Systemic Taxonomy Of Some Pathogenic Aerobic Actinobacteria Strains In Pulmonary Infection

Objectives: 1.To study the distribution characteristics of pathogenic aerobic actinomycetes in pulmonary infection.2.To compare and evaluate the stability and reliability of 16 S rRNA,hsp65 and rpoB genes in the classification of Non-tuberculosis mycobacteria.3.To design and develop the specific probe for identification of Nocardia strains,and to improve the clinical classification and diagnosis capability on Nocardia infection.Methods: 1.The samples of aerobic actinomycetes infected with lung infection in Guizhou province and other cities were collected,purified and observed.2.The pathogenic strains of Nocardia and neighbor species were cultured on brain heart infusion agar medium,and the Mycobacterium strains were cultured and identified on PNB / TCH medium.3.DNA was extracted,then the Mycobacterium strains were identified by PCR-fluorescent probe and gene chip method,the 16 S rRNA gene was amplified by PCR and sequenced.4.The bacterial DNA was extracted and its 16 S rRNA,hsp65 and rpoB genes were amplified by PCR,followed by sequencing,then the phylogenetic tree of Non-tuberculosis mycobacteria was constructed by Neighbour-Joining method and verified by the Maximum Likelihood analysis.5.The molecular beacon probe was designed based on the partial secA1 gene sequences of Nocardia strains,and the fluorescence signals were detected after real-time fluorescence quantitative PCR.Results: 1.A total of 43 pathogenic strains of aerobic actinomycetes were collected in Guizhou and other areas,the result showed that 11 of the above strains belonged to Nocardia,1 of them belonged to Gordonia bronchialis and 31 strains were Non-tuberculosis mycobacteria.2.A total of 31 cases of non-tuberculosis mycobacteria infection were found in Guizhou province,the rate was 2.06%,the isolated strains were classified into the species of Mycobacterium avium,Mycobacterium intracellulare,Turtle/Mycobacterium abscessus,and Mycobacterium fortuitum,Mycobacterium scrofulaceum,Kansas Mycobacterium tuberculosis and Mycobacterium xenopi,the proportion of male to female was 1.21:1,and patients at age of 30-59 years was 61.29%.The gender and age ranges pointed out that men were between 75 to 89 years old and women were between 45 to 59 years old.3.In other areas,total 11 strains of aerobic actinomycetes strains were collected from the lung infection patients,including 4 strains of Nocardia otitidiscaviarum,3 strains of Nocardia cyriacigeorgica,1 strains of Nocardia wallacei and 3 strains of Nocardia farcinica.4.There was a Gordonia bronchialis strain isolated from the patient's sputum samples,which had been misdiagnosed as Mycobacterium tuberculosis infection from a local hospital.5.Two clusters can be observed in the phylogenetic trees based on 16 S rRNA and hsp65 genes,and the bootstrap values of two branches of A and B were 63 and 64,78 and 97;And the tree based on the rpoB gene showed three branches of A,B and,the bootstrap values were 100,100 and 99 respectively.6.The amplified secA1 gene of Nocardia strains was detected fluorescence signal in RT-PCR,but other experimental group showed no fluorescence signal.Conclusion: 1.The pathogenic strains of Gordonia and other neighbor species have been misdiagnosed frequently in local clinical laboratories because of improper identification protocols.2.hsp65 and rpoB genes are better than 16 S rRNA for observing the micro-evolution of non-tuberculosis mycobacteria strains,and phylogeny of rpoB gene is the most suitable method for differentiation the relevant strains on the inner-species level.3.The molecular beacon probe designed in this study can precisely identify Nocardia strains.