Determining the limits of the evolutionary potential of an antibiotic resistance gene

The AAC(6') enzymes inactivate aminoglycoside antibiotics by acetylating their substrates at the 6' position. Based on functional similarity and size similarity, the AAC(6') enzymes have been considered to be members of a single family. Our phylogenetic analysis shows that the AAC(6') enzymes instead belong to three unrelated families that we now designate as [A], [B], and [C] and that aminoglycoside acetylation at the 6' position has evolved independently at least three times. AAC(6')-Iaa is a typical member of the [A] family in that it acetylates tobramycin, kanamycin, and amikacin effectively but acetylates gentamicin ineffectively. The potential of the aac(6')-Iaa gene to increase resistance to tobramycin, kanamycin, or amikacin or to acquire resistance to gentamicin was assessed by in vitro evolution. Libraries of PCR mutagenized alleles were screened for increased resistance to tobramycin, kanamycin, and amikacin, but no isolates that conferred more resistance than the wild-type gene were recovered. The library sizes were sufficient to conclude with 99.9% confidence that no single amino acid substitution or combination of two amino acid substitutions in aac(6')-Iaa is capable of increasing resistance to the antibiotics used. It is therefore very unlikely that aac(6')-Iaa of S. typhimurium LT2 has the potential to evolve increased aminoglycoside resistance in nature. The practical implications of being able to determine the evolutionary limits for other antibiotic resistance genes are discussed.