Study On The New Regimen For Treating Mycobacterium Abscessus Infection

In recent years,Mycobacterium abscess(Mab),an emerging pathogens with natural multiple drug resistance,shown the rapid increase of the number of infection patients.Only few antibiotic can be used in the treatment,Mab infection has become a difficult clinical problem.Unfortunately,the research progress of anti-Mab drug screening and detection system are obsolete,which seriously restricts the development of new drugs.The main objective of this study was to build a new model for the screening of anti-Mab drugs in vivo,and screen out a new treatments that can effectively treat Mab infection based on these models.First of all,we established a new type of macrophage infection model for high throughput drug screening based on builded marker freer autoluminescent Mab(UA1Mab).The model can be continuous detection of the same sample,test process is simple and quickly,only put plates in enzyme standard to test(a 96-well plates only need 10 seconds)without the traditional complicated operation such as,cleaning,rupture,centrifugal,plate count and so on.More over the test cost is very low(without pancreatic enzyme,culture medium,tablet and other consumables).By comparing the growth curves of UA1Mab and WtMab in nude mice,it was confirmed that UA1Mab could replace WtMab in vivo model construction.Light from a live infected mouse can be detected daily(or even hourly if needed)within one minute including anaesthesia time.The obvious advantages of this model are:1)This model can predict the relative activities of regimens quickly within 2 to 4 days.2)It is convenient,economic as tail vein injection needs not special aerosol machine,the same batch of live mice can be monitored easily and the light detection device is inexpensive.3)The number of animals used deduced while the power of statistical analysis was enhanced.4)BALB/c mice are widely used,easy to be handled with and much more economical than immunocompromised mice.In order to better simulate the clinical Mab pulmonary infection patients,we adopt inhalation exposure system(Glas-Col)to infected NSI and Nude mouse by aerosol way,the experimental results shown that both two kinds of mice can be used to build Mab pulmonary infection mice model.Compared with the traditional method,the aerosol infection system can simulate the respiratory infection and the dose of infection can be controlled well.After using UA1Mab for aerosol infection in mice,it was possible to determine the bacterial load by detecting the luminescence value from the t mice lung homogenates.Without plate count,it can largely reduce the workload and the experiment cost experimenters,the whole experiment cycle shorten at least a week,at the same time avoid the risk of pollution during the plate culture.Through the treatment outcome of chronic lung infection mice,we found Clofazimine(CFZ)has delayed antimicrobial activity against Mab in vivo(the short-term treatment has no obvious effect,only gradually show efficacy after a long treatment).However,long-term treatment with CFZ can cause severe pigmentation in patients,and even patients who suffer from depression commit suicide.Through the use of established drug screening model,we found a new anti-Mab drug combination:CFZ,TB47 and Roxithromycin(ROT).CFZ can significantly inhibit bacterial regeneration in ROT treatment.The new compound TB47 had no obvious therapeutic effect on Mab in vitro when it use alone,but it could significantly improve the efficacy of CFZ in vitro and in vivo.Three drugs used in combination with antibiotics not only effectively overcome the macro lides poor long-term efficacy in patients with lung infection,and active faster.It shorted the required treatmen of CFZ,which can effectively reduce the side effects of CFZ.In addition,in order to verify whether the combination of drugs selected in this study has broad spectrum activity,we used the Alarm Blue method to detect the drug sensitivity of 20 different clinical Mab strains.The results showed that the growth inhibition rate of three drugs was more than 84 percent,indicating that the combination of the three drugs could be an effective treatment for Mab pulmonary infection.In conclusion,we have successfully developed M.abscessus macrophage infection model,both acute and chronic M.abscessus murine infection models using AlMab.We showed that the RLUs were correlated with the CFU counts very well in organ homogenates.Being verified using these models,the new triple therapy(TB47+ clofazimine + roxithromycin)showed promising in vivo activity and should be further evaluated in clinical trial.