Identification Of Hep In Type Ⅵ Secretion System Of Acinetobacter Baumannii And Its Correlation With Drug Resistance

Objective To analyze the clinical distribution characteristics and changes in drug resistance rate of Acinetobacter baumannii(A.baumannii),to observe the relationship between drug resistance and the hcp gene of type Ⅵ secretion system(T6SS)in A.baumannii,then to study the extraction method of Haemolysin co-regulated protein(Hcp)and bacterial competition,and to provide further determine the basis the drug resistance and the pathogenesis of Hcp in A.baumannii.Methods We use WHONET 5.6 software to do statistical analysis of clinical strain of A.baumannii in hospital to analyze the change in prevalence and drug esistance of A.baumannii.PCR was performed to screen for the presence of hcp gene of A.baumannii in respiratory tract,to analyze its relationship and drug resistance.Chemical precipitation,ultrafiltration and ultratracentr-ifugation were used to extract Hep of A.baumannii cultured supernatant and cell lysate,we analyze extraction rate of Hep by SDS-PAGE and mass spectrometry.And to research the bacterial competition in A.baumannii and Escherichia coli.Results 1.Analysis resistance to antibacterial drugs of A.baumannii and detection the presence of hcp gene of A.baumannii isolated from respiratory tract.A total of 45744 strains of bacteria were detected in our hospital during 2015 to 2019 in hospital,including 2001 strains of A.baumannii,accounting for 4.38%.The detection rate of A.baumannii showed a downward trend from 5.01%(2015)to 3.90%(2019).In addition,A.baumannii infection was mainly distributed in the Intensive Care Unit(22%)and the emergency department(14%).In our hospital,A.baumannii had the highest resistance rate to cefoxitin(97.4%to 99.1%),and it had good sensitivity to tigecycline,the resistance rate was less than 10%.The resistance to beta lactams(ceftriaxone,ceftazidime,cefepime),carbapenems(imipenem,meropenem)and quinolones(ciprofloxacin,levofloxacin)of A.baumannii all beyond 45%.In the eighty-one clinical strains of A.baumannii were isolated from respiratory tract in our hospital,55.6%(45 of 81)was detected positive for hcp.The hcp gene has improved the drug resistance of A.baumannii,the difference was statistically significant(P<0.001).2.Assay of Hcp ScretionThe cell lysate and supernatant proteins of A.baumannii ATCC17978,A.baumannii hcp knock out strain(ATCC17978△hcp),A.baumannii complemented strain(ATCC17978 △ hcp+)and twenty clinical strains of A.baumannii were separated and extracted by chemical precipitation,ultrafiltration and ultracentrifugation,ultrafiltration was the best method.Hcp could be expressed in A.baumannii ATCC17978,A.baumannii ATCC1797△hcp+and ten clinical isolates of A.baumannii.However,Acinetobacter baumannii ATCC17978 △hcp was detected negative for Hcp.3.Bacterial Competition AssayA.baumannii ATCC17978,A.baumannii ATCC17978 △ hcp,A.baumannii ATCC17978 △ hcp+were co-cultured with Escherichia coli.A.baumannii ATCC17978,A.baumannii ATCC17978△hcp,A.baumannii ATCC1797△hcp+had no killing effect on Escherichia coli,A.baumannii clinical strains could kill Escherichia coli.Conclusion 1.The clinical detection rate and drug resistance antibiotics(except tigecycline)of A.baumannii showed a downward trend in our hospital,and it has indicated the infection of A.baumannii was controlled in recent years..The hcp gene exists in A.baumannii improved drug resistance rate to antibiotics,and it had indicated the hcp gene was correlated with the resistance of A.baumannii.2.Ultrafiltration as a highly efficient extraction method for Acinetobacter baumannii Hcp.The expression of Hcp was different in different strains of A.baumannii.3.Different strains of A.baumannii have different killing effects on Escherichia coli.And Hcp played a competitive role in A.baumannii clinical resistant strain.