Inhibition of Protein Synthesis on the Ribosome by Tildipirosin Compared with Other Veterinary Macrolides

Abstract

Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC 50 ], 0.23 ؎ 0.01 M) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site. Recent approval has been given in Europe and the United States for the use of tildipirosin (20,23-dipiperidinyl-mycaminosyltylonolide; Zuprevo) in combating bovine and swine respiratory tract infections. Tildipirosin is a derivative of the natural compound tylosin with two piperidine rings and no mycarose sugar (Fig. 1). Despite its wide use as a veterinary macrolide, tylosin is not particularly effective at penetrating the outer membrane of Gram-negative pathogens (Table 1), and the substitutions in tildipirosin were made to improve efficacy against Mannheimia haemolytica and Pasteurella multocida, which are the two main etiological agents of bovine respiratory disease (8,22). Tildipirosin has additionally proven effective against Histophilus somni, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, and Haemophilus parasuis (6), which can also be associated with animal respiratory diseases. Different arrangements of hydrophobic and basic substituents are present in an earlier tylosin derivative, tilmicosin (20-dimethylpiperidinyl-mycaminosyltylonolide; Micotil) and the 15-membered triamilide tulathromycin (Draxxin) (Fig. 1), which are used for similar indications.Here we quantified the inhibitory activity of tildipirosin (MSD Animal Health) on protein synthesis and compare it with tylosin, tilmicosin (Sigma), and tulathromycin (extracted from Draxxin [Pfizer]). The concentration of each macrolide that inhibits 50% of protein synthesis (IC 50 ) was determined in an in vitro transcription/translation assay, and the effects of methylations and mutations at rRNA nucleotides within the macrolide binding site were evaluated in cell cultures. The data have been visualized using computationally calculated models of the binding site and reveal subtle differences in macrolide contacts, indicating how changes in the rRNA target have distinct effects on drug efficacy.An in vitro transcription/translation system based on cell extracts containing susceptible, wild-type Escherichia coli ribosomes (Promega) was adapted to translate the 27-kDa green fluorescent protein (GFP). GFP mRNA was transcribed from 3 g of plasmid pIVEX (Roche) in 50 l of 100 mM piperazine-N,N=-bis(2-ethanesulfonic acid) (PIPES)-KOH (pH 7.9), 5 mM MgCl 2 , 0.5 mM CaCl 2 , 100 mM KCl, 5 mM NH 4 Cl, 1 mM dithiothreitol, and 1 mM spermidine and translated to produce [35 S]methioninelabeled protein. Synthesis was followed over 155 min with macrolide an...

Antimicrobial Sensitivity Testing of Mycoplasma bovis Isolates Derived from Western Canadian Feedlot Cattle

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In vitro activity of tylvalosin against Spanish field strains of Mycoplasma hyopneumoniae

“…Reduced susceptibility to spiramycin but not tylosin was previously reported to be associated with the substitution U2504G ( Escherichia coli numbering) in the 23S rRNA-encoding DNA in one mutant of an M. smegmatis strain (Bei-Bei and others 2011). Likewise, other mechanisms of macrolide resistance could also result in resistance to tylosin but not spiramycin or tylvalosin (Leclercq 2002, Andersen and others 2012). The appearance of resistance to tilmicosin, tylosin and lincomycin was previously described in Belgian field strains of M. hyopneumoniae (Vicca and others 2004, Stakenborg and others 2005) and, more recently, in Thai field strains (Thongkamkoon and others 2013).…”

Effect of subtherapeutic vs. therapeutic administration of macrolides on antimicrobial resistance in Mannheimia haemolytica and enterococci isolated from beef cattle

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