A Clinical Investigation Of Bloodstream Infection In Patients With Hematopoietic Stem Cell Transplantation And The Study Of CP-PGN Anti-infection Treatment

Objective: To analyze the incidence and microbiological features of blood stream infections(BSI)of patients with hematopoietic stem cell transplantation(HSCT)and to provide laboratory data for empirical use of antibiotic for the HSCT patients with blood stream infections.And to investigate application potential of peptidoglycan of Corynebacterium pyruviciproducens(CP-PGN)as a new immunomodulator against blood stream infections of patients with hematopoietic stem cell transplantation,in order to reduce the excessive use of antibiotics and try to treat BSI without clear pathogenic bacteria or failed to cure.Methods:(1)The incidence of bloodstream infection,the positive rate of blood culture,bacterial spectrum and drug resistance were analyzed in 1265 HSCT recipients during 2013 and 2015 were retrospectively studied.(2)According to the above pathogenic analysis,three representative bacteria,methicillin-resistant Staphylococcus aureus(MRSA),carbapenenes producing Klebsiella pneumoniae(KPN)and Candida albicans(CAL),were selected to establish the blood flow infection model.The possibility of using in anti infection was evaluated by the survival state,survival time and survival rate of animals.(3)Animal models were established using MRSA to estimate the effects of CP-PGN on prevention or treatment of MRSA infection alone or combined with antibiotic.The objective indexes included survival status,bacterial load in the body,important organ inflammation injury,peripheral blood white blood cell count and so on,to evaluate the potential of CP-PGN as a new immunomodulator in the treatment of bloodstream infections.(4)The activation of CP-PGN on macrophages as one critical cell of inherent immunity was detected through the experiments of phagocytic test,nitric oxide secretion,transwell test,cytokine production and macrophage typing,that was to explore the mechanism of CP-PGN to play as an anti infection immunomodulator.(5)The significantly elevated TLRs after stimulatition by CP-PGN were knocked down by interfering with small Interfering RNA technology,to find and verify the pattern recognition receptor combined with CP-PGN.Results:(1)Of 1265 patients undergoing HSCT,1422 cases of suspected BSI occurred in 784 patients(61.98%),and 464 patients(59.2%)were in the stage of agranulocytosis(ANC<0.5×109/L).The detection rate of pathogens in 2013-2015 was about 20% and increase year after year.Of the 401 strains detected,221 were Gram-negative(G-)bacteria(55.1%),165 Gram-positive(G+)bacteria(41.2%)and 15 fungi(3.7%).Escherichia coli(16.0%),Staphylococcus epidermidis(15.5%)and Klebsiella pneumoniae(11.2%)were listed the top three.The proportion of multidrug resistant Acinetobacter Bauman and Stenotrophomonas maltophilia was 64.70% and 63.64% respectively,and methicillin resistant Staphylococcus aureus(MRSA)was more than half(57.14%).The ratio of vancomycin resistant Enterococci(VRE)and carbapenem resistant Enterobacteriaceae(CRE)was 14.29% and 6.78% respectively.More than 40% Enterobacteriaceae bacteria were resistant to three or four generation cephalosporin antibiotics,and less were resistant to the carbapenems(6.4%).However,many non-fermentative bacteria were highly resistant to these antibiotics and showed diversity among different strains,with a rate of 47.8% resistance to carbapenems.All the Staphylococcus was sensitive to vancomycin,teicoplanin and linezolid.(2)Compared with the untreated control group infected by MRSA,CP-PGN significantly improved survival state of mice including the mental state,activity,diet,body weight,hair gloss,prolonged the survival time and survival rate.And for carbapenemase-produced Klebsiella pneumoniae bacteria(KPN)and Candida albicans(CAL),CP-PGN showed preventive effects of different degree.It is suggested that CP-PGN has a specific effect resistance to common pathogens causing blood flow infection.(3)In the mouse model of MRSA bloodstream infection focused on the prevention function of CP-PGN,CP-PGN promoted the aggregation and release of peripheral blood neutrophil and reduced bacterial load of MRSA in peripheral blood and vital organ,indicating that CP-PGN directly enhanced the ability of mice to clear the invading pathogen.The inflammatory injury in the lungs of mice in CP-PGN treated group was significantly alleviated,and the levels of inflammatory factors in peripheral blood were also decreased dramatically,demonstrating that CP-PGN significantly alleviated the inflammatory injury of vital organs in MRSA bloodstream infection.At the same time,the spleen of the mice in the CP-PGN experimental group increased obviously,indicating that CP-PGN strengthened the immunoreactivity of mice.(4)In the CP-PGN alone or combined with vancomycin treatment mice model of BSI caused by a small or large amount of MRSA,CP-PGN showed the similar therapeutic effect tothe prevention test above,with better improved survival state,the greater survival rate and the prolonged survival time,than the control group.(5)CP-PGN enhanced the macrophage phagocytosis rate of pathogenic bacteria,the intent release of nitric oxide,the production of cytokine synthesis and the chemotactic function.In addition,CP-PGN skewed the macrophages to M1 phenotype with obviously anti infection effect.In a word,CP-PGN played an important role in all aspects of the innate immune cell against infection.(6)CP-PGN played a positive immunoregulatory function through TLR2 pathway,but with down myeloid differentiation factor88(My D88).Conclusion:The incidence of BSI in patients with HSCT was high,and the pathogens were mainly bacteria.In addition to Enterobacteriaceae,the proportion of non-fermentative bacteria was quite high.No G+ bacteria detected were resistant to vancomycin,teicoplanin and linezolid.CP-PGN could be used as a new immunomodulator with great potential at anti BSI through the TLR2 pathway.Firstly,CP-PGN had certain preventive effects on pathogens of bloodstream infections caused by Staphylococcus aureus,Klebsiella pneumoniae,Candida albicans and other common bacteria.Second,CP-PGN alone also showed good treatment effect on BSI caused by a small amount of bacterial infection,and it showed similar function against infection by a large number of bacteria combined with antibiotic.At last,CP-PGN may play as a candidate drug to cure unclear pathogen infection or with failure of antibiotic use.It was believed that through a series of experimental research to improve Clinical application strategy,in certain circumstances,CP-PGN would significantly ameliorate the therapeutic effect of anti infection,while reducing the excessive reliance on antibiotics,alleviating global antibiotic crisis.