The Adjuvant Theraputic Effect Of Alkaline Solution Inhalation In The Treatment Of Resistant Baumanneous Pneumonia And The Underlying Mechanism

BackgroudAcinetobacter baumannii has become the common strains of nosocomial infections due to its survival on surfaces and medical equipments for long periods.It has become the cause most frequently associated with hospital acquired pneumonia,catheter infections and meningitis,especially in the intensive care unit?ICU?.Due to acinetobacter baumannii inclined to antibiotic resistant and clinical antibiotic abuse,the drug resistance of acinetobacter baumannii is increasingly common,and the emergence of resistance in Acinetobacter genomic species to Carbapenems lead to the choice of clinical medicine is very limited.Domestic epidemiology survey showed that carbapenems resistant Acinetobacter baumannii?CRAB?is one of the most common HAP and VAP pathogens in intensive care unit.The patients showed poor basic viscera function and heavy lung infection,but less antimicrobial agents can be chosen.Higher doses,combined use and long period of treatment is often required,but clinical therapy is still not optimistic.In February 2017,the world health organization?WHO?also listed CRAB as one of the most important bacteria in the resistant bacteria.Polymyxin is still not popular in China.There is still controversy about the dosage of the cycloelement,and soon the drug resistance is progressing rapidly.Cefoperazone sodium/sulbatam sodium+minocycline is one of the commonly used joint treatment regimens in China,however,the clinical efficacy still lacks large-scale randomized controlled study.Thus finding a solution to improve antibiotic susceptibility is a feasible solution.Study confirms that airway surface liquid?ASL?in healthy human is alkalescent,and bacterial infection turns the environment to acidulous.Foreign studies have found that many antimicrobial activity of ASL were sensitive to pH and its antibacterial activity decreased significantly in the acid environment.Our previous in vitro experiment showed the pH of culture has an evident effect on bacteria proliferation,and CRAB proliferation was higher in acidulous environment than in alkalescent environment.Further study found that different environmental pH regulate the susceptibility of CRAB to antibiotic and in acidulous environment the activity of cefoperazone/sulbactam was inhibited and alkalescent environment enhanced the activity.Therefore,to maintain normal pH of ASL is vital in defense bacteria invasion,removing bacterial infection and enhance the efficacy of antibiotics.The purpose of this study is to verify the auxiliary effect of restoration of weak base environment on treatment of the CRAB by in vivo and in vitro studies,further it can remove the drug-resistant bacteria in antimicrobial therapy play a supplementary role,and to explore the underlying molecular mechanism.The aim is to provide a theoretical basis for the new clinical treatment of drug-resistant bacterial lung infection.Aims1.To establishment of a murine model of Acinetobacter baumannii pneumonia with a new intubation method.2.To confirm the effect of maintain the pH of airway on the treatment for CRAB infected pneumonia mice with antibiotic.3.To investigate the influence of environmental pH on the antimicrobial activity in ASL.Methods1.CRAB was collected,isolated and identified from the sputum samples of Xijing hospital.2.CRAB infection pneumonia mice were built by implanting the bacterium solution into the airway with a new intubation method.Certain quantity of bacteria solution was injected into the trachea of C57BL/6 mice and the bacteria load of the lung tissue was measured to confirm the success of the model.3.To evaluate the effect of combination of atomization inhalation alkaline solution ?5%NaHCO3?with the antimicrobial agents?sodium cefoperazone sodium/shu ba jotham+minocycline?on treatment of CRAB pneumonia in mice.4.The influence of environmental pH on the antimicrobial activity in ASL was measured by K-B spot.5.Real-time Quantitative PCR was performed to detect the effect of pH on mRNA expression of efflux pumps6.The effect of different pH environment on CRAB efflux capacity was investigated by rhodamine 6G efflux experiment.7.The software SPSS 20.0 was used to analyze the data.Enumeration data were compared using analysis of chi-square test and measurement data were compared by one-way ANOVA.The results expressed as mean±SEM and P<0.05 represent the difference is significant.Result1.Establishment of a murine model of Acinetobacter baumannii pneumonia with a new intubation method.1.1 The experiment was performed with the technique of endotracheal intubation,10 C57BL/6 mice were used as the study subjects,and 10?l bacterium with concentration of 3.9×10⁸ CFU/mL was injected for one mice?one of the mice died due to improper manipulation.?After tracheal intubation,all mice were executed immediately,and the lungs were removed and weighed.The colony count was 4.18×10⁷±0.91×10⁷?3.34×10⁷⁴.42×10⁷?CFU/g lung,indicating that the method was reproducibility.1.2 The mice were randomly divided into the normal control group,the immunodeficiency group and the immunodeficiency infection group.The immune deficiency mice were prepared by intraperitoneal injection of cyclophosphamide, and the final airway injection was 2.5×10⁸ CFU/mL,which was observed at 48h. Compared with the control group,the average number of blood cells and neutrophils in immune deficiency group and immunodeficiency infection group have obviously decreased?P<0.05?,the average number of bacteria in immunodeficiency infection group can reach the 1.03×10⁶ CFU/g lung.Compared with 0 h,the amount of bacteria in the lungs of 4 h immunodeficiency mice infection group increased to 0.92×107 CFU/g per lung?P<0.05?and all kinds of inflammation factor and cell count in peripheral blood also increased?P<0.05?.It was proved that this method was successful in the establishment of a mouse model of Acinetobacter baumannii.2.The effect of aerosol inhalation of alkaline solution on the efficacy of antibiotics in CRAB mice.2.1 Inhalation of alkaline solution can restore the normal pH of ASL in the CRAB mice.Airway liquid pH measured by fluorescent dye method in CON was about 6.3,compared with CON,the airway liquid pH value of CRAB pneumonia mice decreased significantly? <0.05?,and the inhalation of alkaline solution can restore the airway ASL pH.2.2 Combined use of inhalation of alkaline solution and antibiotic treatment can further reduce the mortality of CRAB pneumonia mice.In mice infected with CRAB,30%mice were dead 8h later,and the morality rate of mice in the antibiotic treatment group?IS+IF+AB?was 20%,while that of the mice treated with the inhalation of alkaline solution combined with the antibiotic treatment group?IS+IF+AB+AI?had a morality rate of 10%mice.48h later,the mortality rate of IS+IF was 80%,and the mortality rates in IS+IF+AB and IS+IF+AB+AI were about 60%and 50%,respectively.Aerosol inhalation of alkaline solution increased the survival rate of mice treated with antibiotics.2.3 The combination of aerosol inhalation and antibiotics can reduce the bacterial load of lung tissue and bronchoalveolar lavage fluid in CRAB mice.8h after modeling, bacterial load in lung and bronchoalveolar lavage fluid?BALF?of IS+IF and IS+IF+AB were 85.3×10⁵ and 79.3×10⁵CFU,antibiotic treatment can significantly reduce bacterial load in lung tissue and BALF?P<0.05?.Alkaline solution inhalation alone can't reduce bacterial load,but combined antibiotics with aerosol inhalation can further reduce the bacterial load.2.4 The combination of inhalation of alkaline solution and antibiotics can further reduce inflammatory response.Compared with IF,white blood cell count in BALF and the content of inflammatory cytokines in blood was significantly reduced,combined use of antibiotics and inhalation can further decrease inflammatory cytokines and alveolar leukocyte infiltration.2.5 The combination of inhalation of alkaline solution and antibiotics can further alleviate the pathologic changes of lung tissue in CRAB pneumonia mice.Leukocyte filtration,alveolar collapse and bacterial colonization were observed in lung tissue of CRAB pneumonia mice,and the pathological changes of the lung tissue sections of IS+IF+AB was less evident,and lung tissue sections of IS+IF+AB+AI showed the mildest pathological changes.3.Effects of environmental p H on antibacterial activity of ASL3.1 The strong acid environment reduces the antibacterial effect of ASL on CRAB,and the weak base environment can improve the antibacterial effect of ASL.K-B experiment showed that the maximum radius of bacteriostatic ring was13.5± 2.6mm when the p H was set at 7.1,and bacteriostatic ring radius 12.8mm±2.3mm when p H set at 7.5,no statistical difference was found between the two.But when p H was set at 6 the radius of bacteriostatic ring decreased significantly?P<0.05?,was 6.3 ± 0.8mm.3.2 The environment of weak base can reduce the expression level of the CRAB pump gene.Compare the gene expression of bacterial efflux pump genes at p H6.3,p H7.1 and p H7.5,respectively.The results showed that the expression levels of ade B and ade J were significantly different in different p H environment.Compared with the faintly acidic environment at p H 6.3 the efflux pump genes ade B and ade J decreased significantly at p H 7.1,and the alkaline environment of p H 7.5 could significantly reduce the expression of ade B and ade J.3.3 The environment of weak base can reduce the expulsion ability of CRAB to rhodamine.Rhodamine 6G excretion test is performed to detect residual rhodamine discharged from bacteria.In this study,we found that ES was 765 ± 48.2 and 536 ± 37.5 in p H 6.3 and p H 7.1 cell suspension,respectively?n = 8?.The ES was 968 ± 35.4 in p H7.5 cell suspension?n = 8?.The effluent of Rhodamine was negatively correlated with the environmental p H.ConclusionThe treatment of CRAB pneumonia is an intractable problem in clinic,and antibiotic therapy is not satisfactory.We successfully established CRAB pneumonia mice model which was used to investigate the auxiliary effect of inhaled alkaline solution?5%Na HCO₃?on the effect of antibiotic treatment of pneumonia and explore the underlying mechanism.The experiment in vivo showed that the normalization of ASL p H by inhalation of alkaline solution can reduce the mortality rate of mice,the bacterial load and inflammation,and enhance the efficacy of antibiotics in the treatment of CRAB pneumonia in mice.The experiment ex vivo showed that the effect of inhalation on antibiotic treatment in CRAB pneumonia may be related to the recovery of antibacterial activity of ASL in airway by normalizing the p H.Mechanically,the alkaline environment could significantly reduce the expression of efflux pump genes ade B and ade J.The study will provide new potential adjuvant therapy scheme for the clinical treatment of CRAB pneumonia.