Drug Resistance Rates Of Non-tuberculosis Mycobacteria To 29 Kinds Of Antibiotics In China Mainland-a Meta Analysis And The Research Of Intracellular Drug Susceptibility Test For Mycobacterium Abscess

PART I Drug resistance rates of non-tuberculosis mycobacteria to 29 kinds of antibiotics in China mainland----a meta analysis BackroundExcept for mycobacterium tuberculosis and mycobacterium leprae,the other species of mycobacterium are called non-tuberculosis mycobacterium(NTM).The NTM positive rate in positive acid fast stain samples is getting higher.Most NTM are highly resistant to first-line anti-TB drugs leading to shortage of antibiotics for NTM infection patients.It's necessary to conduct an in vitro drug susceptibility test before the course of treatment to find out the sensitive antibiotics.The popular NTM species in different countries and areas are different,and different NTM species are sensitive to different antibiotics.The pity is,a large-scale drug resistance evaluation all over the nationwide is still in need.ObjectivesTo analysis the in vitro drug resistance rates of NTM to common 29 kinds of antibiotics in China mainland.MethodsTo collect literatures about drug susceptibility of NTM in Chinese mainland by searching databases like Pubmed,the Cochrane Library,Embace,Chinese National Knowledge Infrastructure(CNKI),WANGFANG DATA(CECDB),China Science and Technology Journal Database(CQVIP).Inclusion and exclusion criteria are worked out and we evaluate qualities,extract data,recognize and eliminate duplicated data with the included literatures.To detect heterogeneity and publication bias of included literatures and to merge the drug resistant rates by Stata software.Some subgroups are set to analyze drug resistant rates within different area,different NTM species and different detection methods.ResultsIn all,152 literatures containing 11175 strains of NTM are finally included.Among them,there are 1605 strains of mycobacterium avium and mycobacterium intracellulare,1276 strains of mycobacterium chelonei and mycobacterium abscessus,218 strains of mycobacterium fortuitum,430 strains of mycobacterium kansasii,97 strains of mycobacterium smegmatis,41 strains of mycobacterium gordonae and 5 strains of mycobacterium marianum,and the others are NTM without identifying to species.The antibiotics with the lowest resistance rate are clarithromycin(CLA)9.5%(6.6%-12.4%),cefoxitin(CXT)15.9%(5.8%-26.1%),azithromycin(AZM)17.9%(4.0%-31.9%),tobramycin(TOB)21.0%(11.4%-30.5%),linezolid(LIN)28.1%(20.0%-36.2%),clofazimine(CLO)31.0%(8.8%-53.3%),rifabutin(RFB)37.7%(16.5%-58.9%),amikacin(AMK)39.3%(28.8%-49.8%),moxifloxacin(MFX)40.0%(22.2%-57.9%)?Mycobacterium chelonei and mycobacterium abscessus are most susceptible to CXT,AZM,CLA,TGA,CLO,AMK,TOB and LIN with drug resistance rate of 7.6%(2.6%-12.5%),8.3%(1.4%-5.3%)?8.8%(4.7%-12.9%)?19.7%(12.6%-26.7%)?20.9%(9.3%-32.4%)?21.1%(16.2%-26.0%)?22.4%(6.2%-38.5%)?27.2%(11.8%-42.6%)in sequence.Mycobacterium avium and mycobacterium intracellulare are most susceptible to SMZ,CLA,RFB,AZM,TOB,MFX,AMK and LIN with resistance rate of 1.9%?4.1%(1.7%-6.4%)?20.5%(7.0%-33.9%)?22.2%(0%-53.5%)?27.7%(0%-74.3%)?29.8%(16.0%-43.7%)?30.0%(7.1%-52.8%)and 37.3%(15.2%-59.3%)in sequence.ConclusionThe most susceptible antibiotics for NTM are CLA,CXT,AZM,TOB,LIN,CLO,RFB,AMK and MFX.Those for mycobacterium chelonei are AZM,CXT,CLA,LIN,AMK and TOB.And for mycobacterium abscessus are CXT,CLA,AZM,TGA,CLO,AMK,TOB and LIN.The most susceptible antibiotics for mycobacterium avium are CLA,RFB,MFX,AMK,LIN and AZM.Those for mycobacterium intracellulare are SMZ,CLA,MFX,AZM,TOB,RFB and LIN.And for mycobacterium kansasii are CLA,LIN,RFB,PTO,AMK,LVFX,MFX,RFP,EMB,IMP,CXT and SMZ.PART ? The research of intracellular drug susceptibility test for mycobacterium abscessBackgroundMycobacterium abscess is one of the most common species of non-tuberculosis mycobacteria(NTM)and belong to intracellular bacterium.They are engulfed by macrophages at the first time when they invade into human body,and most of the mycobacterium will be killed.But through a variety of mechanisms to escape from killing,a small number of bacilli can survive in macrophages.Clinical and Laboratory Standards Institute(CLSI)Currently recommend liquid Broth micro-dilution based method to test in vitro drug susceptibility,but they also point out that in vitro drug susceptibility test has much limitation and is not very accurate for intracellular bacterium,especially in pulmonary mycobacterium abscess infection patients.The drugs which mycobacterium abscess show high susceptibility to in vitro test may not practice well in clinical treatment.ObjectivesTo research the intracellular drug susceptibility test method for mycobacterium abscess.Problems to be solved are as follows:To select the suitable cell as host.Find out the condition for cell differentiation.Find out the best MOI,the suitable time for bacteria to infect cells and for antibiotics to kill bacteria.And finally,compare the bacteria inhibition rates(BIR)between extracellular and intracellular.MethodsFind out the best concentrations and time for PMA inducing cell differentiation by concentration and time gradient.Cell differentiations are measured by microscope and flow cytometer.The best MOI and time for infecting are detected by bacterial colony counting at time of 2,12,24,48 hours later with MOI scale of 0.001,0.01,0.1,1,10,100 and 1000.Detect the best time for antibiotics inhibiting bacteria by colony counting on day 0,1,2,3,4 and 5.Compare intracellular BIR and extracellular BIR by t test.ResultsThe best concentration and time for PMA to induce cell differentiation is 100ng/ml and 48 hours.Mean differentiation rate is 87.0%±5.1%and positive rates of CD11b and F4/80 are 89.78%and 87.54%.The best MOI and time for infecting are 1 and 24 hours.The best time for antibiotics to inhibit bacteria is 24 hours.The intracellular clarithromycin concentration is 2 folds of that of extracellular,and the intracellular linzolid concentration is 1/10 of that of extracellular.When the drug concentration is 256ug/ml,128ug/ml,64ug/ml,32ug/ml,16ug/ml,8ug/ml,4ug/ml,2ug/ml,1ug/ml,0.5ug/ml,0.25ug/ml,0.125ug/ml,the mean intracellular BIR of clarithromycin is 94.11%±3.77%,93.00%±4.30%,91.78%±9.83%,91.33%±8.47%,93.46%±4.83%,90.80%±7.82%,89.50%±15.66%,86.76%±11.27%,87.67%±10.54%,84.87%±15.51%,75.46%±24.01%,74.94%±15.95%in sequence,and the mean extracellular BIR is 98.68%±2.19%,98.68%±2.31%,99.32%±0.67%,99.44%±1.06%,98.96%±2.00%,98.87%±3.07%,97.83%±2.99%,98.76%±2.34%,97.48%±5.02%,98.51%±2.30%,95.58%±9.15%,88.89%±21.78%in sequence.At every concentration,the intracellular BIR is lower than extracellular BIR with statistic difference(p<0.05).When the drug concentration is 256ug/ml,128ug/ml,64ug/ml,32ug/ml,16ug/ml,8ug/ml,4ug/ml,2ug/ml,lug/ml,0.5ug/ml,0.25ug/ml,0.125ug/ml,the mean intracellular BIR of linzolid is 88.97%±15.09%,92.56%±9.07%,89.70%±2.21%,84.06%±16.58%,67.24%±22.54%,47.67%±23.55%,40.65%±29.30%,35.55%±24.05%,31.92%±24.85%,26.59%±21.13%,25.67%±23.24%,17.850%±20.39%in sequence,and the mean extracellular BIR is 99.63%±0.46%,99.72%±0.42%,94.75%±20.24%,99.23%±0.82%,95.84%±7.99%,95.55%±6.08%,88.80%±16.03%,79.53%±22.56%,73.09%±28.75%,59.84%±30.85%,50.71%±28.54%,34.21%±20.99%in sequence.At every concentration except 64ug/ml,the intracellular BIR is lower than extracellular BIR with statistic difference(p<0.05).ConclusionWe successfully establish the intracellular drug susceptibility test method for mycobacterium abscess.The best MOI,the best co-culture time,and the best time for antibiotics inhibiting is 1,24 hours and 24 hours.The intracellular clarithromycin concentration is higher than extracellular concentration,and the intracellular linzolid concentration is lower than extracellular concentration,but intracellular BIR of both antibiotics are significantly lower than extracellular BIR.