Drug Susceptibility Testing And Molecular Mechanism Of Resistance To Clarithromycin In Mycobacterium Abscessus Complex

ObjectivesTo explore the prevalence of Mycobacterium abscessus complex (MABC) among non-tuberculous mycobacteria (NTM) of China, and to make clear the drug susceptibility profiles of MABC, further to investigate the drug resistance mechanism to clarithromycin. So as to provide evidence for the treatment of MABC lung diseasesMethods1. Multi target gene PCR-sequencing of 16S rRNA, hsp65, rpoB and ITS was performed to identify 472 clinical isolates primarily identified as NTM by PNB.2. Minimal inhibitory concentrations (MIC) were tested by microplate Alamar blue assays to assess the drug susceptibility of 16 drugs to MABC.3. Synergistic activities of two drugs were tested by checkerboard dilution method for part isolates of MABC which were randomly selected.4. Incubation was prolonged from 3 days to 14 days to detect induced resistance of clarithromycin. rrl and erm(41) genes were PCR-sequencing to compare the phenotype with genotype of clarithromycin resistance.5. Genotyping was performed by using variable number of tandem repeats(VNTR) for all the MABC isolates.Results1.452 isolates were identified as NTM. The number of MABC isolates was 100(22.1%), ranking the second position. Among the 100 MABC strains,75 strains were M. abscessus and 25 strains were M.massiliense.2. In vitro drug susceptibility testing, five drugs including linezolid, clarithromycin, azithromycin, amikacin, tigecycline showed perfect antimicrobial activities, the proportion of drug resistanceagainst which were 2%,7%,9%,15%,20%, respectively. Other five drugs including meropenem, levofloxacin, gatifloxacin, minocycline, cotrimoxazole showed poor antimicrobial activities with resistant rates of 85%,79%,73%,70%,43%, respectively. The activities of moxifloxacin, cefoxitin, imipenem, tobramycin were intermediate. Clofazimine showed lower MIC.3.28 strains (19 strains of M. abscessus,9 strains of M.massiliense) were tested for combined drug susceptibility testing. It’s demonstrated thatclarithromycin combined with moxifloxacin showed more favorable synergistic effects against M.massiliense(88.9%) than M.abscessus (5.2%). For all the strains, good synergistic activities of linezolid combined with amikacin, tigecycline combined with moxifloxacin were observed, with the rates of synergistic effects 42.9%,57.1%, respectively.4. Among 75 strains of M.abscessus,51 strains(68%) showed clarithromycin induced resistance, but none of the 25 strains of M.massiliense showed clarithromycin resistant. The difference was significant.Among 9 strains of clarithromycin acquired resistant strains,6 strains(66.7%) had rrl mutation.All isolates of M. abscessus harbored an intact enn(41) gene and all isolates of M. massiliense had two deletions in erm(41).Furthermore, M. abscessus strains had a T/C polymorphism at the 28th nucleotide:57 isolates were T28 sequevars confering inducible clarithromycin resistance, while 18 isolates were C28 sequevars confering susceptible to clarithromycin.5. Each VNTR loci has obvious polymorphism and HGI was 0.996. Cluster analysis showed all the isolate were divided into 3 genetic groups.ConclusionsMABC is one of the commonly encountered NTM in china, with the notable characteristic of high resistance and limited available drugs. The real species that is refractory to treatment is M. abscessus because of clarithromycin induced resistance. Accurate identificationan and reliable durg susceptibility testing is essential to guide clinical treatment of MABC lung diseases.