Relationship Between Antibiotic Resistance,Efflux Pumps,and Biofilm Formation In Klebsiella Pneumoniae

Objective:To investigate the potential association of antibiotic resistance and the biofilm formation ability of Klebsiella pneumoniae in our hospital.Explore distribution of efflux pump genes acr A,kex D,oqx B,kde A,kpn E,and the expre ssion levels of kex D,kde A,kpn E in biofilm.Study the effect of efflux pump in hibitor cyanocarbonyl-3-chlorophenylhydrazone(CCCP)on the production of Kleb siella pneumoniae biofilms.Further understand the mechanisms of biofilm and dr ug resistance for exploring new anti-biofilm therapy.Methods:1.Collect 110 clinical isolates of Klebsiella pneumoniae from the First Hosp ital of Dalian Medical University between January 2019 to July 2019.Isolates id entification and antimicrobial susceptibility testing were performed by Micro Scan Walk Away 96 Plus automatic bacterial identification system.2.The biofilm formation assays were performed in microtiter plate assay.Ev aluate the biofilm ability of Klebsiella pneumoniae using crystal violet staining method and confocal laser scanning microscope(CLSM).3.Use PCR to dectect the distribution of efflux pump genes acr A,kex D,o qx B,kde A,kpn E in Klebsiella pneumoniae.And the relative expression levels of kex D,kde A,kpn E in biofilm were measured with a reverse transcription-PCR(RT-PCR)assay.4.The effect of CCCP on the biofilm formation of Klebsiella pneumoniae were explored by the method of crystal violet staining in vitro.Results:1.The results of drug susceptibility show that 110 strains of Klebsiella pne umoniae all were naturally resistant to ampicillin,above 70% of isolates were re sistant to cefazolin,cefotaxime,ceftriaxone,cefuroxime,above 60% to ampicillin/sulbactam,cefepime,aztreonam,ceftazidime,ciprofloxacin,cefoperazone,above of50% to tazobactam,cefoxitin,levofloxacin,imipenem,meropenem,ertapenem,a nd compound sinopol,46% to gentamicin,while the resistance rates to tobramyci n,amikacin,and tigecycline were lower,which were 32%,25%,and 2% respectiv ely.Of the 110 clinical isolates of Klebsiella pneumoniae,58 were carbapenem-r esistant,and 52 were non-carbapenem-resistant.2.Klebsiella pneumoniae have different biofilm formation capabilities.Amon g the 110 isolates,63 of these isolates have strong biofilm formation capabilities,20 of them were medium biofilm producers and 19 of them were weak biofilm producers.Eight strains were identified as negative.Observations with CLSM sh ow that the strong biofilm producers distribute densely,get together and form an obvious membrane structure.There was a significant difference of biofilm forma tion ability between Klebsiella pneumoniae carbapenem-resistant and sensitive stra ins.3.The genes acr A,oqx B,kde A,and kpn E were detected in 110 Klebsiella pneumoniae strains with a high rate of over 90%,while the lowest detection ra te for the kex D efflux pump gene was 72.73%.Statistical analysis show that the ability of Klebsiella pneumoniae biofilm formation was not significantly related to the type of efflux pump genes.However,there was a significant difference of the carried efflux pump gene types of Klebsiella pneumoniae between carbapene m-resistant and sensitive groups.4.The relative expression levels of kpn E and kex D genes in Klebsiella pne umoniae biofilm and plankton both were significantly different(kpn E: P<0.05,ke x D: P<0.01).But no significant difference was found in the kde A expression und er these conditions(P>0.05).5.When the CCCP concentration reached 8ug/ml,it could decrease the for mation of most Klebsiella pneumoniae biofilms significantly.Conclusions:1.The antibiotic resistance of clinical Klebsiella pneumoniae isolates from o ur hospital is severe especially to the carbapenem antibiotics.More than half of Klebsiella pneumoniae are multi-drug resistant.The resistance rate of meropenem,ertapenem and imipenem is high to 54%,only tobramycin,amikacin,and tigecyc line are low.2.Klebsiella pneumoniae is capable of forming strong biofilm,and the ability of biofilm-forming between carbapenem-resistant and sensitive isolates is signifi cantly different.3.The detection rate of Klebsiella pneumoniae efflux pump genes is high.Most of Klebsiella pneumoniae have more than 4 efflux pump genes.There are differences of efflux pump gene types carried by carbapenem-resistant Klebsiella pneumoniae and sensitive strains.4.The relative expression levels of kex D and kpn E are increased under the condition of biofilm.A certain concentration of efflux pump inhibitor CCCP can inhibit the formation of Klebsiella pneumoniae biofilm.So the efflux pump gene may be one of biofilm mechanisms in Klebsiella pneumonia.