Development And Validation Of A Model For Predicting The Risk Of Death In Patients With Acinetobacter Baumannii Infection And In Vitro And In Vivo Antibacterial Activity Of Combinations Of Polymyxin B With Other Antimicrobials Against Carbapenem-resistanc

Purpose:This study aimed to develop and validate a personalized prediction model of death risk in patients with Acinetobacter baumannii(A.baumannii)infection,identify the carrying status of the resistance genes and virulence genes of different carbapenem-resistant Acinetobacter baumannii(CRAb)and verify the virulence of strains from different sources in the larvae of Galleria mellonella(G.mellonella).In addition to study activity of polymyxin B(PB)monotherapy and combined imipenem(IMP),meropenem(MEM),tigecycline(TGC),sulbactam(SUL)and rifampicin(RIF)against CRAB in vitro and vivo,to guide clinical research and support clinical decision-making.Methods:1.The development group is comprised of 350 patients with A.baumannii infection admitted between January 2013 and December 2015 in The First Affiliated Hospital of Anhui Medical University.Further,272 patients in the validation group were admitted between January 2016 and December 2018.The univariate and multivariate logistic regression analyses were used to determine the independent risk factors for death with A.baumannii infection.The nomogram prediction model was established based on the regression coefficients.The discrimination of the proposed prediction model was evaluated using the area under the curve(AUC)of the receiver operating characteristic(ROC)curves and decision curve analysis(DCA).The calibration diagram was used to evaluate the calibration degree of this model.2.Polymerase chain reaction(PCR) was used to detect the distribution of drug resistance genes(bla OXA-23,bla OXA-24,bla OXA-51,bla OXA-58,bla IMP,bla VIM,bla ADC,bla SHV,bla TEM)and virulence genes(omp A,csu A,csu E,pga A,aba I)in the 10 non-repetitive CRAB strains selected in this study.3.A broth micro-dilution method was used to determine the minimum inhibitory concentration(MIC)of 10 strains of CRAB against 6 antibacterial drugs and the checkerboard method was used to determine the combined inhibitory index(FICI),to evaluate antibacterial activity of PB monotherapy and the combination of other 5 drugs against CRAB in vitro.4.Inject the same strain with different concentration of bacteria or different strains with the same concentration of bacteria into the larvae of G.mellonella through stomach and foot to detect the difference in larval mortality caused by the bacterial virulence of different strains of the same concentrations and different concentrations of the same strains,establishing a larvae of G.mellonella infection model to verify whether the virulence of A.baumannii isolated from different sources is different.5.Intranasal instillation of Ab5075 in Balb C mice to establish a mouse pneumonia model,to analyze the general conditions of the mice after infection and after medication,lung pathology,lung colony count and inflammation indicators,to evaluate antibacterial activity of PB monotherapy and the combination of other 5 drugs against CRAB in vitro.Results:1.The infectious source,CRAB,hypoalbuminemia,Charlson comorbidity index(CCI),and mechanical ventilation were independent risk factors for death.The AUC of the ROC curve of the two groups was 0.768 and 0.792,respectively.The net income was higher when the probability was between 30%and 80%,showing a strong discrimination capacity of the proposed model.The calibration curve swung around the45°oblique line,indicating a high degree of calibration.2.10 CRAB strains all carry the resistance gene bla OXA-23 and bla OXA-51,3 strains carrying bla VIM,9 strains carrying bla ADC,7 strains carrying bla TEM.Among them,10 strains did not detect bla OXA-24,bla OXA-58,bla IMP and bla SHV genes.10 CRAB strains all carry the virulence genes omp A,csu A,csu E,pga A and aba I.Among the tested Ab resistance genotypes,the proportions in descending order are bla OXA-23(100%),bla OXA-51(100%),bla ADC(90%),bla TEM(70%),bla VIM(30%),bla OXA-24,bla OXA-58,bla IMP and bla SHV are all 0%.3.The resistance rate of 10 CRAB to IMP,MEM and SUL was 100%,and the resistance rate to PB,TGC and RIF was 0%.The order of MIC₅₀ from low to high is:PB<TGC<RIF<IMP=MEM=SUL.The order of MIC₉₀ from low to high is:PB<TGC<RIF<IMP=MEM=SUL.FICI distribution:PB+IMP/MEM/TGC/SUL/RIF:FIC?0.5:50%,60%,10%,30%,80%;0.5<FIC?1:50%,40%,60%,50%,20%;1<FIC?4:0%,0%,30%,20%,0%;FIC>4:0%,0%,0%,0%,0%.After PB is used in combination with 5 other antibacterial drugs,it basically shows synergy and additive effect on CRAB,with little irrelevant effect and no antagonistic effect.PB and RIF have the strongest synergy,followed by MEM and IMP,and the synergy with SUL and TGC is weak.4.The lethality of 10 CRAB strains to the larvae of G.mellonella showed a concentration-dependent,that is,the higher the concentration of the inoculated bacterial solution,the lower the survival rate of the larvae of G.mellonella.Under the conditions of injection of the same bacterial solution concentration,the mortality of the larvae were:10^8,Ab5075=Ab02>Ab09>Ab08>Ab01>Ab03>Ab07>Ab04>Ab06>Ab05.10^7,Ab5075>Ab02>Ab04>Ab03>Ab07>Ab08>Ab09>Ab05>Ab06>Ab01.10^6,Ab5075=Ab04>Ab05>Ab09>Ab03>Ab08=Ab07=Ab01=Ab02=Ab06.The survival curve shows that the virulence of strains derived from blood and cerebrospinal fluid is generally higher than that derived from ascites and respiratory tract.5.The minimum lethal dose of Ab5075 to mice was 10^7.Compared with the control group,the body weight and clinical scores of mice in the modeling group at 24h and48h were significantly different,P<0.05.After inoculation in the model group,a large number of macrophages and neutrophils infiltrated the alveoli,and secretions were seen in some alveolar cavities.Compared with the model group,the degree of inflammation in the treatment group was reduced.Compared with the corresponding monotherapy group,the inflammatory cells and exudate of each group in the combination group were significantly reduced.The results of lung bacterial load showed that compared with the model group,the treatment group reduced the lung bacterial load,and most of the differences were statistically significant(P<0.05).Comparing the inflammatory index between the treatment group and the model group,the serum inflammatory factors(IL-1?,IL-6,TNF-?)index values were significantly decreased,and most of them were statistically significant(P<0.05).Compared with the two drugs alone,the IL-6 and TNF-?values in the PB combined with RIF group were significantly different(P<0.05).Conclusion:1.The proposed model helped predict the risk of death from A.baumannii infection,to improve the early identification of patients with a higher risk of death,and guide clinical treatment.2.The 10 CRAB strains screened in this study mainly carry drug resistance genes including bla OXA-23,bla OXA-51,bla ADC and virulence genes including omp A,csu A,csu E,pga A,aba I.None of the 10 CRAB carry bla OXA-24,bla OXA-58,bla IMP and bla SHV genes.3.The combination of PB and RIF,IMP,MEM has a strong in vitro synergistic effect on CRAB isolates.4.Among the 10 clinical strains selected,Ab5075 has the strongest virulence.The lethality of clinical strains to larvae of G.mellonella is concentration-dependent.The virulence of strains derived from blood and cerebrospinal fluid is generally higher than that from ascites and respiratory tract.5.The combination of PB and other 5 drugs in the mouse pneumonia model has a better effect than the single agent,and the combination of PB and RIF has the best effect.