Analysis of biofilm genes and quorum sensing genes of carbapenem resistant Acinetobacter baumannii in the wounds of diabetic foot patients
Objective To investigate the biotilm genes and quorum sensing genes of carbapenem resistant Acinetobacter baumannii(CRAB)in the wounds of diabetic foot patients.Methods This study was a retrospective observational study.The 233 strains of Acinetobacter baumannii were cultured from 177 inpatients(128 males and 49 females,aged(56±10)years)with diabetic foot admitted to the Department of Diabetic Foot of Tianjin Medical University Chu Hsien-I Memorial Hospital from October 2020 to September 2023.Two hundred and thirty-three Acinetobacter baumannii strains were detected by bacterial culture from the diabetic foot wounds of the aforementioned patients.All Acinetobacter baumannii strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,followed by analysis of their resistance rate using kinetic turbidimetric assay by a fully automated microbiological analysis system.Ten CRAB strains(from 10 patients,9 males and 1 female,aged(63±13)years)and 10 carbapenem sensitive Acinetobacter baumannii(CSAB;from 10 patients,8 males and 2 females,aged(63±9)years)strains were randomly selected,and the whole DNA genome was extracted and sequenced.The antibiotic resistance genes were annotated using a comprehensive antibiotic resistance gene database,and a phylogenetic tree was drawn to analyze the homologous relationship between CRAB and CSAB.The 7 housekeeping genes of Acinetobacter baumannii was entered into the PubMLST website to analyze the multi-locus sequence typing of CRAB and CSAB.All the measured genes were put into the PubMLST website to search for the biofilm genes bap,csuA,csuB,csuA/B,csuC,csuD,csuE,pgaA,pgaB,pgaC,pgaD,bfmR,bfmS,ompA carried by each Acinetobacter baumannii,as well as the quorum sensing genes abaI and abaR,and flagellar gene pilA.The differences in carrying these genes between CRAB and CSAB were compared.The biofilm genes and quorum sensing genes carried by CRAB and CSAB carrying oxacillinase(OXA)resistance gene blaOXA were analyzed.Gross observation was performed to check if there was gel-like membrane-like substance in the diabetic foot wounds infected with CRAB and CSAB,and if so,the microstructure was observed by scanning electron microscope.Results Among the detected Acinetobacter baumannii,the positive detection rates of CSAB,CRAB,multi-drug resistant Acinetobacter baumannii,and pan-drug resistant Acinetobacter baumannii were 16.7％(39/233),83.3％(194/233),95.3％(222/233),and 34.3％(80/233),respectively,and no fully drug-resistant Acinetobacter baumannii was detected.Among 233 strains of Acinetobacter baumannii,the resistance rate to carbapenem antibiotics exceeded 80％;the resistance rate of cefoperazone/sulbactam was relatively low,at 37％;the resistance rates to the other cephalosporin antibiotics(cefotaxime,ceftazimide,cefotetan,and cefuroxime)were more than 80％;the resistance rates to all penicillin antibiotics were greater than 80％;the resistance rates to quinolone antibiotics were all over 60％;the resistance rate to minocycline was only 12％;the resistance rates to tigecycline and colistin did not exceed 1％.The phylogenetic tree showed that 10 CRAB strains were highly homologous,while 10 CSAB strains had low homology.The analysis of multi-locus sequence typing showed that 10 CRAB strains were all the same type;among the 10 CSAB strains,except 1 strain without typing,the remaining 9 CSAB strains had 7 types.Eight of 10 CRAB strains contained complete biofilm genes and quorum sensing genes.The biofilm genes from the strains of CSAB were incomplete and none carried the bap gene.Neither CRAB nor CSAB carried the flagellar gene pilA.Compared with that carried by CRAB,biofilm genes bap,csuA,csuC,and csuD and quorum sensing genes abaI and abaR carried by CSAB were significantly decreased(P＜0.05).The main blaOXA categories carried by CRAB were blaOXA-23-like(specifically BlaOXA-167)and blaOXA-51-like(specifically blaOXA-66),both of which had carbapenase activity.Eight of 10 CRAB strains carried both blaOXA-66 and blaOXA-167,and all of them had relatively complete quorum sensing genes and biofilm genes.The main blaOXA categories carried by CSAB were blaOXA-51-like and blaOXA-213-like.Although they had carbapenemase activity,clinical drug sensitivity test showed that they were all sensitive to carbapenem antibiotics.Gel-like and membrane-like substance could be seen in wounds infected with CRAB,which were biofilm;no gel-like and membrane-like substance was found in the wound infected with CSAB.Conclusions CRAB and CSAB in diabetic foot wounds are significantly different in terms of multi-locus sequence typing,carrying biofilm genes,quorum sensing genes,and blaOXA gene,leading to differences in antibiotic resistance between the two.
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