• View in gallery

    Schematic diagram of an IVPM used to evaluate the impact of oxytetracycline exposure on horizontal transfer of an antimicrobial resistance plasmid from donor (Salmonella enterica subsp enterica serovar Typhimurium) to recipient (Escherichia coli) bacteria.

  • View in gallery

    Protein binding in BHI broth for various concentrations of oxytetracycline.

  • View in gallery

    Concentrations of Salmonella Typhimurium (donor bacteria) cultured for up to 36 hours under various conditions as follows: starting inoculum (horizontally striped bar), LC-LHL control replicate (diagonally striped bars), LC-LHL regimen (black bars), HC-SHL control replicate (white bars with black dots), and HC-SHL regimen (white bars).

  • View in gallery

    Concentrations of transconjugant bacteria (recipient E coli) cultured for up to 36 hours under various conditions as follows: LC-LHL control replicate (diagonally striped bars), LC-LHL regimen (black bars), HC-SHL control replicate (white bars with black dots), and HC-SHL regimen (white bars).

  • 1.

    Spellberg B. Dr. William H. Stewart: mistaken or maligned? (lett) Clin Infect Dis 2008; 47: 294.

  • 2.

    Phillips I, Casewell M, Cox T, et al. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J Antimicrob Chemother 2004; 53: 2852.

    • Search Google Scholar
    • Export Citation
  • 3.

    Tenover FC. Development and spread of bacterial resistance to antimicrobial agents: an overview. Clin Infect Dis 2001; 33 (suppl 3): 108115.

    • Search Google Scholar
    • Export Citation
  • 4.

    Blaser JM, Stone BB, Groner MC, et al. Comparative study with enoxacin and netilmicin in a pharmacodynamic model to determine importance of ratio of antibiotic peak concentration to MIC for bactericidal activity and emergence of resistance. Antimicrob Agents Chemother 1987; 3: 10541060.

    • Search Google Scholar
    • Export Citation
  • 5.

    Jumbe N, Louie A, Leary R, et al. Application of a mathematical model to prevent in vivo amplification of antibiotic-resistant bacterial populations during therapy. J Clin Invest 2003; 112: 275285.

    • Search Google Scholar
    • Export Citation
  • 6.

    Thomas JK, Forrest A, Bhavnani SM, et al. Pharmacodynamic evaluation of factors associated with the development of bacterial resistance in acutely ill patients during therapy. Antimicrob Agents Chemother 1998; 42: 521527.

    • Search Google Scholar
    • Export Citation
  • 7.

    AVMA. Judicious therapeutic use of antimicrobials. Available at: www.avma.org/issues/policy/jtua.asp. Accessed Aug 27, 2009.

  • 8.

    Courvalin P. Can pharmacokinetic-pharmacodynamic parameters provide dosing regimens that are less vulnerable to resistance? Clin Microbiol Infect 2008; 14: 989994.

    • Search Google Scholar
    • Export Citation
  • 9.

    Firsov AA, Smirnova MV, Lubenko IY, et al. Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to daptomycin and vancomycin in an in vitro dynamic model. J Antimicrob Chemother 2006; 58: 11851192.

    • Search Google Scholar
    • Export Citation
  • 10.

    Olofsson SK, Marcusson LL, Lindgren PK, et al. Selection of ciprofloxacin resistance in Escherichia coli in an in vitro kinetic model: relation between drug exposure and mutant prevention concentration. J Antimicrob Chemother 2006; 57: 11161121.

    • Search Google Scholar
    • Export Citation
  • 11.

    Zhao X, Drlica K. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. J Infect Dis 2001; 185: 561565.

    • Search Google Scholar
    • Export Citation
  • 12.

    Schwarz S, Chaslus-Dancla E. Use of antimicrobials in veterinary medicine and mechanisms of resistance. Vet Res 2001; 32: 201225.

  • 13.

    Doucet-Populaire F, Trieu-Cuot P, Dosbaa I, et al. Inducible transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes in the digestive tracts of gnotobiotic mice. Antimicrob Agents Chemother 1991; 35: 185187.

    • Search Google Scholar
    • Export Citation
  • 14.

    Giles WP, Benson AK, Olson ME, et al. DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones. Antimicrob Agents Chemother 2004; 48: 28452852.

    • Search Google Scholar
    • Export Citation
  • 15.

    Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals: approved standard. M31A2. 2nd ed. Wayne, NJ: Clinical and Laboratory Standards Institute, 2002.

    • Search Google Scholar
    • Export Citation
  • 16.

    Grasso S, Meinardi G, DeCarneri I, et al. New in vitro model to study the effect of antibiotic concentration and rate of elimination on antibacterial activity. Antimicrob Agents Chemother 1978; 13: 570576.

    • Search Google Scholar
    • Export Citation
  • 17.

    Murakawa T, Sakamoto H, Hirose T, et al. New in vitro model for evaluating bactericidal efficacy of antibiotics. Antimicrob Agents Chemother 1980; 18: 377381.

    • Search Google Scholar
    • Export Citation
  • 18.

    Sanfilippo A, Morvillo E. An experimental model for the study of the antibacterial activity of the sulfonamides. Chemotherapy 1968; 13: 5460.

    • Search Google Scholar
    • Export Citation
  • 19.

    Pijpers A, Schoevers EJ, Van Gogh H, et al. The pharmacokinetics of oxytetracycline following intravenous administration in healthy and diseased pigs. J Vet Pharmacol Ther 1990; 13: 320326.

    • Search Google Scholar
    • Export Citation
  • 20.

    El Korchi G, Prats C, Arboix M, et al. Disposition of oxytetracycline in pigs after i.m. administration of two long-acting formulations. J Vet Pharmacol Ther 2001; 24: 247250.

    • Search Google Scholar
    • Export Citation
  • 21.

    Blondeau JM, Hansen G, Metzler K, et al. The role of PK/PD parameters to avoid selection and increase of resistance: mutant prevention concentration. J Chemother 2004; 16 (suppl 3): 119.

    • Search Google Scholar
    • Export Citation
  • 22.

    MacGowan AP, Bowker KE, Noel AR. Pharmacodynamics of the antibacterial effect and emergence of resistance to tomopenem, formerly RO4908463/CS-023, in an in vitro pharmacokinetic model of Staphylococcus aureus infection. Antimicrob Agents Chemother 2008; 52: 14011406.

    • Search Google Scholar
    • Export Citation
  • 23.

    Ryback MJ. Pharmacodynamics: relation to antimicrobial resistance. Am J Infect Control 2006; 34: 3845.

  • 24.

    Shoemaker NB, Vlamakis H, Hayes K, et al. Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon. Appl Environ Microb 2001; 67: 561568.

    • Search Google Scholar
    • Export Citation
  • 25.

    Schjørring S, Struve C, Krogfelt KA. Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine. J Antimicrob Chemother 2008; 62: 10861093.

    • Search Google Scholar
    • Export Citation
  • 26.

    Showsh SA, Andrews RE Jr. Tetracycline enhances Tn916-mediated conjugal transfer. Plasmid 1992; 28: 213224.

  • 27.

    Torres OR, Korman RZ, Zahler SA, et al. The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis. Mol Gen Genet 1991; 225: 395400.

    • Search Google Scholar
    • Export Citation
  • 28.

    Loftin KA, Adams CD, Meyer MT, et al. Effects of ionic strength, temperature, and pH on degradation of selected antibiotics. J Environ Quality 2008; 37: 378386.

    • Search Google Scholar
    • Export Citation
  • 29.

    Oxytetracycline monograph. In: Budavari S, ed. The Merck index. 11th ed. Rahway, NJ: Merck and Co Inc, 1989; 1104.

  • 30.

    Hirt H, Schlievert PM, Dunny GM. In vivo induction of virulence and antibiotic resistance transfer in Enterococcus faecalis mediated by the sex pheromone-sensing system of pCF10. Infect Immun 2002; 70: 716723.

    • Search Google Scholar
    • Export Citation

Advertisement

Effects of two simulated oxytetracycline dosing regimens on horizontal transfer of antimicrobial resistance plasmids in an in vitro pharmacodynamic model

View More View Less
  • 1 Departments of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506
  • | 2 PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66506.
  • | 3 Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506
  • | 4 PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66506.
  • | 5 Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506
  • | 6 PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66506.
  • | 7 PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66506.
  • | 8 Departments of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506
  • | 9 PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66506.
  • | 10 Departments of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506
  • | 11 PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66506.

Abstract

Objective—To evaluate the impact of oxytetracycline exposure on horizontal transfer of an antimicrobial resistance plasmid.

Sample—Populations of Salmonella enterica subsp enterica serovar Typhimurium and Escherichia coli.

Procedures—Mixed populations of plasmid donor (Salmonella Typhimurium) and recipient (E coli) bacteria were assigned to 1 of 2 simulated oxytetracycline dosing regimens (high peak concentration-short elimination half-life [HC-SHL] or low peak concentration—long elimination half-life [LC-LHL]) or served as untreated control replicates. Donor, recipient, and transconjugant (E coli that acquired the plasmid) bacteria populations were quantified at 12, 24, and 36 hours after oxytetracycline administration by use of culture on selective bacterial growth media.

Results—The ratio of transconjugant to donor bacteria was significantly reduced in the oxytetracycline-exposed replicates, compared with the ratio for the control replicates, at 12 hours. At 24 and 36 hours, results for the HC-SHL regimens were not significantly different from results for the respective control replicates, and results for the LC-LHL regimens also were not significantly different from results for the respective control replicates. The oxytetracycline concentration at these time points (12 hours in the HC-SHL regimen and all 3 time points in the LC-LHL regimen) were in excess of the minimum inhibitory concentration of the recipient bacteria.

Conclusions and Clinical Relevance—Transfer of antimicrobial resistance plasmids may be suppressed in vitro by oxytetracycline exposure at concentrations greater than the minimum inhibitory concentration of the recipient bacteria.

The remarkable adaptive abilities of bacteria have led to the phenomenon of antimicrobial resistance. Currently, there is a global pandemic of reemerging infectious diseases attributable, in part, to antimicrobial resistance.1,2 Three key factors have been recognized in the emergence of bacterial resistance: mutation, bacterial genetic exchange, and selective pressure in health-care and community settings.3 Of these factors, the primary focus has been investigating methods to decrease the selective pressure within health-care and community settings by focusing on prudent use of drugs.4–6 The importance of prudent antimicrobial use has also been recognized in veterinary medicine, as indicated by the AVMA policy statement on judicious therapeutic use of antimicrobials.7 For veterinarians, the prevention of antimicrobial resistance has important implications in their obligations to public health and the successful treatment of patients. This is particularly true in production animal medicine, for which the goal of successful treatment of patients is often depicted as contradictory to public health.

The roles of mutation and dosing strategies to minimize development of antimicrobial resistance have been studied extensively,8–11 which has given rise to theories such as the mutant prevention concentration. There is also a plethora of information in regard to the mechanisms of bacterial gene exchange12; however, relatively little has been published regarding the influence of drug exposure on the rates of conjugative transfer.13

The issue of antimicrobial resistance has resulted in a paradigm shift with regard to investigation of antimicrobial therapeutics. Since the late 1990s, researchers have sought dosing regimens that impart clinical efficacy and also minimize the development of antimicrobial resistance. Consistent with these goals, the purpose of the study reported here was to evaluate the impact of oxytetracycline exposure on horizontal transfer of an antimicrobial resistance plasmid in an IVPM. Our hypothesis was that different oxytetracycline exposures would result in differential frequency of plasmid transfer between bacterial species.

Materials and Methods

Bacterial species and strains—A clinical isolate of Salmonella enterica subsp enterica serovar Typhimurium strain No. 5678 that contained a 100-kilobase type A conjugative plasmid was used in the study reported here as the donor bacteria. This Salmonella Typhimurium strain has been used previously in plasmid transfer experiments.14 This low copy number plasmid contained a blaCMY-2 ESBL gene (with resistance to ampicillin, ceftriaxone, and cefoxitin) as well as resistance markers for chloramphenicol, streptomycin, sulfisoxazole, trimethoprim-sulfamethoxazole, and tetracycline. The resistance gene that supplies the resistance to tetracycline and oxytetracycline is present on an IS26-like portion of the plasmid and was found by use of PCR amplification to be positive for the tetA efflux pump (data not shown). Monitoring of transconjugants indicated that this plasmid transfers all resistance genes, as indicated by the ability to screen by the use of ampicillin. Additional tests indicated that other antimicrobial markers were also present (data not shown). The recipient bacterium, Escherichia coli C600N, was a laboratory strain that carries a chromosomally encoded resistance marker for nalidixic acid. Aliquots of stock bacteria were stored separately at −80°C.

Determination of MIC—The MIC for each of the study bacteria was determined via modification of procedures recommended by the Clinical and Laboratory Standards Institute.15 Fresh cultures of donor and recipient bacteria were grown separately overnight on a rotary shaker (37°C) in 10 mL of fresh Luria-Bertani broth.a Ten microliters of a standardized bacterial suspension (optical density [measured at 600 nm] = 0.03) was pipetted into 11 mL of Luria-Bertani broth. An aliquot (100 μL) of the bacterial suspension was added to each well of a 96-well plate.b Each well of the plate also contained 100 μL of oxytetracycline solution; the final concentrations of oxytetracycline tested were 0, 1, 5, 25, 50, 75, 100, 125, 150, 175, 200, 250, 500, 750, and 1,000 ng/mL. Testing was performed in duplicate for the donor and recipient strains.

The plates were placed in an incubated spectrophotometerc set to measure absorbance at 600 nm; measurements were obtained hourly. The MIC was defined as the lowest concentration at which there was a reduction in the optical density value.

Experimental stock culture—Single frozen aliquots of Salmonella and E coli organisms were thawed at 21°C. The bacteria were isolated by culture on plates that contained Luria-Bertani agar.a A single colony of each bacteria was transferred to separate flasks that contained 10 mL of sterile BHI brothd; flasks were incubated overnight at 37°C on a rotary shaker.e The entire 10 mL of each culture was transferred into separate flasks that contained 90 mL of sterile BHI broth. Cultures of both bacteria were grown to logarithmic phase (optical density [measured at 600 nm] = 0.6).f Once the desired optical density was achieved, the Salmonella Typhimurium and E coli were transferred to syringesg in a volumetric ratio of 1:5 (1 part of the solution containing Salmonella Typhimurium to 5 parts of the solution containing E coli). Five milliliters of the bacterial mixture was inoculated into each central reservoir of the IVPM system. A 1-hour growth interval was used in the IVPM system before oxytetracycline administration.

IVPM—Variations of the IVPM system used in the study reported here have been described by others.15–18,h The system consisted of a 500-mL central reservoir,i a 4-L fresh media reservoir,j a 4-L waste collection reservoir,j reservoir caps,k connecting tubing,l and peristaltic pumps (Figure 1). All components were autoclaved before the system was assembled. For each experiment, 4 of the systems (2 for oxytetracycline regimens and 2 for the associated control replicates) were assembled. During an experiment, the central reservoir of each system was housed in an incubatorm at 37°C. Medium in the central reservoir was stirred constantly by use of a stir rod and plate configuration.n

Figure 1—
Figure 1—

Schematic diagram of an IVPM used to evaluate the impact of oxytetracycline exposure on horizontal transfer of an antimicrobial resistance plasmid from donor (Salmonella enterica subsp enterica serovar Typhimurium) to recipient (Escherichia coli) bacteria.

Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.877

Speed of the peristaltic pump for the fresh media reservoir that would achieve a specified half-life was determined by use of the following equation:

article image

Speed of the peristaltic pump for the waste media reservoir was set slightly faster than the pump speed for the fresh media, with the exit port set just above the fluid line in the central reservoir to maintain a constant volume in the central reservoir. Dual pump heads were used to control the flow from the fresh and waste media for each oxytetracycline regimen and associated control replicate. To eliminate residual effects in the samples, a single port in the central reservoir was designated for bacterial inoculation and administration of oxytetracycline. A separate port was designated for collection of samples for bacterial and antimicrobial analysis. Three replicates of each oxytetracycline regimen (HC-SHL or LC-LHL) and the associated control replicate were conducted.

The 2 dosing regimens were modifications for IV19 and IM20 doses of oxytetracycline in swine. The IV dosing regimen (ie, HC-SHL) was selected so that initial peak concentrations were > 1,000 ng/mL and time within the range of 150 to 1,000 ng/mL would be minimized. The IM dosing regimen (ie, LC-LHL) was selected so that drug concentrations would remain within the range of 150 to 1,000 ng/mL for a comparatively longer period than for the HC-SHL. Each of the 3 replicates included HC-SHL and its control replicate and LC-LHL and its control replicate.

Antimicrobial regimens—Analytic grade oxytetracycline (in the form of the hydrochloride salt) was diluted in fresh BHI broth before administration in the IVPM. Corrections for salt and purity were made to achieve oxytetracycline concentrations.

Bacterial quantification and determination of plasmid transfer—Samples were collected from the central reservoir of the IVPM at 12, 24, and 36 hours after oxytetracycline administration. Each sample (approx 1 mL) was centrifuged at 5,000 × g for 10 minutes. Supernatant was pipetted into cryovials and stored at −70°C until analyzed for oxytetracycline quantification.

The bacterial pellet was resuspended in PBS solution; serial dilutions (10−1 to 10−8 cells/mL) of the bacterial resuspension were then made. Transconjugant bacteria were quantified via duplicate plating of 50 μL of the 10−1, 10−4, 10−6, and 10−8 dilutions on Hektoen enteric agar that contained 50 μg of ampicillin/mL and 12 μg of nalidixic acid/mL. Salmonella Typhimurium (donor bacteria) were quantified by plating 50 μL of the 10−4, 10−6, and 10−8 dilutions on Hektoen enteric agar that contained 50 μg of ampicillin/mL. Escherichia coli (recipient bacteria) were quantified by plating 50 μL of the 10−4, 10−6, and 10−8 dilutions on Hektoen enteric agar that contained 12 μg of nalidixic acid/mL. Concentrations for transconjugant, donor, and recipient bacteria were determined by use of the following equations:

article image

Oxytetracycline quantification—All chemical reagents were analytic-grade products. Briefly, 50 μL of internal standard (doxycyclineo; 2,000 ng/mL) was added to 50 μL of sample. The analyte and internal standard were extracted by use of 10 μL of concentrated phosphoric acid,p which was followed by the addition of 150 μL of deionized water. The samples were loaded on hydrophilic-lipophilic balanced solid-phase cartridgesq for the extraction procedure. The first elution was performed with a solution of methanol:water (5:95). Columns then were washed with 500 μL of the methanol:water (5:95) solution and dried under high pressure (approx 508 mm Hg) for 10 minutes. Oxytetracycline was recovered from the solid-phase extraction cartridge by use of a methanol wash (300 μL). A 150-μL sample was then transferred to an HPLC vial and stored at 4°C in the sample carousel until analyzed.

Quantification of oxytetracycline was performed by use of HPLC and triple quadrupole mass spectrometry. Chromatographic separation was achieved by use of a gradient elution of 100% (0.2% glacial acetic acidr in water) adjusted to 5%:95% (0.2% glacial acetic acid in water:0.2% glacial acetic acid in acetonitriles) on an HPLC systemt with a C18 analytic column.u Injection volume was 2 μL, flow rate was 0.35 mL/min, and total run time was 5 minutes. Retention times for oxytetracycline and doxycycline (internal standard) were 2.12 and 2.15 minutes, respectively. Mass spectrometry included an electrospray ionization source.v The instrumentw was set to operate in positive ion mode. Transitions were monitored at a mass-to-charge ratio of 461:426 for oxytetracycline and mass-to-charge ratio of 445:321 for doxycycline (internal standard). Standard curves were prepared daily and consisted of 7 nonzero points ranging from 20 to 14,000 ng/mL.

A run was accepted when the concentrations of the standards were within 15% of the expected concentration and the fit of the curve was at least 0.99. Two low-concentration (350 ng/mL) and 2 medium-concentration (6,000 ng/mL) quality-control samples were included in each run; results for 1 low-concentration quality-control sample differed by > 20% from the expected value. For the remaining quality-control samples, accuracy was within 19% of the expected value, and the coefficient of variation was 17%.

Corrections for protein binding—Protein binding was determined in BHI broth via ultrafiltration and centrifugation. Triplicate 200-μL aliquots of the low-(20 ng/mL), medium- (500 ng/mL), and high- (14,000 ng/mL) concentration calibration solutions from the standard curve were pipetted into centrifugal filtration vials.x The vials were centrifuged at 14,000 × g for 30 minutes. The preparation and quantification procedures were performed as described previously, with the following exception: the standard curve for the protein binding estimate was fit with a quadratic equation (R2 = 0.9998) that consisted of 6 points across the range of concentrations. Accuracy for the standards was within 3% of the expected concentrations. The analytic run consisted of only the standard curve and 9 ultrafiltered samples. Protein binding was calculated by use of the following equation:

article image

For the low calibration standards, concentrations for the centrifuged and filtered samples were above the limit of detection but below the lower LOQ. For these samples, the concentration was calculated by dividing the area ratio of the sample by the area ratio of the standard and multiplying the quotient by the known concentration of the standard. The percentage of protein binding was calculated by use of the following equation:

article image

Statistical analysis—Statistical analyses were performed by use of a commercial software package.y Transconjugant ratios were logarithmically transformed (base10) prior to statistical analysis. The lower LOQ for the transconjugant ratio was calculated by use of the following equations:

article image

Transconjugant ratios were analyzed for each time point by use of a 1-way ANOVA, with treatment as the independent variable. Significant treatment differences were further evaluated by use of 2-way contrast statements. Values of P ≤ 0.05 were considered significant.

Results

Determination of MIC—Reductions in bacterial growth generally were detected after incubation for 8 to 12 hours. The oxytetracycline MIC for the E coli (recipient bacteria) was 125 ng/mL. The MIC for Salmonella Typhimurium (donor bacteria) was 60,000 ng/mL.

Oxytetracycline concentrations and protein binding in BHI broth—Protein binding in BHI broth was nonlinear for oxytetracycline concentrations between 20 and 14,000 ng/mL (Figure 2). For concentrations > 700 ng/mL, protein binding was estimated at < 15% and was not corrected. For the LC-LHL regimen, 1 value measured was 751 ng/mL; however, for consistency, all data for this regimen were corrected for protein binding. For the HC-SHL regimen, the concentration in samples obtained after incubation for 12 hours was > 700 ng/mL, but the concentration in samples obtained after incubation for 36 hours was below the lower LOQ of the assay; thus, no corrections were applied. The oxytetracycline concentration in samples obtained after incubation for 24 hours for all replicates of the HC-SHL regimen was corrected on the basis of protein binding.

Figure 2—
Figure 2—

Protein binding in BHI broth for various concentrations of oxytetracycline.

Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.877

Determination of transconjugant ratios—Mean CFU counts for Salmonella Typhimurium (donor bacteria) remained stable throughout the experiment (Figure 3). Four replicates within time points for the antimicrobial regimens had transconjugant ratios of 0 (ie, no transconjugant colonies). These values were mathematically adjusted to equal the lower LOQ of the transconjugant ratio. Three of these replicates were obtained after incubation for 12 hours (2 LC-LHL and 1 HC-SHL), and the fourth replicate was an LC-LHL replicate obtained after incubation for 36 hours (Figure 4; Table 1).

Figure 3—
Figure 3—

Concentrations of Salmonella Typhimurium (donor bacteria) cultured for up to 36 hours under various conditions as follows: starting inoculum (horizontally striped bar), LC-LHL control replicate (diagonally striped bars), LC-LHL regimen (black bars), HC-SHL control replicate (white bars with black dots), and HC-SHL regimen (white bars).

Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.877

Figure 4—
Figure 4—

Concentrations of transconjugant bacteria (recipient E coli) cultured for up to 36 hours under various conditions as follows: LC-LHL control replicate (diagonally striped bars), LC-LHL regimen (black bars), HC-SHL control replicate (white bars with black dots), and HC-SHL regimen (white bars).

Citation: American Journal of Veterinary Research 72, 7; 10.2460/ajvr.72.7.877

Table 1—

Mean ± SD log (base10) transconjugant ratios for oxytetracycline treatment (LC-LHL and HC-SHL regimens) and control replicates after incubation for 12, 24, and 36 hours for mixed cultures of donor (Salmonella enterica subsp enterica serovar Typhimurium) and recipient (Escherichia coli) bacteria.

Time (h)TreatmentLog (base10) transconjugant ratioFree oxytetracycline concentration (ng/mL)
12LC − LHL*−8.80 ± 0.86a514.16 ± 126.77
 LC − LHL control−6.19 ± 1.21b
 HC − SHL−8.40 ± 0.81a636.56 ± 30.34
 HC − SHL control−5.64 ± 1.31b
24LC − LHL−7.83 ± 0.64383.04 ± 86.05
 LC − LHL control−6.90 ± 0.58
 HC − SHL−8.00 ± 0.5562.83 ± 16.76
 HC − SHL control−7.46 ± 0.03
36LC − LHL−8.66 ± 0.65222.51 ± 41.13
 LC − LHL control−7.74 ± 0.60
 HC − SHL−7.95 ± 0.34Below lower LOQ
 HC − SHL control−7.96 ± 0.43

Values reported represent the mean ± SD for 3 replicates for each treatment at each time point.

The HC-SHL regimen was designed such that initial peak concentrations of oxytetracyline were > 1,000 ng/mL and time within the range of 150 to 1,000 ng/mL would be minimized. The LC-LHL regimen was designed such that oxytetracycline concentrations would remain within the range of 150 to 1,000 ng/mL for a comparatively longer period than for the HC-SHL.

Two replicates had zero transconjugant colonies; therefore, the ratio was adjusted to −9.3 (lower LOQ) for those 2 replicates.

One replicate had zero transconjugant colonies; therefore, the ratio was adjusted to −9.3 (lower LOQ) for that replicate.

— = No oxytetracycline treatment.

Within a time point, ratios with different superscript letters differ significantly (P = 0.01).

The mean transconjugant ratios for the 2 control simulations were not significantly different at any of the 3 time points. After incubation for 12 hours, the transconjugant ratios for the HC-SHL and LC-LHL regimens were significantly (P = 0.01 for both contrasts) less than the ratios for their respective control replicates. After incubation for 24 and 36 hours, the transconjugant ratios for the HC-SHL regimens were not significantly (P = 0.24 and 0.98, respectively) different from the ratios for the respective control replicates. The transconjugant ratios of the LC-LHL regimen after incubation for 24 and 36 hours differed, but not significantly (P = 0.057 and 0.062, respectively), from the ratios for the respective control replicates. After incubation for 12, 24, or 36 hours, the transfer rate for the HC-SHL and LC-SHL regimens did not differ significantly (P = 0.65, 0.70, and 0.13, respectively; Table 1).

Oxytetracycline concentrations—In the HC-SHL simulations, mean free (nonprotein bound) oxytetracycline concentrations were approximately 750 and 65 ng/mL after incubation for 12 and 24 hours, respectively. Antimicrobial concentrations were below the limit of detection after incubation for 36 hours for these treatment simulations. For the LC-LHL simulations, mean free concentrations were 515, 383, and 225 ng/mL after incubation for 12, 24, and 36 hours, respectively (Table 1).

Discussion

A growing body of in vitro evidence suggests that the development of antimicrobial resistance within a population of bacteria can be suppressed by extrapolating the pharmacokinetic and pharmacodynamic indices associated with clinical efficacy.21–23 The primary focus of these studies has been on the growth of resistant bacteria that originated from genetic mutation or that preexisted within the population. In contrast, the study reported here focused on the development of antimicrobial resistance attributable to emergence of resistant organisms following the acquisition of a horizontally transferred plasmid. The importance of this mechanism of resistance development in bacterial populations has been discussed in other reports.2,24,25

The IVPM is an ideal laboratory tool for use in evaluating the most basic interactions between antimicrobials and pathogens. A limitation to the 1-compartment IVPM used in the present study was the dilution effect on the bacteria caused by inflow of fresh medium to the central reservoir. Because the transconjugant ratios of the HC-SHL and LC-LHL control replicates were not significantly different at any time during the experiments, the effect of dilution rate was considered negligible. Another limitation of the IVPM for the described conditions was the favorable advantage given to the bacterial population. Type of growth medium, constant inflow of nutrients and removal of waste products, inoculum size, timing of treatment and temperature at which the experiments were conducted, and lack of a functional immune system give every conceivable advantage to the bacterial pathogen. For the present study, conjugative events may also have been favored, compared with conditions in filter-mating studies, given the absolute number of donor and recipient bacteria present in the culture system and the constant stirring in the central reservoir of the IVPM. Results of the present study should be interpreted as the best-case scenario for plasmid transfer given the in vitro conditions of the experiment.

The transfer rates in the present study are in agreement with the conjugative rates found in another study.26 The authors in that study used 2 Bacillus strains and reported filter-mating transfer rates ranging from 1 × 10−1 to < 1 × 10−8 when the donor was grown in the presence of tetracycline (10 μg/mL) prior to mating. Those authors also found that the conjugative frequency was enhanced at low tetracycline concentrations during mating. Although transfer rates in that study26 were achievable in the present study, the conclusions differed with regard to the effects of drug exposure on plasmid transfer. The conclusions from that other study26 suggest that tetracycline exposure during the growth period prior to mating or during the mating period increases the frequency of conjugation; however, in the study reported here, oxytetracycline exposure suppressed transfer rates. The authors of that other study26 hypothesized that enhanced conjugation was a direct effect on the donor strain and not a reflection of antibiosis of the recipients.

In 1 study,27 conjugal transfer rates for Escherichia faecalis were enhanced in the presence of tetracycline. Filter matings were performed at static concentrations (10 μg/mL) of tetracycline, compared with the dynamic pharmacokinetics in liquid culture in the present study. Because of the static drug exposures in the aforementioned studies26,27 and the dynamic drug exposures in the study reported here, it is difficult to make direct comparisons of the results. In fact, it could be argued that the oxytetracycline exposures in those other studies26,27 were not truly static exposures because of oxytetracycline degradation. Investigators in 1 study28 determined that static concentrations of oxytetracycline at 35°C and a pH of 7 degraded with a half-life of 19 hours. The product monograph29 indicates a stability estimate with a half-life of 26 hours under conditions similar to those used in the study reported here. This is in agreement with results of experiments conducted by our research group that revealed a mean half-life of 24 hours for static concentrations. Failure to account for the actual drug exposure limits the conclusions that can be drawn from experiments that involve the use of static concentrations. To the authors' knowledge, there have been no studies conducted to investigate the effects of oxytetracycline on conjugative plasmid transfer with simulated in vivo antimicrobial exposures.

Previous static concentration experiments with the donor and recipient bacteria used in the study reported here revealed that plasmid transfer was most efficient at oxytetracycline concentrations up to 1,000 ng/mL. Results of the present study indicated that conjugation was suppressed when antimicrobial concentrations exceeded the MIC of the recipient strain. In contrast to results of the aforementioned studies,26,27 at no time point in the present study did oxytetracycline exposure enhance the conjugation rates. The discrepancy may be a result of differences in the laboratory conditions (filter vs liquid culture) or the drug exposure profiles (static vs dynamic).

Use of the transconjugant ratio and the equation for its calculation has been described elsewhere.30 The transconjugant ratio is a hybrid of 2 separate measures, which both require examination to make inferences about the ratio. One limitation of the present study was that the plating procedures were not standardized on the basis of sample volume; however, use of the transconjugant ratio rather than actual numbers of transconjugant bacteria addressed this limitation. The Salmonella Typhimurium population in the control replicates was numerically greater than that in the treated replicates. However, the potential impact on the transconjugant ratio attributable to this was approximately a 0.5-log decrease for the oxytetracycline-exposed populations. The decreases in the transconjugant ratio (> 1 log at some time points) were driven by relatively fewer transconjugants formed in the treated populations and not by a comparative increase in the number of donor organisms.

Results of the present study revealed significant (P = 0.01) suppression of conjugative transfer in both oxytetracycline-treated systems after incubation for 12 hours, compared with values for the respective control replicates. Although not significantly different, the transconjugant ratio in the LC-LHL regimens was numerically suppressed (compared with the ratio for the control replicates) after incubation for 24 and 36 hours (P = 0.057 and 0.062, respectively). This suppression was not detected after incubation for 24 or 36 hours in the HC-SHL regimen (P = 0.24 and 0.98, respectively). Considered together, transconjugant ratios were suppressed at all times when corresponding oxytetracycline concentrations were above the MIC of the recipient bacteria (Table 1).

Two observations merit discussion: the peak rate of plasmid transfer was detected early in the time course of the study, and the development of transconjugant bacteria was suppressed by exposure to oxytetracycline. Both observations can be related to effects of bacterial growth. If conjugation is a function of bacterial growth, a change in bacterial growth attributable to the inherent growth properties of the bacterial population (stationary or death phase) or the induction of bacterial stasis as a result of the presence of an antimicrobial would suppress horizontal gene transfer, which was evident in the present study for the treated bacterial populations.

It is inappropriate to use low-power estimates to infer differences when significant differences were not detected. However, numeric differences within studies with relatively low power can legitimately be used as an incentive for additional studies with greater power. By use of the difference in means and SDs in the LC-LHL regimens after incubation for 24 and 36 hours, 3 replicates of each treatment yield a power of approximately 0.57 or a probability of > 0.4 for a false-negative result. These findings provide insights into the relationship between drug exposure and the development of antimicrobial resistance attributable to horizontal gene transfer. Additional studies are needed to investigate other antimicrobial-pathogen combinations and to validate in vivo the findings for the study reported here.

ABBREVIATIONS

BHI

Brain-heart infusion

HC-SHL

High peak concentration—short elimination half-life

HPLC

High-performance liquid chromatography

IVPM

In vitro pharmacodynamic model

LC-LHL

Low peak concentration—long elimination half-life

LOQ

Limit of quantification

MIC

Minimum inhibitory concentration

a.

Becton-Dickinson, Franklin Lakes, NJ.

b.

Corning Costar 96-well plates, Sigma-Aldrich, St Louis, Mo.

c.

SpectraMax 190, Molecular Devices, Sunnyvale, Calif.

d.

Sigma Aldrich, St Louis, Mo.

e.

Gallenkamp orbital incubator, Sanyo-Gallenkamp, Loughborough, Liecestershire, England.

f.

Spectronic 20D+, Thermo Scientific Corp, Waltham, Mass.

g.

Monoject 12-mL regular luer syringe, Kendall, Mansfield, Mass.

h.

Findlay I. The pharmacodynamics of antimicrobial agents against clinical isolates of Staphylococcus epidermidis in a multiple dose in vitro pharmacodynamic model. MPharm thesis. Faculty of Pharmacy, School of Pharmacy, University of Manitoba, Winnipeg, MB, Canada, 2001.

i.

Bellco Technologies, Vineland, NJ.

j.

Thermo Fisher Scientific, Pittsburgh, Pa.

k.

Fibercell systems, Frederick, Md.

l.

Masterflex L/S 13 platinum-cured silicon tubing, Cole-Parmer, Vernon Hills, Ill.

m.

NuAire IR autoflow, NuAire Inc, Plymouth, Minn.

n.

IKA Big Squid, Sigma-Aldrich, St Louis, Mo.

o.

Doxycycline hyclate, Sigma-Aldrich, St Louis, Mo.

p.

O-Phosphoric acid, Fisher Scientific, Pittsburgh, Pa.

q.

Waters Corp, Milford, Mass.

r.

Glacial acetic acid, Fisher Scientific, Pittsburgh, Pa.

s.

Acetonitrile, Fisher Scientific, Pittsburgh, Pa.

t.

Shimadzu LC-20AD, Shimadzu Scientific—North America, Columbia, Mo.

u.

Sunfire C18, Waters Corp, Milford, Mass.

v.

Turbo-Ionspray atmospheric pressure ionization source, MDS Analytical Technologies, Concord, ON, Canada.

w.

Sciex API 4000, MDS Analytical Technologies, Concord, ON, Canada.

x.

Microcon YM-10m, Millipore Corp, Bedford, Mass.

y.

SAS, version 9.3.1, SAS Institute Inc, Cary, NC.

References

  • 1.

    Spellberg B. Dr. William H. Stewart: mistaken or maligned? (lett) Clin Infect Dis 2008; 47: 294.

  • 2.

    Phillips I, Casewell M, Cox T, et al. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J Antimicrob Chemother 2004; 53: 2852.

    • Search Google Scholar
    • Export Citation
  • 3.

    Tenover FC. Development and spread of bacterial resistance to antimicrobial agents: an overview. Clin Infect Dis 2001; 33 (suppl 3): 108115.

    • Search Google Scholar
    • Export Citation
  • 4.

    Blaser JM, Stone BB, Groner MC, et al. Comparative study with enoxacin and netilmicin in a pharmacodynamic model to determine importance of ratio of antibiotic peak concentration to MIC for bactericidal activity and emergence of resistance. Antimicrob Agents Chemother 1987; 3: 10541060.

    • Search Google Scholar
    • Export Citation
  • 5.

    Jumbe N, Louie A, Leary R, et al. Application of a mathematical model to prevent in vivo amplification of antibiotic-resistant bacterial populations during therapy. J Clin Invest 2003; 112: 275285.

    • Search Google Scholar
    • Export Citation
  • 6.

    Thomas JK, Forrest A, Bhavnani SM, et al. Pharmacodynamic evaluation of factors associated with the development of bacterial resistance in acutely ill patients during therapy. Antimicrob Agents Chemother 1998; 42: 521527.

    • Search Google Scholar
    • Export Citation
  • 7.

    AVMA. Judicious therapeutic use of antimicrobials. Available at: www.avma.org/issues/policy/jtua.asp. Accessed Aug 27, 2009.

  • 8.

    Courvalin P. Can pharmacokinetic-pharmacodynamic parameters provide dosing regimens that are less vulnerable to resistance? Clin Microbiol Infect 2008; 14: 989994.

    • Search Google Scholar
    • Export Citation
  • 9.

    Firsov AA, Smirnova MV, Lubenko IY, et al. Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to daptomycin and vancomycin in an in vitro dynamic model. J Antimicrob Chemother 2006; 58: 11851192.

    • Search Google Scholar
    • Export Citation
  • 10.

    Olofsson SK, Marcusson LL, Lindgren PK, et al. Selection of ciprofloxacin resistance in Escherichia coli in an in vitro kinetic model: relation between drug exposure and mutant prevention concentration. J Antimicrob Chemother 2006; 57: 11161121.

    • Search Google Scholar
    • Export Citation
  • 11.

    Zhao X, Drlica K. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. J Infect Dis 2001; 185: 561565.

    • Search Google Scholar
    • Export Citation
  • 12.

    Schwarz S, Chaslus-Dancla E. Use of antimicrobials in veterinary medicine and mechanisms of resistance. Vet Res 2001; 32: 201225.

  • 13.

    Doucet-Populaire F, Trieu-Cuot P, Dosbaa I, et al. Inducible transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes in the digestive tracts of gnotobiotic mice. Antimicrob Agents Chemother 1991; 35: 185187.

    • Search Google Scholar
    • Export Citation
  • 14.

    Giles WP, Benson AK, Olson ME, et al. DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones. Antimicrob Agents Chemother 2004; 48: 28452852.

    • Search Google Scholar
    • Export Citation
  • 15.

    Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals: approved standard. M31A2. 2nd ed. Wayne, NJ: Clinical and Laboratory Standards Institute, 2002.

    • Search Google Scholar
    • Export Citation
  • 16.

    Grasso S, Meinardi G, DeCarneri I, et al. New in vitro model to study the effect of antibiotic concentration and rate of elimination on antibacterial activity. Antimicrob Agents Chemother 1978; 13: 570576.

    • Search Google Scholar
    • Export Citation
  • 17.

    Murakawa T, Sakamoto H, Hirose T, et al. New in vitro model for evaluating bactericidal efficacy of antibiotics. Antimicrob Agents Chemother 1980; 18: 377381.

    • Search Google Scholar
    • Export Citation
  • 18.

    Sanfilippo A, Morvillo E. An experimental model for the study of the antibacterial activity of the sulfonamides. Chemotherapy 1968; 13: 5460.

    • Search Google Scholar
    • Export Citation
  • 19.

    Pijpers A, Schoevers EJ, Van Gogh H, et al. The pharmacokinetics of oxytetracycline following intravenous administration in healthy and diseased pigs. J Vet Pharmacol Ther 1990; 13: 320326.

    • Search Google Scholar
    • Export Citation
  • 20.

    El Korchi G, Prats C, Arboix M, et al. Disposition of oxytetracycline in pigs after i.m. administration of two long-acting formulations. J Vet Pharmacol Ther 2001; 24: 247250.

    • Search Google Scholar
    • Export Citation
  • 21.

    Blondeau JM, Hansen G, Metzler K, et al. The role of PK/PD parameters to avoid selection and increase of resistance: mutant prevention concentration. J Chemother 2004; 16 (suppl 3): 119.

    • Search Google Scholar
    • Export Citation
  • 22.

    MacGowan AP, Bowker KE, Noel AR. Pharmacodynamics of the antibacterial effect and emergence of resistance to tomopenem, formerly RO4908463/CS-023, in an in vitro pharmacokinetic model of Staphylococcus aureus infection. Antimicrob Agents Chemother 2008; 52: 14011406.

    • Search Google Scholar
    • Export Citation
  • 23.

    Ryback MJ. Pharmacodynamics: relation to antimicrobial resistance. Am J Infect Control 2006; 34: 3845.

  • 24.

    Shoemaker NB, Vlamakis H, Hayes K, et al. Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon. Appl Environ Microb 2001; 67: 561568.

    • Search Google Scholar
    • Export Citation
  • 25.

    Schjørring S, Struve C, Krogfelt KA. Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine. J Antimicrob Chemother 2008; 62: 10861093.

    • Search Google Scholar
    • Export Citation
  • 26.

    Showsh SA, Andrews RE Jr. Tetracycline enhances Tn916-mediated conjugal transfer. Plasmid 1992; 28: 213224.

  • 27.

    Torres OR, Korman RZ, Zahler SA, et al. The conjugative transposon Tn925: enhancement of conjugal transfer by tetracycline in Enterococcus faecalis and mobilization of chromosomal genes in Bacillus subtilis and E. faecalis. Mol Gen Genet 1991; 225: 395400.

    • Search Google Scholar
    • Export Citation
  • 28.

    Loftin KA, Adams CD, Meyer MT, et al. Effects of ionic strength, temperature, and pH on degradation of selected antibiotics. J Environ Quality 2008; 37: 378386.

    • Search Google Scholar
    • Export Citation
  • 29.

    Oxytetracycline monograph. In: Budavari S, ed. The Merck index. 11th ed. Rahway, NJ: Merck and Co Inc, 1989; 1104.

  • 30.

    Hirt H, Schlievert PM, Dunny GM. In vivo induction of virulence and antibiotic resistance transfer in Enterococcus faecalis mediated by the sex pheromone-sensing system of pCF10. Infect Immun 2002; 70: 716723.

    • Search Google Scholar
    • Export Citation

Contributor Notes

Dr. Lubbers' present address is Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

Dr. Peterson's present address is Kansas Department of Health and Environment, Forbes Field, Building 740, Topeka, KS 66620.

Supported by the Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University; Targeted Excellence Grant, Kansas State University; and the Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University.

Drs. Lubbers and Peterson contributed equally to the report.

Address correspondence to Dr. Apley (mapley@vet.k-state.edu).