Rapid Molecular Diagnostics Of Antibiotic Resistance Genes In Bacterial Pathogens

Antibiotics are the main tools for clinical treatment of bacterial infections.The massive use of antibiotics accelerates bacterial resistance.The emergence and widespread of drug-resistance bacteria pose challenges for clinical anti-infective treatment,and seriously affect human health.Antimicrobial resistance(AMR)has now become a global issue.Therefore,rapid and comprehensive detection of AMR is of great significance for the timely and effective treatment of bacterial infections,the epidemiological study of pathogens,and the reduction of bacterial resistance.Currently,most clinical laboratories use traditional phenotypic testing,i.e.,antimicrobial susceptibility test(AST),for the detection of AMR.This AST process takes 48-72 hours.Thus,it is urgent to develop a rapid and comprehensive AMR detection technology in order to meet clinical diagnosis needs.In this thesis a multi-color melting curve technique(MMCA)was used to establish an AMR genes detection assay for Gram-negative bacterial pathogens producing ?-lactamase and for major resistant Gram-positive bacterial pathogens.In chapter one,we reviewed the global prevalence of AMR and the mechanism of bacterial resistance.Then we briefly described current methods for detecting AMR,and summarized their respective advantages and disadvantages.Finally we put forward the research purpose,content and significance of this thesis.In chapter two,we designed a 2-tube(Tube A and Tube B)AMR detection assay and a SHV/TEM-ESBL typing assay based on MeltArray technology and MMCA,respectively.In the 2-tube assay,Tube A assay detected 28 antibiotic resistance genes of Gram-negative bacterial pathogens that confer resistance to ?-lactams due to 3-lactamase production,which basically covers all types of ?-lactamases;Tube B assay detected 13 AMR genes including methicillin-resistant,vancomycin-resistant,and macrolides-resistant Gram-positive bacterial pathogens.Using plasmid as a reference,the specificity was demonstrated to be 100%,and the analytical sensitivity of both tubes was 5-50 copies/reaction.The repeatability of study showed that the SD value was less than 0.3 and the CV value was less than 0.5%.These results showed that our assays could accurately distinguish all the AMR targets studied.In Chapter 3,we validated our work with a large collection of clinical isolates using our established assay.In total,2108 clinical isolates were tested,including 1491 strains of Gram-negative bacteria and 617 strains of Gram-positive bacteria.The obtained results for both tubes A and B were compared with the results of AST,respectively.Using AST as the gold standard,the sensitivity and specificity were 87.7%and 92.4%for the Gram-negative bacteria test,respectively(Kappa=0.802,P<0.001);For the Gram-positive bacteria test,the sensitivity and specificity were 92.7%and 97.3%,respectively(Kappa=0.809,P<0.001).Taken tagether,these results demonstrated that our assays are in good agreement with the phenotypic testing.In summary,we have established a simple,rapid,sensitive,specific,and comprehensive detection system for major AMR genes.Our assays have the advantages of shorter detection time,less hand-on operation,cost-effective,and high-throughput as ompared to the traditional phenotypic detection method.Clinical study showed that our system had excellent detection capabilities for both Gram-positive and Gram-negative bacteria.Therefore,this newly developed system could provide rapid AMR diagnosis for patients with bacterial infections,assists clinicians to implement effective antibacterial therapy for patients in a timely manner,and could also be a powerful tool for epidemiological study of antimicrobial.