| OBJECTIVE: We did the research to investigate the difference of virulence between the clinically isolated pan drug-resistant Acinetobacter baumannii and sensitive Acinetobacter baumannii,as well as the virulence difference before and after the biofilm formation of pan drug-resistant Acinetobacter baumannii,which might provide theoretical basis for clinical management and control of drug-resistant Acinetobacter baumannii.METHODS: The bacterial strains of different concentrations of live bacteria were injected into Galleria mellonella larvae in the abdominal foot so as to detect the optimal dose of inoculation bacterial toxicity of the pan-resistant Acinetobacter baumannii strain and the sensitive Acinetobacter baumannii strain to Galleria mellonella larvae.In vitro competition experiments,the colony numbers of pan drug-resistant Acinetobacter baumannii and sensitive Acinetobacter baumannii were respectively calculated at 4,8,12,16 and 24 h by plate counting method.The survival line chart of in vitro competition was drawn to compare the difference of growth ability between independence growth and the mixed growth of the two different groups of bacteria in equal concentration.At the in vivo competition experiment model of Galleria mellonella larvae,injected with same dose of pan-resistant Acinetobacter baumannii strains and sensitive Acinetobacter baumannii strains and their mixtures,the Galleria mellonella larvaes were respectively ground at 4,8,12,16 and 24 h.At each time point,the number of colonies was counted by plating,and the bacterial load of hemolymph was calculated.Consequently,the in vivo competition index was calculated and the in vivo competitive survival line chart was drawn to compare the difference in the concentration of the hemolymph of Galleria mellonella larvae between the groups when infected with pan-resistant Acinetobacter baumannii strains and sensitive Acinetobacter baumannii strains respectively as well as their mixtures,.After culturing macrophages RAW264.7,we plated the cells into 96-well plates,and the optimal inoculation concentration of Pan-resistant Acinetobacter baumannii and Acinetobacter baumannii were added to 96-well plates.24 hours later,the values of each group were measured by microplate reader A450,and the cell survival percentage of each group of macrophages RAW264.7 was calculated by formula.Afterwards,the histograms of different groups of Acinetobacter baumannii macrophage survival percentage were plotted to compare the invasive ability of the different groups of Acinetobacter baumannii strain to macrophages.The pan drug-resistant Acinetobacter baumannii liquid in the state of plankton and biofilm were respectively injected into the larva of the borer with the optimal concentration of inoculation through the abdominal foot,and the virulence of the pan drug-resistant Acinetobacter baumannii in the different states to the Galleria mellonella larvaes was tested.The macrophage RAW264.7 was cultured and inoculated with pan drug-resistant Acinetobacter baumannii solution in the state of plankton and biofilm,respectively.The values of each group were determined by microplate A450,the survival rate of macrophages in each group was calculated,and the invasion ability of the two groups on macrophages was compared.RESULTS: The optimal inoculation concentration experiment was concluded that the survival number of Galleria mellonella larvae presented a different degree of decline at different time points,no matter it was infected with pan drug-resistance Acinetobacter baumannii or sensitive Acinetobacter baumannii.And the more the infection concentration increased,the more the survival number decreased significantly.In the control group,no death was found within 96 h after inoculation with PBS.According to the survival curve of Galleria mellonella larvae infected with different groups of Acinetobacter baumannii,the median lethal dose(LD50)of pan drug-resistance Acinetobacter baumannii strains was(1.311±0.213)×105 CFU,and the median lethal dose(LD50)of sensitive Acinetobacter baumannii strains to Galleria mellonella larvae was(1.262±0.112)×104 CFU.In different drug resistance of Acinetobacter baumannii to Galleria mellonella larvae toxicity experiments,it could be observed that their survival rate showed obviously various degrees of decline after injected with the optimal inoculation concentration with Acinetobacter baumannii regardless of sensitive or pan drug-resistance strains.At 24 h and 72 hours,there was a statistically significant difference in the number of survival percentage of Galleria mellonella larvae which were respectively infected with pan drug-resistance Acinetobacter baumannii and sensitive Acinetobacter baumannii strains(P<0.05),and the number of survival Galleria mellonella larvae in the pan drug-resistance Acinetobacter baumannii group was higher than that of in the sensitive Acinetobacter baumannii group.In vitro growth competition experiments showed that the growth curves of the pan drug-resistance Acinetobacter baumannii strains and the sensitive Acinetobacter baumannii strains were similar when they were cultured separately in vitro.The concentration of pan drug-resistance Acinetobacter baumannii was 0.484 Log of CFU/m L lower than that of the sensitive Acinetobacter baumannii after mixed culturing for 24 hours.In the competition experiment in vitro,conclusions can be drawn with statistical analysis that the concentration of the bacterial liquid was statistically different when the two strains were mixed and cultured in vitro within different measurement times and different strains(P<0.05).When the sensitive Acinetobacter baumannii and the pan-resistant Acinetobacter baumannii were inoculated into Galleria mellonella larvae respectively,the bacterial load of the Galleria mellonella larvae hemolymph of the two groups were roughly the same in 24 h.After the mixture of the two strains was infected with the wax larvae,there was a statistical difference between the concentration of sensitive Acinetobacter baumannii and pan drug-resistance Acinetobacter baumannii at 12 h and 24 h after the Galleria mellonella larvaes were infected with the mixture of the two strains(CI12=O.813,CI24=0.872,P<0.05).The statistical analysis of each group concluded that there was a statistically significant difference in lymphocyte load concentration when different strains were mixed and grown in the environment of Galleria mellonella larvaes at the different measurement time(P<0.05).And after respectively infected by sensitive Acinetobacter baumannii or pan drug-resistance Acinetobacter baumannii,the survival percentage of macrophages gradually decreased.24 hours later,the survival percentage of macrophages infected by pan drug-resistance Acinetobacter baumannii was(96.251±1.204)×100%,and the survival rate of macrophages infected by sensitive Acinetobacter baumannii was(99.843±0.992)×100%.Based on the statistical comparison of the two,it could be proved that there was a statistically significant difference in the survival percentage of macrophage between sensitive Acinetobacter baumannii and pan drug-resistant Acinetobacter baumannii.Moreover,the macrophage survival percentage of the pan drug-resistance Acinetobacter baumannii group was lower than that of sensitive Acinetobacter baumannii group.Among the 26 clinically isolated pan drug-resistant strains,16 of them could be induced to produce biofilms,accounting for 61.54% of the pan drug-resistant Acinetobacter baumannii.The survival rate of Galleria mellonella larvae infected by Acinetobacter baumannii in the state of plankton and biofilm showed no statistically significant difference.No death was observed within 96 h after inoculation with PBS,but the interaction at different time points was input into the statistical analysis software,and P=0.000 could be obtained.Therefore,there was a statistical difference in the number of survival of Galleria mellonella larvae after inoculation before and after the biofilm formation of pan drug-resistant Acinetobacter baumannii at different measurement time.After infected by floating and biofilm state strains respectively,the survival rate of macrophage gradually decline.After 24 hours,the results of floating macrophages in the survival rate by statistical analysis was(95.232±1.828)×100%,the survival rate of biofilm group macrophage was(99.691±1.097)×100%,P=0.0346.Therefore,we concluded that the macrophage survival rate difference between floating and biofilm state pan drug-resistant Acinetobacter baumannii was statistically significant.CONCLUSION: Under the condition of individual existence,the virulence of the pan-resistant Acinetobacter baumannii group was stronger than that of the sensitive Acinetobacter baumannii group,finally leading to the decreasing survival percentage of macrophages RAW264.7 and Galleria mellonella larvaes which were infected by the two strains.However,in the mixed case,the biological cost of the pan-resistant Acinetobacter baumannii was mainly manifested in the weakening of the virulence of Galleria mellonella larvaes and macrophage RAW264.7,which was declined in the competitiveness of growth in vitro and in vivo.There was a statistical difference between the virulence of pan drug-resistant Acinetobacter baumannii under plankton state and biofilm state.The virulence of pan drug-resistant Acinetobacter baumannii under plankton state to Galleria mellonella larvaes and macrophage RAW264.7 was stronger than that under biofilm state. |
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