
Strains and antibiotic disk
Culture exposed to 1/2 the MIC of the following drug:
Unexposed culture
Ciprofloxacin Range
Mean + SD
Range
Imipenem
Mean ± SD
Range
Mean ± SD
8 strains of P. aeruginosa showing ciprofloxacinimipenem cross resistance
Ciprofloxacin Imipenem
29.0-35.5
Aztreonam
21.5-29.0 6.0-30.5
Piperacillin Tobramycin
6.0-29.0 25.5-28.0
32.5 26.2 18.8 18.8 26.9
± 2.1 ± 2.7 ± 10.9
30.8 26.9 27.7 26.6 26.0
± 3.6
± 10.8 ± 0.8
20.0-26.5 11.5-21.0
6.0-34.0 6.0-32.0 25.5-32.0
23.4 15.0 21.4 21.0 29.1
± 2.4 ± 3.2 ± 11.7
± 11.9 ± 2.1
27.0-35.5 10.0-15.5 6.0-30.5 6.0-29.5 24.5-29.0
30.6 11.9 18.8 18.9 26.6
± 3.8 ± 2.1 ± 10.3 ± 10.5 ± 1.6
26.5-36.5 8.5-16.5 25.0-33.0
30.7 11.5 27.3 24.7 25.2
± ± ± ±
5 strains of P. aeruginosa showing no cross resistance
Ciprofloxacin Imipenem
26.0-36.0
Aztreonam Piperacillin Tobramycin
24.5-33.0
24.0-28.5 25.0-29.0 24.5-28.0
+ 2.2 ± 3.3 ± 1.6 ± 1.5
10.5-29.0 21.5-31.5
28.0-33.0 26.5-29.0 26.0-31.5
22.0 27.6 30.4 27.8 28.5
± 7.5 ± ± ± ±
4.1 2.5 1.1 2.2
23.0-26.5 23.5-28.0
± 3.7
3 3| 3.2 1.2 1.8
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. MICs of ciprofloxacin, imipenem, aztreonam, piperacillin, and tobramycin for six strains of P. aeruginosa with small inocula (105 CFU/ml) in cultures incubated without antibiotic or with ciprofloxacin MICa (,ug/ml)
Strains Ciprofloxacin
Imipenem
Aztreonam
Piperacillin
Tobramycin
3 strains of P. aeruginosa showing cross resistance LU I LU VIII LU XI
0.25/4 0.25/2 0.25/4
2/32 2/32 1/32
>32/>32 >32/>32 4/4
>32/>32 >32/>32 4/4
2/2 4/4 1/1
3 strains of P. aeruginosa showing no cross resistance LU IV LU V LU IX
4/16 2/8 0.25/4
4/4 8/8 1/1
4/4 16/16 4/2
8/8 >321>32 4/2
1/1 2/2 2/4
a
The values before the shills show the MICs for unexposed cultures, and the values after the shills show the
these strains were grown without antibiotic and with 1/2 the MIC of ciprofloxacin for 24 h. The results of MIC determination of these bacteria with a small inoculum (105 CFU/ml) are shown in Table 3. The MICs of ciprofloxacin were 4 to 16 times higher for cells grown in the presence of 1/2 the MIC of ciprofloxacin than for cells grown without antibiotic for all strains tested (Table 3). Three of the strains (P. aeruginosa LU I, LU VIII, and LU XI) showed MICs for imipenem which increased 16- to 32-fold after exposure to ciprofloxacin, while three strains
MICs for ciprofloxacin-exposed cultures.
(P. aeruginosa LU IV, LU V, and LU IX) did not show an increase in imipenem MIC (Table 3). The MICs of aztreonam, piperacillin, and tobramycin were almost the same (one dilution step difference) for the bacteria from unexposed cultures and for those grown in 1/2 the MIC of ciprofloxacin. These results (Table 3) are in full agreement with the results obtained with disk diffusion (Table 2). Population analysis. Six strains were chosen for population analysis. Three of the strains (P. aeruginosa LU I, LU VIII, and LU XI) showed ciprofloxacin-imipenem cross resistance
(a)
(b)
0
-1
-1-
-2
-2
0
? 0 c 0
=
-3
-4.
0
U..
-5. -6
-6
-7.
-7. 1/8 1/4
1/2
1
2
4
8
16
1/8'1/4 1/2
1
2
4
8
16
Concentration of imipenem in agarplates expressed as multiples of MIC FIG. 1. Frequencies of variants resistant to different multiples of MIC of imipenem in broth cultures of strains of P. aeruginosa incubated without antibiotic (0), with imipenem (1/2 the MIC) (A), and with ciprofloxacin (1/2 the MIC) (-) for 24 h. (a) Mean frequency + standard deviation of three strains (P. aeruginosa LU I, LU VIII, and LU XI) showing cross resistance. (b) Mean frequency ± standard deviation for three strains (P. aeruginosa LU IV, LU V, and LU IX) showing no cross resistance.
QUINOLONE-IMIPENEM CROSS RESISTANCE IN PSEUDOMONAS AERUGINOSA
VOL. 34, 1990
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(b)
(a) 0*
4 -1
-1-
-2
-2-
-3
-3-
-4
-4
co
0
_ 0 0 2
0
U.
-5
-5.
-e
-6
-7.
-7.
1/8
1/4
1/2
2
1
Concentration
of
4
8
16
1/8
1/4
2
1/2
ciprofloxacin in agarplates expressed
as
4
8
16
multiples of MIC
FIG. 2. Frequencies of variants resistant to different multiples of MIC of ciprofloxacin in broth cultures of strains of P. aeruginosa incubated without antibiotic (0), with imipenem (1/2 the MIC) (A), and with ciprofloxacin (1/2 the MIC) (A) for 24 h. (a) Mean frequency + standard deviation for three strains (P. aeruginosa LU I, LU VIII, and LU XI) showing cross resistance. (b) Mean frequency + standard deviation for three strains (P. aeruginosa LU IV, LU V, and LU IX) showing no cross resistance.
(Fig. la) and three strains (P. aeruginosa LU IV, LU V, and LU IX) did not (Fig. lb) after exposure of large inocula (107 CFU/ml) to 1/2 the MIC of ciprofloxacin in broth, when studied by disk diffusion (Table 2) and MIC determination (Table 3). The frequency of variants resistant to different concentrations of ciprofloxacin or imipenem was calculated by dividing the number of colonies on plates with a particular concentration of ciprofloxacin or imipenem by the number of colonies on plates without antibiotic. Results shown in Fig. la are based on pooled population analysis data of three strains showing cross resistance (P. aeruginosa LU I, LU VIII, and LU XI). The frequency of bacteria resistant to the MIC was about 10' in the unexposed cultures. After exposure of large inocula (107 CFU/ml) to 1/2 the MIC of imipenem or 1/2 the MIC of ciprofloxacin, the frequency curve of variants resistant to imipenem was shifted toward higher concentrations of the drug. The frequencies of bacteria resistant to 4, 8, and 16 times the MIC of imipenem were about 10-1, 10-3, and 10-6, respectively, after exposure to both imipenem and ciprofloxacin. The pooled population analysis data of three strains (P. aeruginosa LU IV, LU V, and LU IX) showing no cross resistance is shown in Fig. lb. The frequency of bacteria resistant to MIC was about 10' in the unexposed cultures. After exposure to 1/2 the MIC of imipenem, the frequency curve was shifted toward higher imipenem concentrations. The frequencies of bacteria resistant to 2, 4, and 8 times the MIC of imipenem were 10-2 to 10-1, 10-4 to 10-3, and 10-5 to 10-4, respectively, while the frequency curve after expo-
sure to 1/2 the MIC of ciprofloxacin was similar to the frequency curve for the unexposed cultures (Fig. lb). The phenomenon of development of ciprofloxacin resistance after exposure to imipenem was not detected in any of these tested strains (Fig. lb and 2b). Stability of resistance. The resistant variants selected and enriched after exposure to 1/2 the MIC of ciprofloxacin in broth were colony purified on agar plates containing ciprofloxacin and imipenem. These colony-purified resistant variants were passaged in antibiotic-free broth for 10 passages. The resistant variants of six strains selected and enriched in 1/2 the MIC of ciprofloxacin and purified on ciprofloxacin agar plates were still, after 10 passages, less susceptible to ciprofloxacin, norfloxacin, and ofloxacin than unexposed bacteria (Fig. 3). The stability of the ciprofloxacin-imipenem cross-resistant variants was studied in three strains (Fig. 4). Bacteria exposed to 1/2 the MIC of ciprofloxacin in broth, which were purified on ciprofloxacin agar plates, were still less susceptible to imipenem than unexposed bacteria after 10 passages (Fig. 4). However, the difference in zone diameter between ciprofloxacin-exposed and unexposed bacteria had decreased during the 10 passages. The ciprofloxacin-imipenem cross-resistant bacteria from the 1/2-MIC ciprofloxacin broth culture were more stable during the 10 passages if they were purified on imipenem agar plates before the passages in antibiotic-free broth (Fig. 4). A proper determination of the growth rates was not made, but the resistant variants caused about the same turbidity as unexposed bacteria during the passage in antibiotic-free
2146
RADBERG
ANTIMICROB. AGENTS CHEMOTHER.
ET AL. 40-
40
(a)
(b)
Imipenem
Imipenem disc
(a)
E
30-
30-
E E
E
C
I
C
0
0
20-
20c
c 0
0
N
N
10-
0
L--j
m
Ciprofloxacin disc
[aALi
I
Nort loxacin disc
Ofloxacin disc
40 (b)
30E
E
1:-
disc
FIG. 4. Mean inhibition zone diameters given by 10-,ug disks of imipenem for broth cultures of three strains of P. aeruginosa incubated without antibiotic (E) and with ciprofloxacin (1/2 the MIC) after purification of resistant variants on ciprofloxacin agar ). Ciprofloxacinplates (DEmI) and on imipenem agar plates ( imipenem cross resistance before passages in antibiotic-free broth (a) and after 10 passages in antibiotic-free broth (b).
0
.Q20C
0
0
N
10A
Ciprofloxacin disc
Norfloxacin disc
Ofloxacin disc
FIG. 3. Mean inhibition zone + standard deviation given by disks of ciprofloxacin, norfloxacin, and ofloxacin for broth cultures of six strains of P. aeruginosa incubated without antibiotic (El) and with ciprofloxacin (1/2 the MIC) after purification of resistant variants on ciprofloxacin agar plates (Em). Quinolone cross resistance before passages in antibiotic-free broth (a) and after 10 passages in antibiotic-free broth (b). 10-lg
broth. The growth rate of the resistant variants seems to be comparable with the growth of the wild-type cells.
DISCUSSION This study shows that ciprofloxacin-resistant variants were selected in large inocula (107 CFU/ml) of P. aeruginosa by subinhibitory concentrations of ciprofloxacin (1/2 the MIC). This has been described in earlier reports (1, 2, 4-7, 10-12). The frequencies of variants resistant to ciprofloxacin in P. aeruginosa at different multiples of the MIC were similar to the frequencies of variants resistant to aminoglycosides (9) and to imipenem (8; also this study). In all P. aeruginosa strains tested, the selected resistant variants showed cross resistance to quinolones, which is in agreement with earlier reports (1, 2, 4-7, 11, 12). Development of resistance to ciprofloxacin in P. aeruginosa after therapy with enoxacin has been described previously (10). These posttherapy isolates showed cross resistance between quinolones and ureidopenicillins (carben-
icillin, mezlocillin, and piperacillin) but no resistance to imipenem (10). In another study, ciprofloxacin-resistant variants of P. aeruginosa showed cross resistance to aminoglycosides and moxalactam (5). The quinolone-resistant variants selected in our study did not show such a cross resistance to piperacillin, tobramycin, or aztreonam. In another study, posttherapy isolates showed resistance to quinolones after ciprofloxacin therapy and no cross resistance to aminoglycosides, moxalactam, and ureidopenicillins (piperacillin and ticarcillin) (3). Similar results were obtained with P. aeruginosa in a study in which resistance to quinolones was obtained after selection with quinolones, but no resistance to aminoglycosides or 3-lactam antibiotics was demonstrated (12). In our study, ciprofloxacin selected and enriched imipenem-resistant variants in a similar way to imipenem in 8 of 13 strains of P. aeruginosa tested. The opposite phenomenon of ciprofloxacin-imipenem cross resistance after exposure to imipenem was not detected. The development of ciprofloxacin-imipenem cross resistance after exposure to ciprofloxacin has been shown earlier in an experimental model of P. aeruginosa infection in mice (7). The variants selected in that study (7) did not show cross resistance to aztreonam, piperacillin, or aminoglycosides, which is in agreement with the results obtained in our study. The mechanism of resistance to ciprofloxacin has been evaluated in several studies (3, 5, 7, 10-12). Most researchers describe two major mechanisms of resistance: changes in the target subunit A of DNA gyrase (4, 11) and alteration in permeability of the bacteria for the quinolones (3-5, 12). When dealing with two chemically different antibiotics, such as quinolones and imipenem, the most likely explanation for decreased susceptibility would be changed drug permeation. To test the stability of quinolone resistance and quinoloneimipenem cross resistance, the bacteria were passaged in antibiotic-free broth. The quinolone-resistant variants were still, after 10 passages, less susceptible to ciprofloxacin, norfloxacin, and ofloxacin than the bacteria from unexposed cultures. The ciprofloxacin-imipenem cross-resistant vari-
VOL. 34, 1990
QUINOLONE-IMIPENEM CROSS RESISTANCE IN PSEUDOMONAS AERUGINOSA
ants purified on ciprofloxacin agar plates were less stable, but after 10 passages these variants were still more resistant than the unexposed bacteria. However, the ciprofloxacinimipenem cross-resistant variants were more stable if they were purified on imipenem agar plates before passages in antibiotic-free broth. This phenomenon can be explained by the facts that the cross-resistant population purified on ciprofloxacin agar plates is not homogeneous and contains a smaller part of the original more susceptible population and that the remaining susceptible bacteria will outgrow the resistant ones during the incubation in antibiotic-free broth. In conclusion, this study indicates that widespread and uncritical use of ciprofloxacin gives a potential risk of development of resistance in P. aeruginosa not only to quinolones but also to another unrelated useful agent, imipenem. Furthermore, the differences between strains in development of resistance to imipenem after exposure to ciprofloxacin might justify in vitro evaluation of this phenomenon in isolates from patients with P. aeruginosa infections.
LITERATURE CITED 1. Barry, A. L., and R. N. Jones. 1984. Cross-resistance among cinoxacin, ciprofloxacin, DJ-6783, enoxacin, nalidixic acid, norfloxacin, and oxolinic acid after in vitro selection of resistant populations. Antimicrob. Agents Chemother. 25:775-777. 2. Culiman, W., M. Stieglitz, B. Baars, and W. Opferkuch. 1985. Comparative evaluation of recently developed quinolone compounds-with a note on the frequency of resistant mutants. Chemotherapy (Basel) 31:19-28. 3. Daikos, G. L., V. T. Lolans, and G. G. Jackson. 1988. Alterations in outer membrane proteins of Pseudomonas aeruginosa associated with selective resistance to quinolones. Antimicrob.
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Agents Chemother. 32:785-787. 3a.Eriksson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. B Suppl. 217:1-90. 4. Kaatz, G. W., and S. M. Seo. 1988. Mechanism of ciprofloxacin resistance in Pseudomonas aeruginosa. J. Infect. Dis. 158:537541. 5. Legakis, N. J., L. S. Tzouvelekis, A. Makris, and H. Kotsifaki. 1989. Outer membrane alterations in multiresistant mutants of Pseudomonas aeruginosa selected by ciprofloxacin. Antimicrob. Agents Chemother. 33:124-127. 6. Limb, D. I., D. J. W. Dabbs, and R. C. Spencer. 1987. In-vitro selection of bacteria resistant to the 4-quinolone agents. J. Antimicrob. Chemother. 19:65-71. 7. Michea-Hamzehpour, R. Auckenthaler, P. Regamey, and J.-C. Pechere. 1987. Resistance occurring after fluoroquinolone therapy of experimental Pseudomonas aeruginosa peritonitis. Antimicrob. Agents Chemother. 31:1803-1808. 8. Nilsson, L., M. Nilsson, and J. Jendle. 1988. Subpopulations of variants resistant to imipenem in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 22:643-649. 9. Nilsson, L., L. Soren, and G. R&dberg. 1987. Frequencies of variants resistant to different aminoglycosides in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 20:255-259. 10. Piddock, L. J. V., W. J. A. Wjnands, and R. Wise. 1987. Quinolone/ureidopenicillin cross-resistance. Lancet ii:907. 11. Robillard, N. J., and A. L. Scarpa. 1988. Genetic and physiological characterization of ciprofloxacin resistance in Pseudomonas aeruginosa PAO. Antimicrob. Agents Chemother. 32: 535-539. 12. Sanders, C. C., W. E. Sanders, Jr., R. V. Goering, and V. Werner. 1984. Selection of multiple antibiotic resistance by quinolones, P-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes. Antimicrob. Agents Chemother. 26:797-801.
