| BackgroundNontuberculous mycobacteria(NTM)refers to all Mycobacterium species besides Mycobacterium tuberculosis complex and Mycobacterium leprae.With the increased numbers of HIV patients and immunosuppressed people,the incidence and prevalence of NTM disease have been rising in the worldwide,also in China.NTM has become an important threaten to human's health and life.Previous researches show that there is unequally geographical distribution of NTM distribution in China,at the same time,the pathogenicity and drug sensitivity of different NTM strains signified sharp difference.Understanding this diversity difference is important to clinical treatment However,the situation of lacking of epidemiology systemic data of geographic distribution is still existed in China,especially in regions with high prevalence of NTM diseases.Identification of bacterial species,drug susceptibility testing and clinical data analysis of NTM are of great significance to the comprehensive understanding the biological characteristics of mycobacteria and the making clinical strategy.In this study,body fluid specimens was first collected from patients with suspected tuberculosis in China;second,NTM was cultured through traditional biochemical tests;third,strains were further identified by by multi-target gene sequencing;finally,the differences of NTM strains distribution in different regions was analyzed according to information above.Based on the drug sensitivity characteristics,the potential drug resistance mechanisms of dominant bacteria of nontuberculous mycobacteria were investigated in this research in order to provide the evidence for showing the epidemiology feature of NTM and making the treatment strategy in clinical practice.Part ? Diversity distribution of nontuberculous mycobacteria in eastern and southern China:across-sectional studyObjective:To analyze the distribution charactersistics of NTM strain in China,especially in the areas with high incidence,across-sectional study was conducted in this research.Methods:Clinical isolated strains were collected from Guangzhou Chest Hospital in southern China and Shanghai Chest Hospital in eastern China from January 2012 to December 2014.Identification of these clinical isolated strains were performed by sequencing on 16S rRNA,rpoB,hsp65 and ITS.Results:(1)A total of 1450 isolated strain were collected by continuous recruitment.Five of the most common isolated strains were Mycobacterium abscess composite group(605 strains,41.7%),Mycobacterium intracellular complex group(330 strains,22.8%),Mycobacterium gordon(244 strains,16.8%),Mycobacterium kansasii(179 strains,12.3%)and Mycobacterium fortuitum(50 strains,3.4%).(2)The distribution of NTM strains were different in Shanghai and Guangzhou.A total of 512 NTM strains were isolated in Shanghai.The most common of isolated strains of NTM were Mycobacterium intracellulare,Mycobacterium abscess,Mycobacterium kansasii,Mycobacterium avium and Mycobacterium marseille.A total of 938 NTM strains were isolated in Guangzhou.The most common of isolated strains of NTM were Mycobacterium abscess,Mycobacterium marseille,Mycobacterium gordon and Mycobacterium intracellulare.Part ? Distribution of Mycobacterium kansasii in China and correlation analysis of drug resistance characteristics to clarithromycinObjective:To analyze the distribution of Mycobacterium kansasii in Chinese and evaluate the drug sensitivity characteristics among different subtypes,this research was conducted.Methods:Mycobacterial strains were collected during 2008-2015 by National Drug-resistant Tuberculosis Control Center.All of Mycobacterium kansasii strains were divided into different subtypes by restriction fragment analysis.The drug sensitivity test of different subtypes of Mycobacterium kansasii was performed in vitro.Results:A total of 78 strains of Mycobacterium kansasiensis were collected.Amikacin was shown with the highest antimicrobial activity against Mycobacterium kansasii.The antimicrobial activity of quinolones to Kansas showed a wide range.The tolerance of levofloxacin was significantly higher than that of moxifloxacin(16.7%,P =0.001)and gatifloxacin(19.2%,P =0.005).For antimicrobial activity of rifamycin,the proportion of rifampicin resistant strains was significantly higher than that of the rifabutin resistant strains(P=0.006).There were 4 different subtypes of Mycobacterium kansasii which were analyzed by PCR-restriction enzyme analysis(PRA)in these 78 strains,i.e.subtypes I,II,III and IV.Subtypes I was the most common(56/78,71.8%).The rate of clarithromycin resistance in subtype I was significantly higher than its in other subtypes(26.8%;vs4.5%;P =0.031).Among the 16 strains of Mycobacterium kansasii with clarithromycin resistance,9(56.2%)of them were shown with 23 S rRNA gene mutation.Part ? Clinical manifestation of Mycobacterium avium-intracellulare complex disease and the analysis of its drug sensitivity test's resultObjective:NTM strains was collected from the tuberculosis specialist hospital in China.The clinical characteristics of Mycobacterium avium-intracellulare was summarized and the characteristics of the drug susceptibility analyzed.Methods:Mycobacterial strains were collected from 4 tuberculosis hospital from 2011 to 2013 and the strains identification were performed by 16S rRNA,rpoB,hsp65 and ITS sequencing.The clinical data of patients with Mycobacterium avium-intracellulare infection was collected.Vitro Drug susceptibility test on the common used 12 types of antibacterial drugs was performed for the identified Mycobacterium avium-intracellulare strains.Results:(1)253 strains of Mycobacterium avium-intracellulare were obtained in this study.Among them,188 strains of Mycobacterium intracellular(41.6%)and 65 strains of Mycobacterium avium(14.4%)was isolated.(2)The rate of patients ?65 years old(41.5%)with M.intracellulare infection was significantly higher than that of M.avium infection(23.1%)(OR=2.36,95%CI:1.25 to 4.46,P =0.008);the numbers of patients diagnosed of chronic obstructive pulmonary disease in M.intracellulare disease group were significantly higher than those of patients in M.avium disease group(OR = 2.84,95%CI:1.56?5.19,P = 0.001).(3)Larithromycin,crispycin and rifambutin showed strong antimicrobial activities with Mycobacterium intracellular and Mycobacterium avium,by the rate of drug resistance below 5%.However,azithromycin,levofloxacin,gatifloxacin and streptomycin showed little antimicrobial activities with Mycobacterium intracellular and Mycobacterium avium by the rate of drug resistance rate above 70%.(4)Among the isolated strains,mycobacterium avium strains were with significantly stronger resistant characteristics to moxifloxacin and linezolid than Mycobacterium intracellular strains(10.8%,1.6%,?2=10.71,P<0.05;40.0%,8.5%,x2=44.71,P<0.05).However,the resistance rate of Mycobacterium avium to rifampin was significantly lower than that of Mycobacterium intracellular(38.5%,66.0%,?2=9.01,P<0.05).Part ? A survey of the prevalence of Mycobacterium abscessus and its antimicrobial susceptibility in a general hospital,ChinaObjective:Mycobacterium abscesses isolated from respiratory airway tract samples in patients from general hospitals was collected in this research and drug sensitivity tests were performed to analyze the characteristics of drug sensitivity of Mycobacterium abscesses.Methods:Mycobacterial strains from Beijing General Hospital were collected from 2012.4-2015.3.Thees strains were identified by gene sequencing of 16S rRNA,rpoB,hsp65 and ITS.After identified as Mycobacterium abscessus,these strains were obtained for drug susceptibility testing on 13 antibacterial drugs,which were commonly used in treatment of Mycobacterium abscessus complex infection.Results:(1)A total of 61 NTM isolated stains were included in the study for further molecular identification.The detection rate of Mycobacterium abscesse ranked second(22/61,36.1%).(2)Clarithromycin had high antibacterial activity against Mycobacterium abscessus strains with MIC50 and MIC90 of 0.06 ?g/mL and 8?g/mL,respectively.For fluoroquinolones,moxifloxacin and levofloxacin showed different drug resistance profiles,and the drug resistance rates of moxifloxacin(6/22,27.3%)were significantly lower than those of levofloxacin(17/22,77.3%)(P<0.01).(3)All the clarithromycin-resistance isolates of Mycobacterium abscessus had 23 S rRNA mutations associated with clarithromycin resistance.Conclusion(1)Distribution of NTM strains in China has significant geographical differences.Mycobacterium abscessus is mainly distributed in the southern part of China.Mycobacterium intracellular is mainly distributed in eastern China.This difference in NTM distribution may reflect the strains in the environment..(2)In vitro antimicrobial drug susceptibility test of Mycobacterium kansasii,Amikacin was the most active antibacterial drug.While among these strains,the drug resistance of clarithromycin was high.In the molecular subtypes of Kansas in China,the dominant subtype was subtype I,which is with significantly higher resistance to clarithromycin than other subtypes.(3)Mycobacterium avium complex group is one of the most common NTM in China,and there is a significant difference in the drug resistance spectrum between Mycobacterium intracellulare and Mycobacterium avium.In clinical observation,mycobacterial pulmonary disease is more likely to occur in elderly and patients with chronic obstructive pulmonary disease.(4)Clarithromycin,amikacin,and linezolid show excellent antibacterial activity against Mycobacterium abscessus in vitro,while levofloxacin,imipenem,and sulfamethoxazole have poor antibacterial activity against Mycobacterium abscessus.Gene mutations on the 23 S rRNA can be used as a predictor of clarithromycin resistance in Mycobacterium abscessus. |
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