In Vitro Susceptibility Study of Two Porcine Mycoplasmas Isolated from Lung Lesions of Slaughtered Pigs in Thailand

Metta Makhanon1, Pacharee Thongkamkoon2, Nuvee Prapasarakul1 1 Depart. of Veterinary Microbioogy, Fact. of Veterinary Science, Chulalongkorn University, Bangkok, Thailand 2National Institute of Animal Health, Depart. of Livestock Development, Ministry of Agriculture and Cooperative, Bangkok, Thailand metta.makhanon@novartis.com, thongkamkoon@msn.com, nuvee.p@chula.ac.th

Introduction
Antimicrobial resistant bacteria isolated from livestock is dramatically increasing as well as the development of antibiotic resistance to Mycoplasma spp. The drugs of choice for anti-mycoplasmas include pleuromutilins (tiamulin and valnemulin), tetracyclines (doxycycline), macrolides (tylosin), lincosamides (lincomycin), and fluoroquinolones (enrofloxacin)1,4,6,7. The study of porcine mycoplasmal susceptibility is necessary for veterinary practitioners to choose the drugs of choice for control and treatment of mycoplasmal infection in pig farms. The objective of this study is to evaluate the MICs of Mycoplasma hyopneumoniae (MH) and M. hyorhinis (MHR) isolated from lung lesions of slaughtered pigs in Thailand.

Material and methods

Twenty-six isolates of MH and twentyone isolates of MHR derived from lung lesions of slaughtered pigs were used as the tested samples. The location of slaughterhouses were in North Eastern, Northern, Western and Eastern provinces of Thailand. The reference strains of MH and MHR were J-strain and BTS 7, respectively. Counting the organisms to 107-108 cfu/ml in BHL for MH and Modified Hayflicts for MHR. Twelve serial broth dilution of antimicrobials were 100 g/ml to 0.049 g/ml for doxycycline and enrofloxacin and 12.5 g/ml to 0.006 g/ ml for lincomycin, tiamulin, tylosin and valnemulin. The MIC test was conducted by the microtiter dilution test. Diluting the organisms by appropriated broth to be 5x105 cfu/ml. Dipping 25 l of

antimicrobial broth dilution and 175 l of organism dilution into microtiter plate size 96 pores. After incubating at 37C for 7 days, mycoplasmal growth was observed by color change in the broth. The lowest antimicrobial concentrations for inhibiting mycoplasmal growth were recorded and MICs values calculated from these data.

Results
The MIC range and values are detailed in Tables 1 and 2 below.

Discussion
In previous studies, MH showed high resistance to lincomycin, enrofloxacin, and tylosin, while there was no incidence of resistance to spectinomycin, oxytetracycline, doxycycline, gentamicin,

Table 1. MIC values of 26 Mycoplasma hyopneumoniae strains

MIC-MH (g/ml) Antimicrobials Range Doxycycline Enrofloxacin Lincomycin Tiamulin Tylosin Valnemulin 0.781-6.25 0.391-3.125 0.098-0.781 0.024-0.19 0.012-0.391 <0.006 MIC50 1.563 0.781 0.195 0.097 0.073 <0.006 MIC90 3.125 3.125 0.391 0.097 0.195 <0.006 J-Strain 0.683 0.144 0.290 0.097 0.144 <0.006

Table 2. MIC values of 21 Mycoplasma hyorhinis strains

MIC-MHR (g/ml) Antimicrobials Range Doxycycline Enrofloxacin Lincomycin Tiamulin Tylosin Valnemulin 1.563-6.25 0.781-50 0.781->12.5 0.144-0.781 1.563->12.5 <0.006-0.036 MIC50 1.563 1.563 1.563 0.391 6.250 0.012 MIC90 3.125 9.375 2.340 0.781 9.375 0.024 BTS7 0.781 3.125 1.563 0.195 1.563 <0.006

florfenicol and tiamulin. MHR was reported to have resistance to macrolides2,5,9. The resistance to macrolides occured in several species of mycoplasmas as a result of point mutation at the genes encoding domain II and V of 23S ribosomal RNA and also exhibited multi-resistance to lincomycin3,10. Tiamulin targeted the peptidyl transferase center (PTC) of the 23S rRNA during protein synthesis. Its resistant mechanism developed slowly because of its complicated structure7. This study confirmed the previous studies that pleuromutilins (tiamulin and valnemulin) illustrated the lowest MIC90 of MH and MHR isolated from slaughtered pigs in Thailand as well as in Europe5,9. Furthermore in this study, the MICs of MH field strains for pleuromutilins was the same as J-strain.

Acknowledgement
This study was funded by Novartis (Thailand) Ltd.

7. Schlunzen, F. et al., 2004. Mol. Microbiol. 54:1287-1294. 8. Stipkovit, L., et al., 2001. Can. J. Vet. Res . 65:213-222. 9. Vicca, J., et al., 2004. Antimicrob. Agents and Chemotherapy. 48:4470-4472. 10. Wu, C.M., et al., 2005. FEMS Microbiology Letters. 247:199-205.

References
1. Burch, D. 2005. In Practice. 27:3743-1. 2. Kobayashi, H., et al., 1996. Antimicrob. Agents and Chemotherapy. 40(4): 1030-1032. 3. Kobayashi, H., et al., 2005. J.Vet.Med Science. 67:795-800. 4. Maes, D., et al., 2008. Vet. Microbiol. 126:297-309. 5. Makhanon, M. et al., 2009. Proce. of 4th APVS Congress, 410. 6. Phillips, I., et al., 2004. J. Antimicrob. Chemotherapy. 53: 28-52.

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