Analysis of the mutant selection window and killing of Mycoplasma hyopneumoniae for doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin

Mycoplasma hyopneumoniae is the major pathogenic microorganism causing enzootic pneumonia in pigs. With increasing resistance of M. hyopneumoniae to conventional antibiotics, treatment is becoming complicated. Herein, we investigated the mutant selection window (MSW) of doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin for treating the M. hyopneumoniae type strain (ATCC 25934) to determine the likelihood of promoting resistance with continued use of these antibiotics. Minimum inhibitory concentration (MIC) values against M. hyopneumoniae were determined for each antimicrobial agent based on microdilution broth and agar dilution methods (bacterial numbers ranged from 105 colony-forming units (CFU)/mL to 109 CFU/mL). The minimal concentration inhibiting colony formation by 99% (MIC99) and the mutant prevention concentration (MPC) were determined by the agar dilution method with three inoculum sizes. Antimicrobial killing was determined based on MIC99 and MPC values for all five agents. MIC values ranged from 0.001 to 0.25 μg/mL based on the microdilution broth method, and from 0.008 to 1.0 μg/mL based on the agar dilution method. MPC values ranged from 0.0016 to 10.24 μg/mL. MPC/MIC99 values were ordered tylosin > doxycycline > danofloxacin > tiamulin > valnemulin. MPC achieved better bactericidal action than MIC99. Based on pharmacodynamic analyses, danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.


Introduction
Mycoplasma hyopneumoniae is the primary pathogen causing enzootic pneumonia, an important chronic respiratory disease in pigs resulting in high morbidity, low feed conversion rate, and considerable economic losses in the swine breeding industry [1]. Additionally, the disease makes pigs more susceptible to infection by secondary bacterial pathogens such as Pasteurella multocida and Actinobacillus pleuropneumoniae [2]. There are several classes of antimicrobial agents that exhibit in vitro activity against M. hyopneumoniae, such as pleuromutilins, fluoroquinolones, macrolides, and tetracyclines [3,4]. However, widespread use of these agents has resulted in acquired resistance of M. hyopneumoniae to fluoroquinolones, lincosamides, and macrolides [5][6][7]. Thus, in order to reduce the risk of drug resistance, it is necessary to develop novel drugs. However, even if new drugs are discovered, re-evaluation of antimicrobial dosing is essential, and is the main method aimed at preventing the emergence and expansion of drug-resistant strains. The mutant selection window (MSW) hypothesis postulates that, for each antimicrobialpathogen combination, an antimicrobial concentration range exists in which selective amplification of single-step, drug-resistant mutants occurs [8]. The upper and lower boundaries of the MSW are the mutant prevention concentration (MPC) and the minimal concentration that inhibits colony formation by 99% (MIC 99 ), respectively. The MPC is the minimum concentration that inhibits colony formation of the least antibacterial drug-susceptible mutant subpopulation. Therefore, when antimicrobial concentrations fall within the range of the MSW, this tends to lead to the enrichment of drug-resistant bacteria. Keeping drug concentrations above the MPC is likely to restrict the emergence of resistance [9]. This hypothesis has been verified by in vitro and in vivo experiments [10-13].
Because M. hyopneumoniae lacks a cell wall and is a small bacterium (0.4-1.2 μm), and it is fastidious and generates small colonies, culture isolation conditions in vitro are a technical challenge. In particular, quantification by the viable count method to determine colony-forming units (CFU) is arduous. Therefore, studies on the pharmacodynamics of M. hyopneumoniae are scarce. In the current study we determined for the first time the MPC and identified the MSW for doxycycline, tylosin, danofloxacin, tiamulin and valnemulin against M. hyopneumoniae in vitro. We also used time-kill tests to determine the relative antibacterial effects at the MIC 99 and the MPC. MSW and MPC are useful parameters for optimizing dosing regimens, reducing the emergence of resistant mutants, and analyzing treatment failure [14]. In addition, using the CFU counting method, changes in the amount of M. hyopneumoniae after antibiotic action can be determined.

Materials and methods
Mycoplasma hyopneumoniae type strain ATCC 25934 was obtained as a freeze-dried powder from the China Institute of Veterinary Drug Control (Beijing, China) and stored at -80˚C. Pig Mycoplasma Base (contains PPLO broth, brain heart infusion, yeast extract, Hank's balanced salt, phenol red; identification of product: HB8572), cysteine, and NADH were purchased from Qingdao Hope Biological Technology (Qingdao, China). Sterile swine serum was bought from Guangzhou Ruite Biological Technology (Guangzhou, China). The initial pH of the medium was 7.7 ± 0.1, and 1% agar was added to solid media. Doxycycline (85.8%), danofloxacin (100%), tiamulin (99%), tylosin (82.6%), and valnemulin (98.3%) were obtained from Guangdong Dahuanong Animal Health Products (Guangzhou, China). The percentages refer to the active ingredients of the drug. These five antibacterial agents were dissolved in Milli-Q water and sterilized by filtration. A 1280 μg/mL fresh stock solution of each antibacterial agent was prepared for each experiment.

Determination of Minimum Inhibitory Concentration (MIC)
MIC values were determined as described previously [15]. Briefly, MIC values were calculated for 10 5 , 10 6 , and 10 7 CFU/mL M. hyopneumoniae cultures in the exponential phase. A 100 μL sample of exponential phase culture was added to an equal volume of drug-containing medium culture in a 96-well plate. A growth control (inoculum without antimicrobials), sterility control (sterile broth at pH 7.8), and end-point control (blank medium at pH 6.8) were included. Plates were cultured at 37˚C with 5% CO 2 in an incubator after being sealed. When the color of the growth control was the same as the end-point control, the MIC was determined as the minimal concentration of antibacterial agent that resulted in no color change.
MIC values were determined by the agar dilution method as described previously [16]. Ten drops of a 10-μL sample of M. hyopneumoniae culture (10 5 , 10 6 , 10 7 CFU) were evenly placed on the surface of a plate containing 1.25−20 μg/mL danofloxacin, 2−32 μg/mL tiamulin, 4−64 μg/mL tylosin, 5−80 μg/mL doxycycline, or 0.16−2.56 μg/mL for two-fold agar dilution analysis. All plates were incubated for at least 8 days. Meanwhile, growth control plates without antimicrobials were set up for each test, and all experiments were repeated three times. The lowest concentration without M. hyopneumoniae growth on agar plates was taken as the MIC value. Each test was repeated three times.

Measurement of MIC 99 and Mutant Prevention Concentration (MPC)
MIC 99 values were measured as reported previously [17] with modifications. MIC 99 drug concentrations were based on linear decreasing dilutions of MIC values. The antibiotic concentration ranged from 1 × MIC to 0.5 × MIC in sequential 10% dilution decreases. The quantity of bacteria in the logarithmic growth phase reached 10 7 CFU/mL. Three 10 μL drops of each diluted suspension were inoculated onto agar plates and cultured for at least 8 days as described above. Colony numbers between 30 and 300 were counted.
The MPC is defined as the lowest drug concentration that prevents bacterial colony formation from a culture containing �10 9 CFU/mL bacteria [18]. We attempted different centrifugal methods for enriching M. hyopneumoniae. Ultimately, an 800 mL stationary growth phase culture was transferred into 20 tubes (each 50 mL), tubes were centrifuged (5000 × g for 20 min), and each bacterial solution was resuspended in 1 mL fresh medium. All 20 enriched cultures were combined into two 15 mL tubes, centrifuged (5000 × g for 20 min), and resuspended in 1 mL fresh medium for counting. The final concentration of M. hyopneumoniae was 8.8×10 9 CFU/mL. MPC values were measured by the agar method as described previously [19]. Briefly, 200 μL samples of each enriched culture were inoculated onto agar plates containing various concentrations of antibiotic (six parallel solid plates per antibiotic concentration). These plates were incubated at 37˚C with 5% CO 2 in a humidified incubator for 8−10 days. The lowest antibiotic concentration that resulted in no colony formation was considered the primary MPC (MPC pr ). After a 20% linear drug concentration decrease in MPC pr , the MPC was tested again, and recorded as the lowest drug concentration preventing bacterial growth. Each test was repeated three times.

Time-kill tests
In vitro time-killing assays were performed as described previously [20]. Briefly, MIC 99 and MPC values for all five agents were tested. After adding 3.5 mL blank medium and 0.1 mL drug solution (40 times the target concentration) to each penicillin bottle (a glass bottle of 10 ml with a rubber stopper), 0.4 mL exponential M. hyopneumoniae suspension with an inoculum size between 10 5 CFU/mL and 10 9 CFU/mL was added. Cultures were incubated at 37˚C with 5% CO 2 for 48 h. Aliquots of 100 μL were collected from each culture at 0, 3, 6, 9, 12, 24, 36, and 48 h. The viable cell number was determined via 10-fold serial dilutions and plating 10 μL of each diluted culture on drug-free agar. Growth controls (M. hyopneumoniae cultures without drugs) and sterility controls (5 mL medium at pH 7.8) were also included. Plates were incubated for at least 8 days at 37˚C with 5% CO 2 in a humidified incubator. Each test was repeated three times.

MIC determination
MICs of danofloxacin, tiamulin, tylosin, doxycycline, and valnemulin against M. hyopneumoniae determined by the microdilution and agar dilution methods are shown in Fig 1. Values determined using the solid MIC method were 8-fold higher (tylosin), 4-fold higher (danofloxacin, doxycycline, and valnemulin), and 2-fold higher (tiamulin) than those determined by the liquid method at an identical inoculum size of 10 5 CFU/mL. In addition, as the inoculum size (10 6 and 10 7 CFU/mL) used in these assays was increased, the MIC values also increased, except for tiamulin and valnemulin. M. hyopneumoniae displayed its greatest sensitivity to valnemulin and its least to doxycycline.

In vitro killing analysis
Time-kill curves of compounds against M. hyopneumoniae were obtained using three different inoculum sizes. Reductions in M. hyopneumoniae count with different inoculum sizes for MIC 99 and MPC are listed in Table 2 and Table 3. Danofloxacin, tiamulin, tylosin, and valnemulin achieved bactericidal activity against 10 5 CFU/mL M. hyopneumoniae with MIC 99 dosage, while doxycycline achieved bacteriostatic activity only. Colony count reductions recorded at the 48-h time point 3.63, 3.68, 3.75, and 3.61 log 10 CFU/mL for danofloxacin, tiamulin, tylosin, and valnemulin, respectively, but only 1.4 log 10 CFU/mL for doxycycline. All five compounds achieved bactericidal activity against 10 5 CFU/mL M. hyopneumoniae with MPC dosage (Fig 2).

Discussion
M. hyopneumoniae is a major respiratory disease-causing pathogen in modern intensive pig farming worldwide [1]. Although vaccine-based immunization is an important preventive measure for enzootic pneumonia, treatment with antibacterial agents is known to accelerate disease recovery and reduce disease-related complications [21]. However, strains resistant to enrofloxacin and tylosin have appeared among clinically isolated strains [6,7]. In particular, many fluoroquinolones are important antibiotics for the treatment of human infections, and are more likely to lead to cross-resistance [22]. Fortunately, some countries have banned fluoroquinolones for use in food animals [23]. Due to the difficulties associated with in vitro MIC values determined by the liquid method were similar to those measured in previous studies [4,5]. Using both liquid and solid agar methods, MIC values increased with increasing inoculum size; values obtained with a large inoculum were two to four times higher than those obtained with small inoculum. It has been reported previously that MIC values increase with increasing bacterial load [24,25]. Among the five antibiotics tested, danofloxacin and tylosin were more sensitive to inoculum size for MIC determination. In an earlier report [26], a larger inoculum of Staphylococcus aureus had a more significant effect on the antibacterial activity of nafcillin and vancomycin than a smaller inoculum. Therefore, in cases of high bacterial inoculation, more careful consideration is required when selecting the MIC reference value for the relevant experiment. In clinical treatment, the MIC value should be determined for different bacterial counts according to the severity of animal infection to establish a better treatment plan.
The MPC is defined as the concentration of antibacterial drug that prevents the growth of large quantities of resistant sub-populations. Because culturing of M. hyopneumoniae is M. hyopneumoniae cultures at cell densities of 10 5 , 10 7 , and 10 9 CFU/mL were exposed to agents at MIC 99 dosage, and colonies were counted on drug-free plates.   Consequently, by combining the in vivo pharmacokinetic parameters and the in vitro pharmacodynamic results, we speculate that danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin. Continued use of first-line antibacterial agents against M. hyopneumoniae according to current dosing regimens may therefore promote drug resistance selection, and hence limit their long-term efficacy in the treatment of endemic pneumonia in pigs. We determined the bactericidal effects of danofloxacin, tylosin, doxycycline, tiamulin, and valnemulin against M. hyopneumoniae at various bacterial densities and drug concentrations. At three different inoculation amounts, doxycycline displayed bacteriostatic activity at MIC 99 dosage and bactericidal action at MPC dosage. At the highest inoculation amount, tiamulin and valnemulin acted as bacteriostatic agents at MIC 99 dosage and as bactericidal agents at MPC dosage. Danofloxacin exhibited the fastest sterilization rate. Moreover, M. hyopneumoniae was highly sensitive to valnemulin, and exerted an obvious bactericidal effect. These results showed that when the concentration of antibiotic equaled or exceeded the MPC, M. hyopneumoniae was rapidly killed. Drug concentrations at the MPC also reduced the chances of bacteria re-growing during drug exposure. These results are similar to those of an earlier report [33]. In that previously study, the bactericidal effect at MIC was slow and incomplete. However, at MPC and maximum serum or tissue drug concentrations, killing was more pronounced than at MIC, and increased with increasing duration of drug exposure. It is worth noting that macrolides and tetracyclines are typical bacteriostatic agents. However, in this study, when the drug concentration reached MPC, it had a bactericidal effect on M. hyopneumoniae. At the dose of MPC, macrolides and tetracyclines have a bactericidal effect, which has also been reported in other studies. According to the study reported by Joseph et al. [33], tilmicosin and tulathromycin had bacteriostatic effect on Mannheimia haemolytica at MIC dose and bactericidal effect at MPC dose. The research reported by Zhang et al. [20], with the increase of the concentration of doxycycline, the antibacterial effect of Mycoplasma gallisepticum was more obvious. The antibacterial type was concentration-dependent. It can also be proved that when the concentration is continuously increased to reach or exceed MPC, it will have a bactericidal effect.
The main limitation of the present study was that the in vitro pharmacodynamic determination of M. hyopneumoniae was carried out for the type strain, and clinical isolates should be assessed to confirm our findings. Nevertheless, the present work represents a meaningful pilot study in this area. A second limitation is that all experiments were performed under ideal conditions in vitro, without considering the complexity of factors in vivo. Thus, in vivo experiments are currently being explored. The third limitation is that this study is only a proof of concept for the MSW hypothesis and does not provide proactive validation to prevent drug resistance.
In conclusion, the present study was the first to establish pharmacodynamic analyses of five antimicrobial agents against M. hyopneumoniae. We determined MPC and MSW parameters to explore the risk of M. hyopneumoniae resistance. The results showed that the bactericidal action of MPC was better than MIC 99 , and the antibacterial effects of these drugs against M. hyopneumoniae are significantly different. These pharmacodynamic results are meaningful in choosing antimicrobials for therapy. Danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.