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Antibiotic Resistance And Azithromycin Resistance Mechanism Of Legionella Pneumophila Serogroup 1 In China

Posted on:2020-12-07Degree:MasterType:Thesis
Country:ChinaCandidate:X Y JiaFull Text:PDF
GTID:2404330578469085Subject:Herbal biology
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Rationales:Legionella disease is an acute respiratory infection,the main symptom is atypical pneumonia.Legionella is the causative pathogen of Legionnaires' disease.The clinical treatment of Legionnaires' disease is mainly macrolides and fluoroquinolone antibiotics,and it is also treated with rifampicin.The resistance of bacterial can lead to difficulties in the treatment of clinical infections,pose a direct threat to patients,and even lead to a cure-free situation when humans face serious infections.At present,there are few reports on the Legionella pneumophila resistant to antibiotics at domestic and abroad.Therefore,it has great significance for the detection of antibiotic susceptibility of Chinese Legionella and the study of strain resistance mechanism in the further step.Research objective:This study intends to study the drug resistance spectrum of Legionella by detecting the antibiotic sensitivity of Legionella to erythromycin,azithromycin,moxifloxacin,levofloxacin and rifampicin and further explored the resistance mechanism of azithromycin-resistant Legionella;the relationship between Legionella resistance and sequence-based typing(SBT)types and the relationship between bacterial resistance and pathogenicity.To provide guidance for the prevention of outbreaks of Legionnaires' disease and provide advices for clinical rational using drugs to effectively treatment of Legionnaires' disease.Research method: 1.E-test was used to detect the antibiotic sensitivity of Legionella,isolated from clinical or environmental,to erythromycin,azithromycin,moxifloxacin,levofloxacin and rifampicin.2.PCR amplification and sequencing of 23 S ribosomal gene rrl,encoding L4 and L22 ribosomal protein genes rpl D and rpl V,compared with wild-type international reference strain ATCC 33152 genotype to detect antibiotic resistance site mutation;PCR amplification of the coding methylase genes erm A,erm B,erm C and erm F,the coding inactivation enzyme genes ere A,ere B,mph A and mph B,and the efflux pump gene mef A/E and lpe AB to detect the antibiotic mechanism of Legionella resistance to azithromycin.SYBR Green ? real-time fluorescent quantitative PCR was used to determine the lpe AB efflux pumps relative expression level of antibiotic-resistant strains to non-resistant strain.The effect of carbonyl cyanide m-chlorophenyl hydrazine,an efflux pump inhibitor,on the change in the minimum inhibitory concentration of drug-resistant strains in efflux pump inhibitors.3.PCR amplification and sequencing of 25 azithromycin-resistant Legionella strains genes of fla A,pil E,asd,mip,momp S,pro A and neu A to SBT typed and compared with internationally resistant strains.4.The mouse macrophage J774 cell-line was infected with azithromycin resistant Legionella,and the infection solution was collected and applied to BCYE medium.After five days of culture in the incubator,the colony count was carried out,and the pathogenicity of the resistant strain was studied.Research results:1.149 strains of serotype 1 Legionella pneumophila were sensitive to erythromycin,moxifloxacin,levofloxacin and rifampin with MICs ranging from 0.064–1.0,0.064–0.38,0.19–0.75 and 0.002–0.023 mg/L.25 strains of Legionella showed resistance to azithromycin,the resistance rate was 16.78%,and the MIC ranged from 1.5-2 mg/L.2.Amplification and sequencing of the 23 S ribosomal RNA domain V region gene rrl,no mutations were detected;no mutations in the L4 ribosomal protein and L22 ribosomal protein amino acid sequence were detected.And there is no ribosomal RNA methylation-modified genes erm A,erm B,erm C and erm F;the inactivating enzyme genes ere A,ere B and mph A;the mef efflux pump gene mef A/E in 25 azithromycin resistant strains.However,the efflux pump gene lpe AB was detected in 25 resistant strains,and the expression level of lpe AB gene was higher than that of non-resistant strain;the MIC of 25 resistant strains in presence of efflux pump inhibitors was lower than that of absence in 4 times or more.3.There are five types of resistant strains: ST1,ST144,ST150,ST154 and ST629.4.Among the 25 strains of azithromycin resistant Legionella,24 strains had strong intracellular growth ability,and 15 strains had stronger intracellular growth ability than the reference strain ATCC 33152.Analysis conclusion:149 strains of serotype 1 Legionella pneumophila were sensitive to erythromycin,moxifloxacin,levofloxacin and rifamycin,and 25 strains of Legionella were resistant to azithromycin..The expression of lpe AB efflux pump confers resistance to azithromycin in Legionella;The ST types of ST144,ST150,ST154 and ST629 are the first international reports.In addition to 15 of the resistant strains are showed characteristic of highly resistant and highly pathogenic.The epidemiological surveillance of Legionella in China and the monitoring of drug sensitivity to azithromycin should be strengthened.
Keywords/Search Tags:Legionella, Azithromycin, Antibiotic resistance mechanism, Squence-based typing, Intracellular growth ability
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