gallisepticum

strains PG31 and S6 in broth medium containing subinhibitory concentrations of tiamulin or valnemulin. A portion of the gene encoding 23S rRNA gene (domain V) and the gene encoding ribosome protein L3 were amplified and sequenced. No mutation could be detected in ribosome protein L3. Mutations were found at nucleotide positions 2058, 2059, 2061, 2447 and 2503 of 23S rRNA gene (Escherichia coli numbering). Although a single mutation could cause elevation of tiamulin and valnemulin MICs, combinations of two or three mutations were necessary to produce high-level resistance. All the mutants were cross-resistant to lincomycin, chloramphenicol and florfenicol. Mutants with the A2058G or the A2059G mutation exhibited see more cross-resistance to macrolide antibiotics erythromycin, tilmicosin and tylosin. Pleuromutilin antibiotics inhibit

protein synthesis by binding to the bacterial 50S ribosomal subunit (Hunt, 2000; Yan et al., 2006). This group of antibiotics is derived from pleuromutilin, which is a natural JQ1 ic50 product of the basidiomycete Pleurotus mutilus (now called Clitopilus scyphoides) (Kavanagh et al., 1951). X-ray crystallographic data (Schlünzen et al., 2004; Davidovich et al., 2007) and biochemical information from chemical footprinting analysis (Poulsen et al., 2001; Long et al., 2006a) have revealed that this class of antimicrobial agents binds at the peptidyl transferase center and inhibits the peptide bond formation. Pleuromutilin antibiotics, such as tiamulin and valnemulin, have been exclusively used in veterinary medicine to treat infections caused by various pathogens in pigs and poultry. However, because of the emergence and spread of pathogenic bacteria resistant to existing antibiotics, there has been a renewed interest in developing novel pleuromutilin derivatives to treat bacterial infections in humans. Retapamulin, the first pleuromutilin derivative used in humans, has recently been approved for the topical treatment of skin infections caused by Staphylococcus aureus or Streptococcus pyogenes

(Jacobs, 2007). Furthermore, pleuromutilins exhibit excellent antibacterial activity against Mycoplasma spp., and valnemulin has been used in isolated cases in human medicine to treat resistant PAK5 mycoplasma infections in immunocompromised patients (Heilmann et al., 2001). Mycoplasma gallisepticum, which causes chronic respiratory disease (CRD) in chickens and sinusitis in turkeys, is one of the most significant pathogens of poultry (Ley & Yoder, 1997). Infections with M. gallisepticum are highly prevalent in almost all poultry-producing areas and cause major economic losses to the poultry industry (Mohammed et al., 1987). Tiamulin and valnemulin have been used in the treatment of M. gallisepticum infection, but the clinical use of these antibiotics could not eradicate the infection probably due to the emergence of resistant isolates.