Exploration Of Early Preventive Measures For Pneumonia Induced By Carbapenemase Resistant Bacteria Perioperative Infection And Development Of NDM-1 Targeted Therapy

Background:Carbapenem antibiotics have been used as a powerful weapon against microbial infection for many years.They have many advantages such as clear effects,controllable toxic and side effects,and acceptable prices.However,the widespread emergence of carbapenem resistant bacteria and genes greatly limited the effectiveness of this weapon,and made many patients with carbapenem resistant bacteria infection fall into the dilemma of medication shortness or even no drug available.Pneumonia caused by carbapenem resistant bacteria infection has become one of the main factors leading to poor prognosis of hospitalized patients.Up to now,safe therapies that can effectively inhibit carbapenem resistant bacteria are still lacking or cannot be used in clinical settings due to various limiting factors.In the absence of effective anti-microbial drugs and treatments,vaccines have become the most powerful weapon for human beings to resist pathogenic microorganisms by activating their own immune system.In fact,the COVID-19 global pandemic further confirmed and emphasized the great potential of human autoimmune system and the role of vaccines in eliminating and controlling lethal pathogenic microorganisms.Vaccine also has other advantages such as precise effect,clear adverse reactions and side effects,and it is easier to control and produce,so it is still highly expected in real use.Carbapenem-resistant bacteria can generally be divided into two categories:serine?-lactamases and metallo?-lactamases,and the metallo?-lactamase gene can easily contaminate between different bacteria in a short period of time through plasmids.At present,the most common carbapenem-resistant bacteria in clinic contain the NDM(New Delhi metallo-?-lactamases)-1 gene—bla _NDM-1.The NDM protein expressed by this gene can hydrolyze the?-lactam ring and renders carbapenems ineffective.At present,common pathogenic bacteria transfected with bla _NDM-1gene in clinical settings include Klebsiella pneumoniae(KP),Pseudomonas aeruginosa(PA),Acinetobacter baumannii,etc.In our previous studies,we found that mice immunized with high temperature inactivated HIKP or HIPA obtain acquired immunity against KP or PA.When these mice were infected with live bacteria of KP or PA,CFU count in the lung decreased significantly,and the levels of interleukin-17(IL-17)and IFN-g titer in serum and spleen cell culture medium increased significantly,suggesting that the bacteria inactivated by high temperature can make the body produce a strong specific immune response and obtain better immune protection when challenged with the same antigen.Since HIKP and HIPA contain a variety of substances that can be recognized by the organism as antigens,we cannot determine which one of the antigenic substance causes the organism to produce acquired immunity,and also the NDM protein expressed by bla _NDM-1gene is one of the antigens.According to this,we propose the following scientific hypothesis:“Mice inoculated with recombinant NDM protein(rNDM-1)as main antigenic substance develop specific immune memory,and will recognize live Klebsiella pneumoniae(KPndm)transfected with bla _NDM-1gene as a specific antigen and eliminate it.”While when KP without transfection of bla _NDM-1gene infects organism,antibiotics can be used to control it.This study is divided into two parts.Firstly,the effect and mechanism of recombinant NDM-1 on respiratory immune response in mice were studied;Secondly,we studied the target cells of drug-resistant bacteria and their immune mechanism.Methods:1.Establish wild-type mice model using blank control,ovalbumin(OVA),HIKPndm and rNDM-1 grouping for immunization and live KPndm bacterial for infection challenge,through colony forming unit(CFU)count,ELISA,flow cytometry technology,real time PCR(RT-PCR)and other methods to observe the role of humoral and cellular immunity in the control of lung inflammation in different groups of mice after live bacterial challenge.The spleen cells of wild-type mice with rNDM-1 immunization were extracted,and injected through tail vein into the Rag2/II2rg double knockout mouse to establish the cell transplantation model.The transplanted Rag2/II2rg mice were subjected to receive live KPndm bacterial challenge to explore whether cell transplantation can provide immune protection for immunodeficient mice,and to further explore the role of cellular immunity after rNDM-1 immunization.2.To study the different expression of key genes at the gene regulation level between wild-type mice immunized with rNDM-1 and unvaccinated mice:establish a live KPndm bacterial infection model using wild-type mice immunized with rNDM-1 and blank control respectively.CD4+/CD8+cells were sorted and collected by flow cytometry,and then sc RNA-Seq was performed to observe the differential expression of key genes of immune cells under multiple inoculate conditions with live bacterial infectionResults:1.Immunization of mice with rNDM-1 can make the mice generate an acquired immune response against live KPndm bacteria.Immunization with rNDM-1 can reduce the bacterial CFU count in the lungs of mice infected with KPndm,play an anti-inflammatory protective effect,and this protective effect can be blocked by IL-17A inhibitors.Wild-type mice immunized with rNDM-1 were infected with KPndm and their lung cells were collected.The fluorescent antibody screen revealed that they significantly have more T cells secreting IL-17A(P<0.05),IFN-g(P<0.05),and IL-22.(P<0.05),proving that the immune protection is mainly mediated by T cell immunity.The spleen cells of mice immunized with rNDM-1 and infected with KPndm secreted more IL-17A(P<0.005)and IFN-g(P<0.01)under the stimulation of rNDM-1 or HIKPndm.At the same time,the expression of Th17/IL17 pathway genes in mice under this condition was significantly up-regulated.2.Single-cell gene sequencing technology proves that when different mice are immunized with OVA or HIKPndm and KPndm is used for infection,mice infected with HIKPndm will have more activated or proliferative T cells accumulated in the lungs during infection,and the number of T cells that secrete Th17/IL17 pathway products is also significantly higher.The lung samples of hikpndm immunized mice had significantly increased gene expression levels of Il17a,Rorc and Rora(P_Il17a<0.01,P_Rora<0.001,P_Rorc<0.05).In addition,immunoactivated T cells were mainly distributed in proliferative T cell subsets and partially activated T cell subsets.Conclusion:The results showed that rNDM-1 immunization reduced the bacterial colonization in the lung of kpndm challenged mice;The expression of IL-17A was up-regulated in mice immunized with rNDM-1;Inhibition of IL17A attenuated the protective effect of rNDM-1on lung infection in mice;rNDM-1 immunization stimulated the production of specific Ig G in mice;T cell mediated immune response plays an important role in the process of rNDM-1immunization;Kpndm infection activated Th17/IL17 pathway in mice immunized with rNDM-1;HIKPndm can promote the activation and proliferation of mouse T cells and induce secondary immune response;Th17/IL17 pathway plays an important role in the immune response of mice to HIKPndm.