Establishment And Application Of Caenorhabditis Elegans-carbapenem Resistance Klebsiella Pneumonia Model

BackgroupKlebsiella Pneumoniae（CRKP）is a gram-negative bacillus,which is a relatively short crude bacillus without spores、flagella、thick decidua,and most bacteria with pilus.Klebsiella pneumoniae is one of the main pathogens in normal flora,and it is an opportunistic pathogen.When the body’s immunity is suppressive,CRKP evades the host immune response and causes the disease.CRKP disrupts normal flora and colones in the gastrointestinal tract while host immunosuppressive.Since the 1970s,the number of multi-drug resistant pathogens has been increasing due to antibiotic abuse.CRKP infection is one of the major causes of nosocomial infection worldwide.Urinary tract infections,pneumonia,and intra-abdominal infections are easily caused in hospitalized patients.It is reported about 15%of pathogenic gram-negative bacteria is multidrug resistant Klebsiella pneumonia in hospital intensive care units.CRKP was first reported in 1996,and CRKP was subsequently discovered in the United States,Britain,Athens,and China.Carbapenemase is the main cause of Klebsiella pneumoniae resistance to carbapenem drugs,which can effectively hydrolyze various antibiotics such as penicillin,cephalosporin,aztreonam and carbapenems.Carbapenemase related drug resistance genes are mainly locate on plasmids and transposons and are easily spread horizontally in the population.Therefore,it is difficult to control the spread of multidrug-resistant bacteria.Bacterial resistance has become a worldwild public health crisis.With more and more multidrug-resistant bacteria,drug development is difficult to cope with the increasing demand for anti-infective treatments,and efficient screening methods are being developed to find new antibacterial drugs.C.elegans is a small nematode which is capable of living on its own.The C.elegans begins to feed and develop into four larval stages（L1-L4）,and40%of genes known to be related to human diseases.There is a clear orthologue in the C.elegans genome with that of human.Therefore,many findings of C.elegans are related to the study of human health and disease.Caenorhabditis elegans is also an animal model for studying human-related diseases.In this study,the establishment of the whole animal infection model of C.elegans can quickly and conveniently screen the antibacterial compounds in vivo.The aim of this study is to investigate an transcriptional response to CRKP infected Caenorhabditis elegans.The model was used to screen for metal ion concentrations on antibacterial effects.Finally,an effective antibacterial active compound screening platform and anti-infection research tool will be established.MethodsThe Klebsiella pneumoniae strains collected from patients in Guangzhou General Hospital of PLA.Klebsiella pneumoniae is obtained from the blood and identified as carbapenem-resistant Klebsiella pneumoniae（CRKP）by microbial susceptibility and PCR.The K.pneumoniae model of C.elegans was established by co-cultivating CRKP and Caenorhabditis elegans to explore the culture conditions,infection time and treatment time.RNA-seq was used to quantify the mRNA level of Caenorhabditis elegans at different infection time points,thereby revealing the response mechanism of C.elegans to CRKP infection.At the same time,an antibacterial ion with anti-CRKP infection was screened using a constructed infection model.Results1 Establishment of Caenorhabditis elegans-carbapenem resistance Klebsiella pneumonia modelAfter co-cultivation of CRKP with C.elegans,we found that the self-infected C.elegans autonomous sinusoidal motion disappeared and the oropharyngeal activity was lost to reach the initial infection state.If polymyxin B is administered after CRKP infection,the infected C.elegans can be recovered.The survival rate of C.elegans after treatment is related to the drug concentration.After drug treatment,the viability of the nematode was observed by a microscope to evaluate the effect of the drug.Fluorescent tracer experiments showed that CRKP colonizes the intestine of the C.elegans through its own growth and secretion of toxins.The optimal culture condition for bacterial infection of C.elegans was20%BHI-M9 medium;the infection concentration was optimized at different infection concentrations（10⁸ cfu/mL,10⁷ cfu/mL,10⁶ cfu/mL,10⁵ cfu/mL,and blank control group）.The survival rate of nematode that eventually became 7.84%,3.448%,6.667%,3.7%,and100%,respectively.There was a statistically significant difference between each infection group and the negative control group（χ2=56.44 P<0.0001）.In order to establish a rapid and effective infection model and reduce the impact of bacterial metabolites,we chose an infection concentration of 10⁶ cfu/mL for studies.Through the exploration of different infection time and treatment time,it was determined that the drug was added after 24 hours of infection,and the treatment effect could be achieved compared with the control group after 12 hours of treatment.2 An transcriptional response to carbapenem-resistant Klebsiella pneumoniae infection in Caenorhabditis elegansAt 24 hours after infection,C.elegans infect with CRKP significantly reduced the expression of genes associated with cell growth.The expression of acy-1,itr-1,pal-1,and ceh-30 genes was up-regulated,which was involved in the cell death-related process,indicating that the bacterial infect with nematodes induced the progression of death.At the same time,the negative regulation involved in cell death is also activated,and daf-7 is a key regulator of metabolic and food-dependent behaviors.Studies of daf-10 may be related to the shortened lifespan of C.elegans.In the early stage of infection,the expression of immune-related genes was up-regulated,but as the infection time prolonged,the expression of related genes gradually decreased.The host’s defense response is based on the response to bacterial infections,but the host’s immune defense ability decreases as the duration of the infection prolongs,and expression of daf-2 lifespan regulators declines in the early stages of infection.3 Screening of antibacterial concentrations of metal ions Using Caenorhabditis elegans-carbapenem resistance Klebsiella pneumonia modelUsing C.elegans-CRKP infection model to screen for antimicrobial metal ions,it was found that silver ions of 10^-6 M have antibacterial and non-toxicity against K.pneumoniae infection,and copper ions of 10^-4 M have antimicrobial activity against CRKP infection without toxicity.ConclusionIn conclusion,it successful set up Caenorhabditis elegans-Carbapenem Resistance Klebsiella pneumonia Model.This model states that CRKP infected with Caenorhabditis elegans lead to cell growth,death,and immune responses.The model was used to screen for metal ion concentrations on antibacterial effects.Finally,an effective antibacterial active compound screening platform and anti-infection research tool will be established.