The Study On The Drug-resistance Of Mycobacterium Tuberculosis To Aminoglycoside And Polypeptide Antibiotics And Relationship Between The Mutations In Gene RpsL, Rrs With The Drug-resistance

Background and objective:Aminoglycoside and polypeptide antibiotics are a class of drugs for anti-TB treatment earlier. Due to the lack of new drugs and the increasing rates of MDR-TB(defined as resistance to at least the two most powerful anti-TB drugs, RMP and INH) the aminoglycosides Kanamycin(KM),amikacin(AM) and the polypeptide Capreomycin(CPM) are key drugs for the treatment of multidrug-resistant tuberculosis(MDR-TB). MTB cross-resistance occurs between aminoglycosides and polypeptides or among different aminoglycosides, Inappropriate use of these drugs may lead to ineffective outcome and increase drug aside effects. To understand the drug susceptibility testing (DST) characteristic of clinical isolates to these drugs will provide the scientific evidences for clinical treatment. To understand the molecular mechanism of drug resistance and cross-resistance is conducive to rapid diagnosis. The aim of this study is to observe the level of drug resistance and cross-resistance of SM,KM,AM,CPM for MTB clinical isolates by DST, and to study the association of mutation in MTB gene rpsL&rrs and the level of these drugs resistance and cross-resistance.Methods:(1) Collect the L-J DST results of SM,KM,AM and CPM of768MTB clinical isolates from hospitalized patients with TB, to analyze the resistance rates and cross-resistance rates of these drugs, and the different drug resistance characteristics between initial treatment and retreatment cases.(2) The liquid dilution method Microdilution alamar blue assay (MABA) was performed to detect MICs of SM,KM,AM,CPM in117MTB clinical isolates.(3) Gene rrs and rpsL of64MTB clinical isolates,52isolates resistant to at least one injectable anti-TB drug and12isolates sensitive to all four injectable anti-TB drugs,were sequenced, to analyze the relationship of the mutation in gene rrs, rpsL and the resistance and cross-resistance of SM,KM, AM,CPM.Results:(1) Of the768MTB clinical isolates,35.3%.16.8%,15.2%,7.8%were resistant to SM,KM,AM and CPM, respectively. The resistance rates of KM and AM were no significant difference.(x2=0.691, P=0.404) SM resistance rate was20.2%and60.9%in initial and retreatment groups, respectively。Of128MDR-TB isolates, SM resistance rate(81.3%) was higher than KM, AM, CPM (7.8%、7.8%、3.1%). Of92XDR-TB isolates, CPM resistance rate (46.7%) was lower than SM, KM, AM (91.3%、96.7%、82.6%).(2) The MICs of SM,KM,AM,CPM in117clinical isolates were determinated by MABA. Of83SM resistant isolates,63isolates (75.9%) were highly SM resitance,19isolates (21.7%) were low resistance,2isolates (2.4%) were moderate resistance.78.3%of KM resistance isolates and94.7%of AM resistance isolates were high-level resistance isolates(MIC≥128μg/ml),21.7%of KM resistance and5.3%AM resistance isolates were low-level resistance, no moderate-level resistance isolates were observed. The MIC range of CPM resistant isolates was5-20μg/ml.18strains were resistant to both KM and AM, and all of these strains were highly resistant to both drugs, accounted for78.26%and94.73%in KM and AM drug-resistant strains, respectively, there was no significantly statistical difference (x2=1.157,P=0.282>0.05).5strains were low level resistant to KM, but sensitive to AM.13strains were resistant to both AM and CPM, accounted for78.3% and94.7%in AM and CPM resistant strains, respectively.(P=0.195>0.05),and all of these13strains were highly resistant to both AM and KM.(3) Gene rrs and rpsL of64isolates were sequenced. Of52SM resistant isolates mutation in codon43or88of rpsL gene were observed in42(80.8%) isolates, mutation in rrs514were observed in8(15.4%) isolates, no mutation were observed in2(3.8%) isolates. The32isolates with mutation in codon43(Lys43Arg) with SM MIC no less thanl28μg/ml.9isolates with mutation in rpsL263A→G (Lys88Arg) and1isolate with mutation in rpsL263A→T (Lys88Met), the SM MICs of these isolates were4-64μg/ml. rrs A514C were observed in9isolates, SM MIC in8of these isolates were4-8μg/ml,1isolate was2ug/ml. Gene rrs A1401G mutation were observed in18isolates which were high-level resistant to both KM and AM. Gene rrs A1401G mutation were observed in all isolates which were resistant to SM, KM, AM three drugs, and mutations in rrsA514C or rpsL were also observed in these isolates.Conclusion:(1) The resistance rates of KM and AM were similar, the resistance rate of SM was significantly higher than KM and AM, CPM resistance rate was significantly lower than other there drugs. The majority of initial treatment TB patients are still sensitive to SM, and most MDR-TB patients are sensitive to KM, AM, and CPM. Most of the retreatment patients and MDR-TB patients were resistant to SM., but were susceptible to KM, AM, and CPM. The resistance rate of CPM in XDR-TB patients was lower than other three drugs.(2) The majority of the clinical isolates of resistant to SM/KM/AM were high-level resistance (MIC≥128μg/ml),the majority of the CPM-resistant clinical isolates were low-level resistance. Isolates of resistant to KM showed high-level of cross-resistance with AM. A small proportion of KM resistant isolates were low-level resistant to KMamycin and susceptible to AM. Almost all CPM resistant isolates were resistant to KM, AM, and CPM. However, a part of isolates of resistant to both KM and AM were susceptible to CPM.(3) Mutation in codon43of rpsL (Lys43Arg) was associated with high-level resistance to SM, mutations in codon88(Lys88Arg/Met) of rpsL were associated with moderate resistance to SM, mutation in rrs514A→C was associated with low-level resistance to SM. Combination of mutations in AM/KM target gene rrs A1401G and SM target gene rpsL, rrs514may be the molecular mechanism of resistant to SM,KM and AM three drugs.