Abstract
Exposure of ciprofloxacin- and cefepime-susceptible Pseudomonas aeruginosa isolates to increasing concentrations of ciprofloxacin selected for ciprofloxacin resistance in 26/27 and cefepime nonsusceptibility in 7/27 isolates. Exposure of the isolates to increasing concentrations of cefepime selected for cefepime nonsusceptibility in 20/27 isolates but not for ciprofloxacin resistance.
Emergence of resistance to one class of antimicrobial agents following exposure to a different class has potential clinical importance. In particular, selection of efflux pump-mediated resistance may compromise not just the selecting agent but others as well. If one agent (or class of agents) is more likely to select for resistance to agents of other classes, reduced use of agents more likely to select such cross-resistance may ultimately prevent emergence of cross-resistance. In this regard, a clinical study has shown that previous quinolone use is a risk factor for development of ventilator-associated pneumonia due to imipenem- or piperacillin-resistant Pseudomonas aeruginosa (10). In a recently published study, ciprofloxacin was identified as a risk factor for acquisition of P. aeruginosa with combined decreased susceptibility to piperacillin, ceftazidime, imipenem, and ciprofloxacin in patients in intensive care units (8).
P. aeruginosa has two mechanisms of resistance to quinolones: modification of DNA gyrase and/or topoisomerase IV and increased expression of efflux pumps. Selection of resistance to nonquinolone antimicrobics by quinolones in P. aeruginosa is likely mediated by increased expression of these efflux pumps. Several resistance-nodulation-division multidrug efflux pumps in P. aeruginosa have been described. MexAB-OprM is expressed in wild-type organisms and overexpressed in mexR mutants. MexCD-OprJ and MexEF-OprN are not expressed in wild-type organisms but are overexpressed in nfxB and mexT mutants, respectively. MexXY-OprM and MexVW-OprM share the same outer membrane protein with MexAB-OprM (4); the former is associated with aminoglycoside efflux, in addition to efflux of other agents. Mutations in the regulatory genes mexR, mexZ, nfxB, and mexT (related to MexAB-OprM, MexXY-OprM, MexCD-OprJ, and MexEF-OprN, respectively) are associated with pump overexpression. Several studies have shown that efflux pumps have a significant effect in conferring resistance to multiple antimicrobial classes when overexpressed and that partial or complete reversal of resistance can occur by efflux pump inhibitors or via efflux pump-associated gene deletion.
The purpose of this study was to compare the frequency of selection of cefepime resistance following ciprofloxacin exposure with that of selection of ciprofloxacin resistance following cefepime exposure in a collection of clinical isolates of P. aeruginosa. Selection of cross-resistance to garenoxacin and tobramycin, following ciprofloxacin exposure, was also examined.
(This work was presented in part at the 12th International Symposium on Infections in the Immunocompromised Host, Bergen, Norway, 2002, and the Swiss Society for Microbiology, Swiss Society for Infectious Diseases, Swiss Society of Tropical Medicine and Parasitology Joint Annual Meeting, Basel, Switzerland, 2003.)
A total of 28 clinical isolates of ciprofloxacin-susceptible (hereafter referred to as wild-type) P. aeruginosa and P. aeruginosa ATCC 25619 were studied. MICs of ciprofloxacin, garenoxacin, cefepime, and tobramycin were determined by broth microdilution according to the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) (7). MIC90 values for the wild-type isolates were 0.5, 2, and 8 μg/ml for ciprofloxacin, garenoxacin, and cefepime, respectively (Table 1). Using the proposed garenoxacin susceptibility breakpoint of ≤4 μg/ml (2), all wild-type isolates would be considered garenoxacin susceptible. One wild-type isolate was intermediate and another resistant to cefepime. One wild-type isolate was resistant to tobramycin.
TABLE 1.
MIC values and susceptibility for 29 P. aeruginosa isolates
| Isolate group and drug | MIC90 (μg/ml) | MIC range (μg/ml) | No. of nonsusceptible isolates |
|---|---|---|---|
| Wild type | |||
| Ciprofloxacin | 0.5 | ≤0.03-0.5 | 0 |
| Garenoxacin | 2 | ≤0.125-4 | 0a |
| Cefepime | 8 | 0.5-32 | 2 |
| Tobramycin | 1 | ≤0.125->128 | 1 |
| Ciprofloxacin exposed | |||
| Ciprofloxacin | 64 | 2-64 | 29 |
| Garenoxacin | 128 | 8->128 | 29a |
| Cefepime | 16 | 1-128 | 8 |
| Tobramycin | 1 | ≤0.125-64 | 1 |
| Cefepime exposed | |||
| Cefepime | 64 | 1-128 | 22 |
| Ciprofloxacin | 1 | ≤0.125-2 | 1 |
Using proposed garenoxacin susceptibility breakpoint of ≤4 μg/ml (2).
For in vitro exposure to ciprofloxacin, isolates were grown in 10 ml of Mueller-Hinton broth containing 0.125 μg/ml of ciprofloxacin for 24 h at 35°C. Following incubation, the culture was concentrated by centrifugation and the bacterial pellet was resuspended in 10 ml of broth containing twice the previous concentration of ciprofloxacin and incubated for 24 h at 35°C. This procedure was repeated daily for seven consecutive days (to a final ciprofloxacin concentration of 8 μg/ml). Thereafter the same procedure was repeated using a fixed 8 μg/ml concentration of ciprofloxacin for an additional 3 days. The isolates were then grown on sheep blood agar plates overnight at 35°C in 5% CO2, and MICs of ciprofloxacin, garenoxacin, cefepime, and tobramycin were again determined.
The MIC90 values for the ciprofloxacin-exposed isolates were 64, 128, and 16 μg/ml for ciprofloxacin, garenoxacin, and cefepime, respectively. After exposure to ciprofloxacin all isolates developed nonsusceptibility to ciprofloxacin; all isolates developed resistance to garenoxacin (using the proposed garenoxacin susceptibility breakpoint of ≤4 μg/ml) (2); seven isolates developed nonsusceptibility (MIC ≥ 16 μg/ml) to cefepime. One of the wild-type isolates which had a cefepime MIC of 16 μg/ml showed an eightfold decrease in susceptibility following exposure to ciprofloxacin; decreases in ceftazidime MICs of nfxC mutants have been previously reported (6). None of the ciprofloxacin-exposed isolates developed resistance to tobramycin.
DNA was extracted from wild-type and ciprofloxacin-exposed isolates (DNA Stat-60; Tel-test B, Inc., Friendswood, TX), and quinolone resistance-determining regions were amplified by PCR using previously published primers (1). All reactions were performed in a final volume of 50 μl with 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems Division, Foster City, CA). The cycling parameters consisted of denaturation at 95°C for 10 minutes followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 52°C, and 60 seconds at 72°C. Bidirectional sequencing of amplified product was performed as previously described (9). Sequence data were analyzed for quinolone resistance-determining region mutations determined using Sequencher 3.0 (Gene Codes Corporation, Ann Arbor, MI).
No GyrA, GyrB, or ParE mutations were noted in the wild-type strains, although three wild-type isolates were not tested for GyrB mutations and two wild-type isolates were not tested for ParE mutations. Quinolone resistance-determining region mutations in the ciprofloxacin-exposed isolates were found in all except one isolate (Table 2). One isolate had a deletion in gyrB and another an insertion in parE. The remaining mutations were point mutations.
TABLE 2.
Quinolone resistance-determining region mutations detected in ciprofloxacin-exposed P. aeruginosa isolatesa
| Isolate | Mutation
|
||
|---|---|---|---|
| GyrA | GyrB | ParE | |
| ATCC 25619 | Thr83Ile | —b | Ala473Val |
| 1 | — | Ser468Phe | — |
| 2 | Thr83Ile | — | — |
| 3 | Thr83Ile | — | Val455 insertion,c Ala473Val |
| 4 | Asp87Asn | — | — |
| 5 | Asp87His | — | Ala473Val |
| 6 | Thr83Ile | — | — |
| 7 | Asp87His | — | Ala473Val |
| 8 | Asp72Gly | Ala436Thr | — |
| 9 | Asp87Tyr | — | — |
| 10 | — | — | Ala473Val |
| 11 | Thr83Ile | — | Ala473Val |
| 12 | Thr83Ile | — | — |
| 13 | — | Ser468Tyr | — |
| 14 | Asp87Gly | — | — |
| 15 | Asp72Gly | — | — |
| 16 | Ala67Ser, Asp87Tyr | — | Pro394Arg |
| 17 | Thr83Ile | — | — |
| 18 | Thr83Ile | — | Ala473Val |
| 19 | — | Ser468Phe | — |
| 20 | — | Ser468Tyr | — |
| 21 | Thr83Ile | — | — |
| 22 | — | Glu470Asp | Not tested |
| 23 | — | Ser468 deletion | — |
| 24 | Thr83Ile | — | — |
| 25 | — | — | — |
| 26 | Asp87Gly | — | |
| 27 | — | Ser468Tyr | — |
| 28 | Asp87Tyr | — | Ala473Val |
No ParC quinolone resistance-determining region mutations were identified in the ciprofloxacin-exposed P. aeruginosa isolates.
—, no mutation identified.
One extra valine was present at the indicated position.
The same set of wild-type isolates was exposed to cefepime to determine whether exposure to cefepime would select for ciprofloxacin nonsusceptibility. The isolates were grown in increasing concentrations of cefepime (0.25 to 64 μg/ml) in Mueller-Hinton broth over 10 days using a method analogous to that used for ciprofloxacin exposure. Using this method, 11 of the 27 cefepime-susceptible isolates became resistant to cefepime and 9 became “intermediate” to cefepime, but none developed resistance to ciprofloxacin or tobramycin (Table 1).
We have shown that, using the technique described, in vitro exposure to ciprofloxacin selects for ciprofloxacin and cefepime nonsusceptibility whereas in vitro exposure to cefepime selects for cefepime nonsusceptibility but not ciprofloxacin resistance. Upregulation of efflux pumps MexAB-OprM and/or MexCD-OprJ (3), selected by exposure to ciprofloxacin, may have simultaneously compromised susceptibility to ciprofloxacin (and to other quinolones, notably garenoxacin, in this study) as well as to cefepime. Tobramycin susceptibility was not substantially impacted in the ciprofloxacin-exposed isolates, suggesting that MexXY-OprM was not affected. In addition to enhancing antimicrobial activity, efflux pump inhibitors have been shown to reduce the frequency of emergence of quinolone-resistant P. aeruginosa mutants (5); whether use of efflux pump inhibitors would likewise reduce the emergence of multidrug resistance remains to be shown but is possible. Besides efflux pump upregulation, other mechanisms of cefepime resistance may additionally or alternatively have been selected by cefepime exposure.
In summary, using the technique described, we have shown that in vitro exposure of P. aeruginosa to ciprofloxacin selects for ciprofloxacin and cefepime nonsusceptibility whereas in vitro exposure to cefepime selects for cefepime but not ciprofloxacin resistance. Our findings may have clinical relevance in terms of the impact of selection of individual antimicrobial agents on the selection of antimicrobial cross-resistance.
REFERENCES
- 1.Akasaka, T., M. Tanaka, A. Yamaguchi, and K. Sato. 2001. Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrob. Agents Chemother. 45:2263-2268. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Fung-Tomc, J. C., B. Minassian, B. Kolek, E. Huczko, L. Aleksunes, T. Stickle, T. Washo, E. Gradelski, L. Valera, and D. P. Bonner. 2000. Antibacterial spectrum of a novel des-fluoro(6) quinolone, BMS-284756. Antimicrob. Agents Chemother. 44:3351-3356. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Lee, A., W. Mao, M. S. Warren, A. Mistry, K. Hoshino, R. Okumura, H. Ishida, and O. Lomovskaya. 2000. Interplay between efflux pumps may provide either additive or multiplicative effects on drug resistance. J. Bacteriol. 182:3142-3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Li, Y., T. Mima, Y. Komori, Y. Morita, T. Kuroda, T. Mizushima, and T. Tsuchiya. 2003. A new member of the tripartite multidrug efflux pumps, MexVW-OprM, in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 52:572-575. [DOI] [PubMed] [Google Scholar]
- 5.Lomovskaya, O., A. Lee, K. Hoshino, H. Ishida, A. Mistry, M. S. Warren, E. Boyer, S. Chamberland, and V. J. Lee. 1999. Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 43:1340-1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Maseda, H., H. Yoneyama, and T. Nakae. 2000. Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:658-664. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—sixth edition. NCCLS document M07-A6. National Committee for Clinical Laboratory Standards, Wayne, PA.
- 8.Paramythiotou, E., J. C. Lucet, J. F. Timsit, D. Vanjak, C. Paugam-Burtz, J. L. Trouillet, S. Belloc, N. Kassis, A. Karabinis, and A. Andremont. 2004. Acquisition of multidrug-resistant Pseudomonas aeruginosa in patients in intensive care units: role of antibiotics with antipseudomonal activity. Clin. Infect. Dis. 38:670-677. [DOI] [PubMed] [Google Scholar]
- 9.Patel, R., K. E. Piper, M. S. Rouse, J. M. Steckelberg, J. R. Uhl, P. Kohner, M. K. Hopkins, F. R. Cockerill, and B. C. Kline. 1998. Determination of 16S rRNA sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates. J. Clin. Microbiol. 36:3399-3407. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Trouillet, J. L., A. Vuagnat, A. Combes, N. Kassis, J. Chastre, and C. Gibert. 2002. Pseudomonas aeruginosa ventilator-associated pneumonia: comparison of episodes due to piperacillin-resistant versus piperacillin-susceptible organisms. Clin. Infect. Dis. 34:1047-1054. [DOI] [PubMed] [Google Scholar]