The Construction Of LuxCDABE Bioluminescence Bacteria For Potency Evaluation Of Antibiotic And Meningitis Mechanism

Several new antibiotic drugs and combination therapies have been developed to treat multidrug-resistant organisms.Hence,the need for appropriate methods to real-time evaluate bacterial antibiotic susceptibility and visualizing colonization and clearance dynamics of bacteria has become of paramount importance.The unique ability of the bacterial luciferase lux CDABE system to express the biosynthetic enzymes for substrate synthesis within the engineered cell to produce a bioluminescent signal without exogenous substrate input has ensured that it is an ideal reporter for real-time monitoring of bacterial infection.The lux CDABE bioreporter also allows researchers to monitor the status of bacterial infections in living animals and enables the infection progression and antibiotic evaluation to be followed longitudinally over time for the same animal.This paper aim was to develop bacteria expressing bacterial luciferase and development a rapid bacterial antibiotic susceptibility test(AST)method by bioluminescence signals.The luciferase expressing bacteria that could be used for real-time monitoring the infection in BALB/c mice of bacterial meningitis.1.Development of bioluminescent bacteriaThe lux CDABE operon is generate bioluminescent light in an autonomous fashion without any light activating chemical substrate that must be repeatedly purchased.The p BBR-lux is derived from the broad host range plasmid p BBR1MCS-2,which is stable in gram-negative bacteria in the absence of antibiotic selection.The p BBR1MCS-lux was successfully transferred to C.kazakii,E.coli O157:H7,S.Typhimurium,and S.flexneri;all of these bioluminescent strains are sensitive bioluminescence reporter that is not impacted by the lux CDABE operon,and the intensity of the bioluminescence signal represents bacteria survival in real time.This construction strategy of reporter system has numerous potential applications for Gram-negative bacteria in monitoring the evolution of infectious diseases in animal models in the absence of antibiotic selective pressure.2.Rapid bacterial antibiotic susceptibility test based on lux CDABE bacteriaThe present study aims to evaluate the influence of different antibiotics on bacterial bioluminescence.In the MIC or up-MIC concentration of the antibiotics,the bioluminescence and CFU of bacteria was inhibited in 2 h.The results obtained with the whole animal optical imaging show that the growth of bacteria was inhibited at a dose of 20 mg/kg in mice infected with E.coli O157:H7-lux.Together,this data demonstrated that the bioluminescent bacteria could be exploited for rapid bacterial antibiotic susceptibility test and MIC determination.What’s more,The results indicated all bioluminescent bacterial strains have similar bioluminescence and CFU enhancement at sub-minimum inhibitory concentration concentrations of six different antibiotics(tigecycline,ciprofloxacin,polymyxin B,amikacin,meropenem and rifampicin)with different modes of antibacterial action.This bioluminescence enhancement was demonstrated with a lux-tagged E.coli O157:H7 infection and a dose of 2 mg/kg of tigecycline treatment animal model.This dose(2 mg/kg)is under the bactericidal activity dose of the drug.the bioluminescent signals and CFU enhancement at sub-MIC antibiotic concentrations in vitro and in vivo should be valued in the research of the bacterial infection and treatment study based on bioluminescence imaging.3.Bioluminescence imaging infection and treatment model of acute bacterial meningitisCronnbacter spp.is an important emerging foodborne pathogen,its can cause necrotizing enterocolitis and meningitis.The infection mechanism of Cronnbacter spp.is a intergrated model for acute bacterial meningitis.But few studies of Cronnbacter spp.have been published.C.muytjensii is one subspecies of Cronnbacter spp.A bioluminescent C.muytjensii strain that can be used for real-time monitoring of infection and treatment of acute bacterial meningitis in BALB/c mice.This study fist shows the procession of acute bacterial meningitis caused by C.muytjensii in vivo.Spna2、Acly Protein acetylation to participate in a variety of biological functions.Over the course of acute bacterial meningitis caused by C.muytjensii infection,the inflammatory cells migrated to the brain by the regulation of vessels at 0-6 h post-infection,which related to the up-regulation of protein acetylation and the chemotactic effect of cytokines,resulting in the inaccessibility of bacteria crossing the blood-brain barrier.Of note,the down-regulation of protein acetylation contributed to the out-of-balance of cell apoptosis or programmed cell death during the infection of C.muytjensii.After 6h post-infection,the bacteria were cleared by inflammatory cells through vesicle formation and endocytosis concerned with the up-regulation of protein acetylation,however,more bacteria colonize the brain owing to the inflammatory cell mitosis is reduced by the down-regulation acetylation,finally resulting deaths.