Most strains of Escherichia coli are considered part of the intestinal microflora in clinically normal chickens. However, some strains of E coli are associated with various infections, such as colibacillosis, air sacculitis, and cellulitis.1 Because of their low toxicity and relatively broad-spectrum activity, fluoroquinolones are commonly used in treatment of infections in humans and other animals.2 Enrofloxacin is one of the antimicrobials typically used to treat colibacillosis in chickens in China.3,4 Unfortunately, because of frequent use, misuse, and unnecessary and preventive uses of antimicrobials,5 E coli organisms have developed resistance to fluoroquinolone drugs worldwide.6-8
Resistance to fluoroquinolones in gram-negative bacteria is thought to develop in a stepwise manner through accumulation of specific point mutations in the genes encoding enzymes that target fluoroquinolones, which eventually renders the bacteria resistant to those drugs. The enzymes DNA gyrase A and topoisomerase IV, which cause a variety of amino acid exchanges in so-called mutational hot spots (ie, QRDRs),9-11 are involved in the development of bacterial resistance to enrofloxacin; bacteria with such mutations are amplified by consecutive steps of clonal enrichment under selective drug pressure during drug treatment.12-14 If therapeutic strategies are not developed specifically to prevent the outgrowth of resistant mutants, bacterial diseases are likely to become untreatable with enrofloxacin. To limit the emergence of resistant bacterial strains and provide an effective dose range for enrofloxacin, we believe that it is necessary to implement new dosing strategies for fluoroquinolones. A new concept, which is based on the in vitro MPC of a drug, appears to provide a strategy for prevention of selective enrichment of resistant variants in vivo by use of more appropriate drug dosages.14,15 The MPC concept is based on the theory that there is a concentration range in which the drug can prevent the growth of resistant mutants.15 The lower limit of this range is approximated by the MIC. The upper limit of this range is the concentration that inhibits growth of most susceptible cells and all organisms with a single-step mutant.14,15 Bacterial cells must acquire 2 or more resistant mutations to enable them to grow in the presence of fluoroquinolone concentrations that are greater than the upper limit of that range.14-16 This antimicrobial concentration is referred to as the MPC.14-16 Thus, if the serum drug concentration is greater than the MPC, the drug will be more effective in treatment of infection and the selection of mutants during antimicrobial treatment can be minimized.11 The MPC is calculated experimentally in vitro and is the lowest drug concentration that allows no colony growth when > 1010 cells are applied to drug-containing agar plates. The agar plates contain > 1010 cells because infections rarely involve > 1010 organisms.16
The purpose of the study reported here was to examine the development of enrofloxacin resistance of E coli isolates obtained from chickens by determining MPCs and comparing those MPCs with the maximum serum drug concentrations in chickens attained after administration of enrofloxacin in doses recommended for treatment of E coli infections. The mutation frequencies of the isolates were also assessed, and the QRDRs of gyrA and parC genes of the selected isolates were sequenced to genetically characterize the isolates.
Materials and Methods
Bacterial strains and growth conditions—Fifteen E coli isolates that represented a wide variety of phenotypes with different levels of susceptibility to enrofloxacin were originally obtained from chickens on several chicken farms. Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213) were used as quality-control strains for susceptibility testing. The liquid and solid medium used for bacterial growth were Mueller-Hinton brotha and Mueller-Hinton agar,a respectively. Strains were incubated at 37°C.
Antimicrobial preparations—Prior to use, enrofloxacinb was dissolved in 0.1M NaOH at a concentration of 2.56 mg/mL before incorporation into Mueller-Hinton agar to the final concentrations used in the MIC and MPC assays.
Measurement of MIC and MPC—Minimal inhibition concentrations were determined via agar dilution and broth dilution methods with inocula of 5 × 105 CFUs/mL, according to guidelines of the CLSI (formerly the National Committee for Clinical Laboratory Standards).17 The performance was controlled by testing in parallel the recommended CLSI quality-control reference strains.
The MPC was determined as described by Zhao and Drlica.13 Briefly, the tested microorganisms were cultured in Mueller-Hinton broth and incubated for 24 hours. The suspension was centrifuged (4,000 × g for 10 minutes), resuspended in Mueller-Hinton broth to yield a concentration of 1010 CFUs/mL, and then plated onto each of 10 plates supplemented with a dilution of enrofloxacin (1X to 128X MIC). Plates were incubated at 37°C for 24 hours; the colonies were counted, and the plates were incubated again for an additional 24 hours. The MPC was recorded as the lowest concentration of antimicrobial that prevented the emergence of any mutants after 24 and 48 hours. For each strain, MPC was determined in at least 3 independent experiments.
Determination of mutation frequencies—Bacteria were grown at 37°C in Mueller-Hinton broth until they reached the logarithmic phase of growth. Aliquots of broth were spread onto selective and nonselective Mueller-Hinton agar plates and incubated at 37°C. After 24 hours, mutation rates were calculated from the number of resistant bacteria divided by the number of bacterial cells on the agar plates (determined in triplicate from each of 3 independent cultures). With 6 agar plates, mutations could potentially be detected at frequencies of 1 to 1011 bacteria. The selection concentrations were 4X MIC for each isolate.
PCR amplification and DNA sequencing of gyrA and parC genes—The QRDRs of gyrA and parC genes were sequenced prior to the onset of the study to genetically characterize the isolates. Chromosomal DNA was prepared from the isolates by use of a genomic DNA kit.c The QRDRs of gyrA and parC were amplified by PCR procedures involving previously published primers.7 Each PCR reaction was performed in a 50-ML volume consisting of 0.25mM of each deoxyribonucleotide, 1.5mM MgCl2, 1 unit of EX Taq DNA polymerase, and 50 pmol of each primer. The temperature profile was as follows: 94°C for 10 minutes, 30 cycles at 94°C for 30 seconds, 55°C for 45 seconds, 72°C for 45 seconds, and a final cycle of 72°C for 7 minutes. Predicted polypeptide products were analyzed for amino acid changes by comparison with wild-type E coli gyrA (GenBank accession No. AE000312) and parC (GenBank accession No. AE000384).
Results
MICs, MPCs, MPC:MIC ratios, and mutation frequencies—To determine the MIC for enrofloxacin, 15 E coli isolates from chickens were analyzed by use of an agar dilution method (Table 1). The MICs of 6 isolates were high; these values were equal to or greater than the CLSI breakpoint for enrofloxacin resistance (4 μg/mL). The MICs of 8 isolates were equal to or less than the CLSI breakpoint for susceptibility (1 μg/mL). Only 1 E coli isolate had intermediate resistance (MIC, 2 μg/mL).
Values of MIC, MPC, and MPC:MIC ratio; mutation frequency for enrofloxacin; and specific mutations in GyrA and ParC of 15 Escherichia coli isolates obtained from chickens.
| Isolate | MIC (μg/mL) | MPC* (μg/mL) | MPC:MIC ratio | Mutation frequency† | Mutations in QRDRs | |
|---|---|---|---|---|---|---|
| GyrA | ParC | |||||
| CE0125 | 0.031 | 0.062 | 2 | 1.73 ± 0.70 × 10.8-8 | NC | NC |
| CE6542 | 0.004 | 0.016 | 4 | 2.10 ± 0.75 × 10.8-8 | NC | NC |
| CE2132 | 0.125 | 2 | 16 | 2.56 ± 1.03 × 10.7-7 | Asp-87→Gly | NC |
| CE9841 | 0.125 | 0.5 | 4 | 2.17 ± 0.87 × 10.8-8 | Asp-87→Asn | NC |
| CE3213 | 0.5 | 1 | 2 | 1.34 ± 0.53 × 10.8-8 | Asp-87→Asn | NC |
| CE1246 | 0.25 | 4 | 16 | 2.12 ± 0.45 × 10.7-7 | Asp-87→Tyr | NC |
| CE7623 | 0.5 | 1 | 2 | 1.77 ± 0.51 × 10.8-8 | Ser-83→Leu | NC |
| CE6523 | 1 | 4 | 4 | 2.34 ± 0.62 × 10.8-8 | Ser-83→Leu | NC |
| CE5513 | 2 | 4 | 2 | 2.21 ± 0.29 × 10.8-8 | Ser-83→Leu | NC |
| CE3543 | 4 | 8 | 2 | 3.19 ± 0.56 × 10.9-8 | Ser-83→Ala | NC |
| CE8761 | 8 | 16 | 2 | 2.81 ± 1.03 × 10.9-8 | Ser-83→Leu | NC |
| CE4543 | 16 | 32 | 2 | < 10.11-11 | Ser-83→Leu + Asp-87→Asn | Ser-80→Ile |
| CE3523 | 128 | ND | ND | < 10.11-11 | Ser-83→Leu + Asp-87→Tyr | Ser-80→Ile |
| CE1567 | 256 | ND | ND | < 10.11-11 | Ser-83→Leu + Asp-87→Asn | Glu-84→Lys |
| CE6758 | 256 | ND | ND | < 10.11-11 | Ser-83→Leu + Asp-87→Asn | Ser-80→Ile |
Concentration of antimicrobial required to prevent growth of any mutants from 1010 CFUs.
Values are mean ± SD.
NC = No change. Asp = Aspartic acid. Gly = Glycine. Asn = Asparagine. Tyr = Tyrosine. Ser = Serine.
Leu = Leucine. Ala = Alanine. Ile = Isoleucine. ND = Not determined. Glu = Glutamic acid. Lys = Lysine.
The MPCs for the 8 enrofloxacin-susceptible isolates ranged from 0.016 to 4 μg/mL (Table 1). Two isolates without mutations in GyrA and ParC had MPCs of 0.016 and 0.062 μg/mL and MPC:MIC ratios of 2 and 4. Among other susceptible isolates with a point mutation in gyrA, 4 had MPCs that ranged from 0.5 to 2 μg/mL, but 2 had MPCs of 4 μg/mL. The latter MPC value exceeds the Cmax of enrofloxacin (2.44 μg/mL) in chickens given 10 mg of enrofloxacin/kg orally18 and cannot be achieved with currently approved dosing practices. One isolate with intermediate resistance and the 6 resistant isolates had MPCs ≥ 4 μg/mL. The mutation frequency for E coli exposed to enrofloxacin ranged from approximately 10−7 to approximately 10−11. The frequency at which mutants were selected decreased as the antimicrobial concentration approached or exceeded the MPC values.
GyrA and ParC point mutations in E coli isolates—Fifteen E coli isolates were tested for amino acid substitutions in GyrA and ParC (Table 1). Six enrofloxacin-susceptible E coli isolates (MIC range, 0.125 to 1 μg/mL) had a mutation at position 83 or 87 in GyrA. The isolate with intermediate resistance (MIC, 2 μg/mL) and 6 resistant isolates (MIC range, 4 to > 256 μg/mL) had a mutation at GyrA or double mutations in GyrA and single mutation in ParC. The isolates with double mutations in GyrA and a single mutation in ParC had higher MICs than the others. Because 1 isolate (MIC, 16 μg/mL) had an MPC value of 32 Mg/ mL, which is several fold higher than the Cmax of enrofloxacin in chickens,18 MPCs and MPC:MIC ratios of the other resistant isolates were not determined.
Discussion
Resistance to antimicrobials has been recognized as an emerging issue worldwide in both human and veterinary medicine.19,20 To minimize or prevent development of antimicrobial resistance, it is important to optimize drug dosage regimens to achieve a desired therapeutic effect without promoting resistance. Assessment of MPCs is intended to provide information regarding the potential for the selection and enrichment of microbial mutants, on the basis of which appropriate changes in dosage regimens can be made.21 In the present study, all E coli isolates obtained from chickens that had MICs greater than the CLSI breakpoint for enrofloxacin susceptibility had MPCs ≥ 8 μg/mL. For these isolates, attaining enrofloxacin concentrations greater than their MPCs at the site of infection via monotherapy is impossible because the MPCs exceed the Cmax of enrofloxacin (2.44 μg/mL18) in chickens that is achievable with currently approved dosing procedures. For the 8 isolates that had MICs less than the breakpoint for enrofloxacin susceptibility, MPC values ranged from 0.016 to 4 Mg/ mL. For most of those susceptible isolates, it would be possible to attain concentrations of enrofloxacin greater than their MPCs at the site of infection with currently approved dosing procedures.18 However, there was an exception among the susceptible isolates. Because of high mutation frequency, 2 enrofloxacin-susceptible isolates (CE1246 and CE6523 that had MICs of 0.25 and 1 μg/mL, respectively) each had an MPC value of 4 μg/mL. The MPCs of those 2 isolates exceed the Cmax of enrofloxacin in chickens. Drlica et al22 have suggested that there was low correlation between MIC and MPC, and the results of the present study have provided confirmation of that assertion.
Prior to MPC assessments, we sequenced the QRDRs of gyrA and parC to characterize genetic changes in E coli isolates. For E coli isolates, gyrase is the primary quinolone target, and first-step resistance mutations often occur in GyrA. The ParC mutations are generated only at drug concentrations that are higher than those required for gyrase mutation. Topoisomerase IV is considered to be a secondary target.23 In our study, 9 isolates with only 1 point mutation in GyrA had relatively low MPCs (0.5 to 16 μg/mL). In 4 isolates that had double mutations in GyrA and a single point mutation in ParC, the MPCs of enrofloxacin increased to values that were dramatically higher than the Cmax of enrofloxacin in chickens. Drlica et al24 have suggested that combination antimicrobial treatment can be used to limit enrichment of resistant mutants when antimicrobial concentrations greater than the MPC cannot be maintained through administration of the maximum recommended drug dosage. In isolates of E coli with high MPCs, especially those containing a mutation in both GyrA and ParC, treatments with combinations of antimicrobials must be adopted. Novel quinolones such as those with a methoxy group at the C-8 position (eg, moxifloxacin and gatifloxacin) have enhanced activity against gram-positive bacteria and anaerobes as well as a reduced rate of selection for resistance. It is believed that the latter, in part, is attributable to DNA topoisomerase IV and DNA gyrase acting as targets for inhibition. Thus, C-8-methoxy fluoroquinolones inhibit gradual stepwise selection of resistance in bacteria as well as the 1-step acquisition of high-level resistance.24 However, these new quinolones have not yet been used clinically in veterinary patients.
ABBREVIATIONS
| QRDR | Quinolone resistance–determining region |
| MPC | Mutant-prevention concentration |
| MIC | Minimal inhibition concentration |
| ATCC | American Type Culture Collection |
| CLSI | Clinical and Laboratory Standards Institute |
| Cmax | Maximum plasma concentration |
Hangzhou Tianhe Microorganism Reagent Co Ltd, Hangzhou, China.
Bayer, Shawnee Mission, Kan.
Bacterial genomic DNA mini-prep kit, V-gene Biotechnology Ltd, Hongzhou, China.
References
- 1↑
Gross WB. Colibacillosis. In:Calnek BW, ed.Diseases of poultry. 9th ed. Ames, Iowa: Iowa State University Press, 1991;138–144.
- 3
Cui HB, Zhu H. Fluoroquinolone susceptibility test in Escherichia coli from chickens. Chin J Vet Sci 2002;38:44–45.
- 4
Fang B, Zeng Z, Feng Q, et al. Efficacy of enrofloxacin against experimentally induced colibacillosis and staphylococcosis in chickens. Chin J Vet Sci 1997;17:157–160.
- 5↑
Solomon DH, VanHouten L, Glynn RJ, et al. Academic detailing to improve use of broad-spectrum antibiotics at an academic medical center. Arch Intern Med 2001;161:1897–902.
- 6
Norbert L, Jutta SH, Theresia K, et al. Characterization of clinical isolates of Escherichia coli showing high levels of fluoroquinolone resistance. J Clin Microbiol 1996;34:597–602.
- 7↑
Everett MJ, Jin YF, Ricci V, et al. Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob Agents Chemother 1996;40:2380–2386.
- 8
Wang H, Joann LD, Chen M, et al. Genetic characterization of highly fluoroquinolone-resistant clinical Escherichia coli strains from China: role of AcrR mutations. Antimicrob Agents Chemother 2001;45:1515–1521.
- 9
Piddock LJV. Mechanisms of fluoroquinolone resistance: an update 1994–1998. Drugs 1999;58:11–18.
- 10
Heisig P. Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli. Antimicrob Agents Chemother 1996;40:879–885.
- 11↑
Oram M, Fisher LM. 4-Quinolone resistance mutations in the DNA gyrase of Escherichia coli clinical isolates identified by using the polymerase chain reaction. Antimicrob Agents Chemother 1991;35:387–389.
- 12
Zhao X, Xu C, Domagala J, et al. DNA topoisomerase targets of the fluoroquinolones: a strategy for avoiding bacterial resistance. Proc Natl Acad Sci U S A 1997;94:13991–13996.
- 13↑
Zhao X, Drlica K. Restricting the selection of antibiotic resistant mutants: a general strategy derived from fluoroquinolone studies. Clinic Infect Dis 2001;33 (suppl 3):S147–S156.
- 14
Drlica K. The mutant selection window and antimicrobial resistance. J Antimicrob Chemother 2003;52:11–17.
- 16↑
Dong Y, Zhao X, Kreisworth BN, et al. Mutant prevention concentration as a measure of antibiotic potency: studies with clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2000;44:2581–2584.
- 17↑
National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. NCCLS M31-A2. 2nd ed. Wayne, Pa: National Committee for Clinical Laboratory Standards, 2003.
- 18↑
Anadon A, Martinez-Larranaga MR, Diaz MJ, et al. Pharmacokinetics and residues of enrofloxacin in chickens. Am J Vet Res 1995;56:501–506.
- 19
Neu HC. The crisis in antibiotic resistance. Science 1992;257:1064–1073.
- 20
Witte W. Medical consequences of antibiotic use in agriculture. Science 1998;279:996–997.
- 21↑
Epstein BJ, Gums JG, Drlica K. The changing face of antibiotic prescribing: the mutant selection window. Ann Pharmacother 2004;38:1675–1682.
- 22↑
Drlica K, Zhao X, Blondeau JM, et al. Low correlation between MIC and mutant prevention concentration. Antimicrob Agents Chemother 2006;50:403–404.
- 23↑
Bagel S, Hullen V, Wiedemann B, et al. Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli. Antimicrob Agents Chemother 1999;43:868–875.
- 24↑
Hawkey PM. Mechanisms of quinolone action and microbial response. J Antimicrob Chemother 2003;51 (suppl 1):29–35.
