Rapid Detection Of Pathogens And Their Drug Resistance And Optimal Drug Combination Inhibition Therapy

Bacterial contamination causes many serious diseases,threatening the survival and development of human beings.In particular,the rapid evolution of antibiotic resistance in bacteria is a major public health threat and a substantial financial burden on the healthcare system.It is a major problem faced by both developing and developed countries.Due to the widespread application of antibacterial drugs,the problem of bacterial resistance has become increasingly prominent,not only producing multi-drug resistant bacteria,but also invalidating a large number of currently in use antibacterial drugs.This will inevitably lead to a drastic decrease of available drugs for clinical use.As the WHO put it in 2011,"Combating drug resistance:no action today,no cure tomorrow".Bacterial detection is the first step in developing an effective treatment plan to delay the process of bacterial resistance.Therefore,it is crucial to find a convenient,rapid,low-cost and high-accuracy bacterial detection method.This not only can effectively reduce the risk of bacterial infection in the environment,medical treatment and food fields,but also provide a basis for subsequent treatment and guide clinical rational drug use.However,current methods,such as plate culture,polymerase chain reaction(PCR),surface plasmon resonance(SPR),enzyme-linked immunosorbent assay(ELISA),surface-enhanced Raman scattering(SERS),and mass spectrometry,etc.cannot meet the growing demand of analysis.Therefore,it is of urgent need to further optimize detection methods and realize rapid and efficient detection of bacteria and multi-drug resistant bacteria.At the same time,on the basis of detection,further search for reasonable and rapid solutions to bacterial resistance needs to be sought after.Antibiotic combination therapy is one of the mainstream methods to overcome the decline in antibiotic efficacy and reduce the occurrence of drug resistance.By combining multiple ineffective antibiotics at different dosages,the synergistic effect between drugs can restore efficacy and even lead to better outcomes.Therefore,it is important to quickly find effective combinations to improve the effectiveness of clinical treatment and reduce the cost of treatment for patients.However,the interaction mechanism of antibiotics and their killing effect on drug-resistant bacteria are systematic and complex.Guidance on combined medication based solely on bacterial action mechanisms is yet able to meet the increasing demand for bacterial resistance.At the same time,the large number of antibiotics also makes it difficult to choose a combination.Faced with hundreds or thousands of antibiotic choices,we cannot explore every potential combination to find the optimal combination for treatment without increasing the dose and biotoxicity.Complex combinatorial optimization experiments and drug screening methods have also slowed down the process of finding possible synergistic antibiotics.Faced with such a complex system,we should choose antibiotics more reasonably,and quickly find the optimal combination of antibiotics with high efficacy and low dose.Based on the above issues,the following research has been carried out in this paper:1)A rapid and broad-spectrum test strip for bacteria based on bacterial inhibition of GOX-catalyzed reaction was designed.Through bacterial metabolism,we realize broad-spectrum detection of live bacteria,which results in a visible color change without any complex instrumentations.The feasibility of this method was proven with the detection of five common clinical bacteria.This method is specific for the detection of live bacteria,and only a few microliters of sample are required and the overall detection time is under 20 minutes.Finally,the practical applicability of the method was verified by detecting the ascites samples from infected mice.2)The identification of E.coli and their antibiotic resistance based on p-benzoquinone-mediated bioassay was realized.Using p-benzoquinone as a redox mediator,a simple and efficient colorimetric and electrochemical combined bioassay method was developed to analyze the presence of E.coli and its relative level of antibiotic resistance.This method can specifically distinguish E.coli from four other common clinical bacteria,and the limit of detection is at 1.0×10~3 CFU/m L.At the same time,the capability of this method for drug resistance detection was proven by the detection of E.coli with different drug resistance.3)A large-scale combination antibiotic screening using algorithms is realized.To overcome the problem of large-scale combination drug screening,a new large-scale drug combination optimization algorithm was developed to realize the optimization of antibiotic combinations for multi-resistant bacteria.We successfully identified effective combinations of 26antibiotics that are not individually effective at inhibiting an artificially induced multi-resistant bacteria.Rather than performing millions of tests,this method accomplished this task with only 120 carefully selected tests.The platform introduced here is a feasible solution to inhibit the growth of multi-resistant bacteria.4)Designed a microfluidics-based drug screening and drug combination optimization chip.In order to achieve rapid and efficient combination drug screening and to solve the time-consuming and laborious problem of traditional drug screening methods.Based on the properties of liquid laminar diffusion in microfluidics,a drug combination screening chip is designed,which can realize drug concentration screening and drug combination optimization on chip,providing a convenient and feasible experimental method for drug screening.In summary,in the face of the increasingly serious problem of bacterial resistance,this thesis first proposed two new methods for rapid detection of bacteria;Then combined with the large-scale drug combination screening algorithm,the rapid screening of antibiotic combinations was realized to inhibit the multi-drug-resistant bacteria;Finally,the personalization and automation of drug combination screening are realized through microfluidic chip.In a word,this thesis proposes a complete solution of bacterial drug resistance step by step from detection to treatment,and then to the optimization of screening methods.