New Methods For Rapid Drug Susceptibility Testing Of Pathogenic Bacteria Based On Nucleic Acid

During the last decades the increased spread of antibiotic-resistant bacterial strains because of its overuse and misuse represents a major global health challenge.Rapid and accurate antibiotic susceptibility testing(AST)of pathogen could contribute to giving appropriate drug treatment,reducing chances to generate new resistance and saving the patient's life.Traditional methods in the laboratory for AST are based on culture,which are labor-intensive and time-consuming.Molecular diagnosis assays detect only the common mutations conferring resistance.In order to overcome these shortcomings,in this study,three new rapid AST methods combining with latest molecular technology had been developed.In the part ?,a culture-dry-reagent-based qPCR method for parallel and highthroughput AST is developed.In this method,culture of bacteria and DNA extraction could be done using a common PCR instrument.A lyophilized 16 S rDNA targeted qPCR reagent based on utilizing trehalose was also developed,which was stable and could be kept at room temperature.This method combining culture and qPCR was based the detection of nucleic acids change using 16 S rDNA universal primers when bacteria was cultured with antibiotic,which coluld overcome the defects in traditional culture method because of time-consuming and molecular method restricted to the common mutations conferring resistance.Thermo-cold lysis in this study could avoid the multiple steps of DNA extraction by either commercial kits or the phenolchloroform method used in the current culture-qPCR method not only prolong the overall test time and might also generate some extra-errors for accurate quantitation of DNA changes by qPCR plus reducing biosafety risk.This method adopts freeze-dried technology could overcome the cost that the fragility of the DNA polymerase needed for qPCR makes it necessary to use cold-chain transportation and storage for the qPCR reagents,which decreases financial burden and broaden its use in under-resourced countries.Testing of P.aeruginosa isolates and S.aureus isolates showed that the method had good agreements with the standard broth microdilution method,with an overall agreement of 97.22 %(95% CI,85.83-99.51)for P.aeruginosa and 96.43 %(95% CI,79.76-99.81)for S.aureus.This method could test 12 samples against a panel of up to 7 antibiotics simultaneously in two 96-well PCR plates within 4 h,which greatly improves the testing efficiency of the culture-qPCR method.In the part ?,a new single-bacterium duplex droplet digital PCR for accurate and rapid identification of methicillin-resistant Staphylococcus aureus(MRSA)was developed based on the key features of ddPCR is its ability to form nanoliter liquid droplets.In this method,bacteria were dispersed into the droplets so that there can be one bacteria in each droplet.Then amplification and detection of the markers(nuc and mecA)using standard TaqMan chemistries with ddPCR.Results were analyzed based on duplexpositive markers in droplets ratios.The method could overcome the defect of the traditional culture method with both labor-intensive and time-consuming.The most common PCR tests are to detect a specific gene of S.aureus and the methicillinresistance marker mecA simultaneously using a duplex real time quantitative PCR.But false-positive results could happen if there exist methicillin-susceptible S.aureus(MSSA)(nuc positive)and other methicillin-resistant coagulase-negative Staphylococci(CoNS).Another PCR method for MRSA detection is to detect the SCCmec right extremity junction(MREJ)region between the mecA cassette and orfX gene.False positive results would also appear when testing mecA dropout strains.The whole process does not require the isolation and purification of bacteria.The nucleic acid release of bacteria was based on the cell lysis of bacteria by Staphylococcus aureus specific chimeric lyase ClyH constructed in our laboratory.The method was able to achieve an absolute limit of detection(LOD)of 2,900 CFU/mL for MRSA in nasal swabs spiked with excess amounts of non-targeted bacteria.Testing of 104 nasal swabs showed the method had 100% agreement with the standard culture method.Results show that the single bacterial duplex ddPCR assay is rapid and powerful for more accurate detection of MRSA directly from clinical specimens within 4 h.In part ?,a new method combining culture and ddPCR(culture-ddPCR)resorting to the foundation of part ? and part ? was developed.The method could accomplish rapid detection Mycobacterium tuberculosis(MTB)and multidrug-resistant tuberculosis(MDR-TB)directly from sputum.The ddPCR assay showed 100%(95%CI = 86.66% to 100%)agreement with MGIT 960 for MTB detection.The agreement of susceptibility testing of the 32 positive specimens against rifampin,isoniazid and streptomycin were between the culture-ddPCR and the standard agar proportion method were 87.5%(95%CI = 71.93% to 95.03%),84.38%(95%CI = 68.25% to 93.14%)and 96.88%(95%CI = 84.26% to 99.45%),respectively.This study demonstrates that the culture-ddPCR method could be used for rapid detection of MTB and MDR-TB directly from sputum within 4 days and shorten the time of current AST substantially.Three new methods for rapid drug susceptibility testing of pathogenic bacteria based on nucleic acid could shorten the time of current drug susceptibility testing and accomplish high throughput drug susceptibility testing.Still,its clinical application prospects need to be further evaluated by massive specimens in the future.