Study Of The Susceptibility Of Aminoglycosides To Modifications By Recombinant Aminoglycoside Modifying Enzymes And The Molecular Mechanisms Of Drug Resistance

Aminoglycoside antibiotics (AGs) are a very important class of drugs for the treatment of infectious diseases and it has been more than 60 years since streptomycin had first been put into clinical use in 1944.During these years, bacteria have evolved and generated effective ways to fight against the selection of the antibiotics, the resistance mechanism can be generally classified into four categories:(1) Aminoglycoside modification enzymes (AMEs) which included three families namely O-phosphotransferase (APH), N-acetyltransferase (AAC), O-adenyltranferase (ANT); (2) 16s rRNA methylases which can modify 16s rRNA;(3) The reduced permeability of the outer membrane; (4) The drug efflux pump.Among these mechanisms the AMEs and 16s rRNA methylases are the predominant resistant mechanisms, the former can modify the amino and hydroxyl groups of the AGs, the later can modify the 16s rRNA. Both mechanisms can make AGs unable to bind with their target, and thus loss the antimicrobial activity.In this study, we evaluated the susceptibility of aminoglycoside antibiotics to AME modifications, and studied the molecular resistance mechanism of Acinetobacter baumannii (a class of predominant clinical pathogen) against AGs, aimed at elaborating resistance mechanisms based on AMEs and 16s rRNA methylases and provided rationale for semisynthetic drug design and also provided more effective drugs for clinical chemotherapy against infectious diseases.In the first part of this work, we obtained 6 recombinant aminoglycoside resistance enzymes through conventional molecular cloning methods and set evaluation methods and models by these enzymes. We evaluated the susceptibility of 8 AGs to predominant AMEs. The selected AGs included four natural products generated by bacterial and four semisynthetic AGs, they were kanamycin, amikacin (1-N-amino-hydroxybutyryl kanamycin A), gentamicin(a mixture of gentamycin Cl(～28.8%), Cla(～32.9%), C2a(～19.5%), C2(～19.7%)), etilmicin (1-N-ethyl gentamicin Cla), sisomicin, netilmicin(1-N-ethyl sisomicin), verdamycin, vertilmicin(1-N-ethyl verdamycin). The selected enzymes included AAC(6’)-Ie-APH(2")-Ⅰa, AAC(6’)-Ie, APH(2")-Ⅰa, ANT(2")-Ⅰa, AAC(6’)-Ib and AAC(6’)-Ib-cr which belonged to the three families of AMEs respectively. We compared different turnover efficiencies between different enzymes and also the substrate specificities for a single enzyme.The turnover efficiency of the acetylatic domain of AAC(6’)-Ie-APH(2")-Ia was the highest with kcat/Km values in the range of 105～107M-1S-1.The turnover efficiency of AAC(6’)-Ie was in the the range of 103～105M-1S-1. The turnover efficiency of AAC(6’)-Ib, AAC(6’)-Ib-cr, APH(2")-Ia and phosphorylatic domain of AAC(6’)-Ie-APH(2")-Ⅰa varied in the the range of 104～105M-1S-1. Generally speaking, Vertilmicin and amikacin was the most stable compounds to AMEs modification, followed by netilmicin and etilmiicin.The semisynthetic antibiotics were more stable compared with the corresponding parental compounds.Vertilmicin was the most stable compound for acetylations by AAC(6’)-Ⅰe and AAC(6’)-Ⅰe-APH(2")-Ⅰa with the lowestκcat/Km values of (1.2±0.1)×103 and (1.2±0.2)×105M-1s-1 for AAC(6’)-Ⅰe and AAC(6’)-Ⅰe-APH(2")-Ia acetylation respectively, which were 45.8 and 39.2 fold lower than those of the parental compound verdamicin, and 69.1-250.0 and 60.0-116.7 fold lower than those of the other six aminoglycosides. For ANT(2")-Ⅰa adenylation, vertilmicin exhibited the lowestκcat/Km values among the seven aminoglycosides (amikacin can not be modified by ANT(2")-Ia), which was 1.8-7.5 fold lower than those of others. The susceptibility of vertilmicin was relatively comparable for AAC(6’)-Ib and AAC(6’)-Ⅰb-cr acetylation, APH(2")-Ⅰa and AAC(6’)-Ⅰe-APH(2")-Ⅰa phosphorylation and did exhibit any advantage.Amikacin exhibited the most stable compound for the all the enzyme modifications except AAC (6’)-Ⅰe-APH (2")-Ⅰa (AAC (6’)-Ⅰe) acetylation, the long side chain of amikacin at the 1-N position can block amikacin binding with these enzymes, and weaken the metabolism of the substrate.In the second part of this work, we studied the molecular mechanism of the aminoglycoside antibiotic resistance. We described the distribution of 13 predominant AME genes and three 16S rRNA methylase genes in clinical isolated Acinetobacter baumannii which exhibited high level AGs resistance [gentamycin and amikacin MIC>1024μg/mL].Among all the AME genes,The detection rate of 16s rRNA methylases acetyltransferase gene aac(3)-I and aac(6’)-Ib were the highest, exhibited 71.1%(37/52)and 57.6%(30/52) respectively, followed by adenyltransferase genes ant(2")-Ⅰa and ant(3")-Ⅰa, the detection rate were 53.8% and 5.7% respectively. Phosphotransferase genes aph(3’)-Ⅲa, aph(2")-Ⅰb, aph(2")-Ⅰc, aph(2")-Id, aph(4’)-Ia and adenyltrasferase gene ant(4’)-Ⅰa were not detect out. The detection rate of armA was the highest among the 3 16s rRNA methylase genes, up to 98%(51/52). rmtB and rmtC were not detected out. The rate for detecting out three or more different genes in a single strain simultaneously is 69.2%(36/52). We constructed genomic libraries and screened for transformants carried resistance genes, the result showed that armA gene mediated the high level resistance to AGs in clinical strains. Vertilmicin did not exhibit any advantages against 16s rRNA methylase producing strains.