| Background and Purpose:Acinetobacter baumannii(A.baumannii)is one of the leading pathogens in hospital infections,especially in burn units.We collect 1658 blood samples from 162 patients during a five years period(from January 2011 to December 2014).Among them,339 samples were positive for bacterium.The first three species are A.baumannii,S.aureus and P.aeruginosa.It’s reported that A.baumannii is the second pathogens following P.aeruginosa.There is one outbreak of A.baumannii in newyork city in 1991.The antibiotic-resistance(especially resistant to Carbapenem antibiotics)of A.baumannii had drawn the public concerns since then.Phage is a virus targeting bacteria,which had been proved useful against infection diseases.Dr.Yu once used phage to treat burn infections caused by P.aeruginosa in 1958.In the last decades,phage therapy was ignored by the widely usage of antibiotics.Recently,phage therapy draws the public concern again for the emergency of antibiotic-resistance.In this study,we test the therapy effect of phages using a mice infection model.Methods:(1)Isolation and storage of A.baumannii phagesPhages were amplified with mixed host bacteria method.The pan-resistant A.baumannii was used as the host strain.Eight phages were obtained in the untreated sewage pool.One of the lytic phages was selected for the following experiment and the titer was 1×108PFU / mL.(2)Establishment of pan-resistant A.baumannii infection mice modelForty-eight 8 to 12 weeks old male BALB / c mice were randomly divided into groups(12 rats in each group)and A,B,C and D groups.The mice were injected with5 × 108 CFU / m L,2.5 × 108 CFU / mL,1 × 108CFU/ mL and 5 × 107 CFU / mL of A.baumannii.After the injection,the mice in each group were kept in normal diet.The vital signs were observed closely.The number of dead mice,the culture results of the peritoneal lavage fluid and the blood culture were collected.(3)Effect of phage therapy on pan-resistant A.baumannii infectionsⅠ.Sixty BALB/c mice were divided into blank control group,sepsis control group,antibiotics treatment group,phage treatment group,and phage control group according to the random number table,with 12 mice in each group.Mice in blank control group were intraperitoneally(the same injection position below)injected with 1 mL normal saline.Mice in sepsis control group,antibiotics treatment group,and phage treatment group were injected with 1 mL extensively drug-resistant Acinetobacter baumannii(the strain was isolated from the blood of a severely burned patient hospitalized in our unit)in the concentration of 5×107 colony-forming unit/mL to reproduce sepsis model.Two hours later,mice in sepsis control group,antibiotics treatment group,and phage treatment group were injected with 1 mL saline,1 mg/mL imipenem/cilastatin,and 1×108 plaque-forming unit(PFU)/mL phages screened based on above-mentioned Acinetobacter baumannii(the same phages below)respectively.Mice in phage control group were injected with 1 mL phages in the titer of 1×108 PFU/mL.The injection was performed continuously for 7 days in each living mouse,and the survival situation of mice was observed each day to calculate the survival ratio in one week.Ⅱ.Another 60 BALB/c mice were grouped and treated as in experiment Ⅰ,and the injection was performed continuously for 5 days in each living mouse.On experiment day 2,4,and 6,3 mice from each group were selected(if the number of survived mouse in any group was less than 3 at sample collecting,all the survived mice were selected),and blood was drawn to determine white blood cell count(WBC,with 3 samples at each time point in each group).On experiment day 2,blood was drawn from the mice that had their blood taken earlier for bacterial culture,and lung,liver,kidney,and spleen tissue was collected from the same mice.The tissue samples were added to the LB solid medium after being homogenized and diluted for bacterial culture.The content of bacteria was calculated after the bacterial colony number was counted.Data were processed Wilcoxon rank sum test,one-way analysis of variance,LSD test and Kruskal-Wallis rank sum test.Results:(1)With 22 host A.baumannii isolates,a total of 8 phages were isolated and they are proved to be lytic phages.Of them,Bp201404072 shows the widest host rang(59%).So we use this phage in our following experiments.These phages were stored at 4 centigrade.(2)The mice in each group were treated with different concentrations of Acinetobacter baumannii,and all the four groups of mice showed typical sepsis-related symptoms.Among them,A and B mice were more severe than C and D groups.A week later,A,B,C,D four groups of mice were died.Of A group,all mice died on the third day of the experiment.Of group B,all mice died on the fourth day of experiment.Of C group,all mice died on the fifth day.Of group D mice were all died on the seventh day of the experiment(one was killed for orbital venous blood bacterial culture).The survival time of each group was analyzed with log-rank test.Compared with group D,the survival time of other groups was shorter(P = 0.002,there was statistical difference).Peritoneal lavage fluids of four groups were positive for bacteria culture.Orbital venous blood culture of group D was also positive,and the bacteria were further identified to be A.baumannii.Compared with other groups,the concentration of group D(5 × 107 CFU / mL)meets the requirements of subsequent animal experiments.So a bacterial concentration of 5 × 107 CFU / m L was used in the following animal experiment.(3)ⅠOn experiment day 7,there were 12,8,10,and 12 mice survived in blank control group,antibiotics treatment group,phage treatment group,and phage control group respectively,while no mouse survived in sepsis control group.Compared with that in sepsis control group,the survival ratio of mice was significantly higher in the other four groups(with Z values from 55.635 to 106.593,P values below 0.05).The survival ratio of mice in phage treatment group was slightly higher than that in antibiotics treatment group,without statistically significant difference(Z=2.797,P>0.05).Ⅱ3,2,and 1 mouse died on the second day in Sepsis control group,antibiotic treatment group and phage treatment group respectively.10,7,and 6 mice died on the fourth day in Sepsis control group,antibiotic treatment group and phage treatment group respectively.12,11,and 2 mice died on the second day in Sepsis control group,antibiotic treatment group and phage treatment group respectively.On experiment day 2,WBC data of mice in blank control group,phage treatment group,and phage control group were close,all significantly lower than the datum in sepsis control group,(P<0.05),and the WBC data in the latter two groups were significantly lower than the datum in antibiotics treatment group(with P values below 0.05).On experiment day 4,WBC data of mice in antibiotics treatment group,phage treatment group,and phage control group were close,all significantly lower than the datum in blank control group(P<0.05),and WBC data in the above-mentioned four groups were all lower than the datum in sepsis control group(with P values below 0.01).On experiment day 6,there was no statistically significant difference in WBC among blank control group,antibiotics treatment group,phage treatment group,and phage control group(χ2=4.128,P>0.05).On experiment day 2,respectively 12,7,and 2 mice were detected as blood bacterial culture-positive in sepsis control group,antibiotics treatment group,and phage treatment group,while no positive result was detected in the other two groups.Positive ratios of blood bacterial culture of mice in blank control group,phage treatment group,phage control group were significantly lower than the ratio in sepsis control group(with χ2 values from-30.000 to 30.000,P values below 0.01).Positive ratio of blood bacterial culture of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group(with χ2 values respectively 17.500 and-17.500,P values below 0.05).On experiment day 2,except for the kidney tissue of mice in phage treatment group,the bacteria load in each viscus of mice in blank control group,phage treatment group,and phage control group was significantly lower than that in sepsis control group(with χ2 values from-9.000 to 9.000,P values below 0.01).The bacteria load in kidney of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group(with χ2 values respectively-7.500 and 7.500,P values below 0.05).Conclusion:In this study,the pan resistant A.baumannii was used as the host bacteria,and the untreated sewage pool of the hospital was a phage source.The phage was used as an antibacterial agent for the treatment of sepsis caused by Pan resistant A.baumannii in mice.Phage treatment can improve the survival rate of sepsis mice and control inflammatory response.It is proved that phage can be used for clinical treatment of bacterial infection. |
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