Study On The Correlation Between The Biofilm Of Klebsiella Pneumoniae And Drug Resistance And Efflux Pump Genes

ObjectTo analyze the clinical characteristics and related drug resistance of Klebsiella pneumoniae in our hospital departments.To investigate the relationship between biofilm formation ability of Klebsiella pneumoniae and antibiotic resistance and efflux pump genes.To analyze the m RNA expression of acr B,oqx B,rar A,ram A in the biofilm state relative to the planktonic state,and the effect of the efflux pump inhibitor Carbonyl cyanidem-chlorophenylhydrazone（CCCP）on biofilm of Klebsiella pneumoniae.In order to provide a relevant theoretical basis for the clinical treatment of infections caused by Klebsiella pneumoniae.Methods1.The 85 strains of non-repetitive Klebsiella pneumoniae isolated from various departments of the Affiliated Chaohu Hospital of Anhui Medical University from August 2018 to March 2022 were collected.Identification and drug sensitivity analysis were conducted by VitekⅡCompact automatic analysis system.The source and distribution of strains and drug resistance to common antibiotics were analyzed.2.Crystal violet staining was used to detect the biofilm formation of Klebsiella pneumoniae,and to study the correlation between biofilm formation and antibiotic resistance of Klebsiella pneumoniae.3.The OD value of the biofilm was dynamically detected by crystal violet staining,and the rule and morphology of the biofilm was observed by light microscope.4.Efflux pump genes acr A,acr B,oqx A,oqx B,tol C,Kex D,Kde A,Kpn E,ket M and regulatory genes rob A,ram A,mar A,sox S,rar A,acr R were detected by PCR.5.RT-PCR was used to detect the relative expression of the Klebsiella pneumoniae efflux pump genes acr B,oqx B,rar A and ram A in two states:biofilm and planktonic,respectively.6.Crystal violet staining was used to detect the effect of CCCP on biofilm of Klebsiella pneumoniae.Results1.The 85 strains clinical isolates of Klebsiella pneumoniae came from sputum（53strains,62.35%）,followed by urine（13 strains,15.29%）.In the distribution of clinical departments,the detection rate of the strains were the highest in the department of neurosurgery（24,28.24%）,followed by the department of critical care medicine（15,17.65%）.The resistance rate of Klebsiella pneumoniae to ampicillin was 100%,and the resistance rate to ampicillin/sulbactam and cefazolin were more than 85%.However,the resistance rate to piperacillin/tazobactam,cefepime,tobramycin,imipenem,ertapenem,cefotetan and amikacin were less than 30%.Among the 85strains of Klebsiella pneumoniae,58 strains（68.24%）were MDR,66 strains（77.65%）were ESBLs-producing.Statistical results showed that there were statistically significant differences between MDR-KP strains and non-MDR-KP strains in the resistance rates of ampicillin/sulbactam,furantoin,cotrimoxazole,ciprofloxacin,levofloxacin,tobramycin,amtrannan,gentamicin and ceftazidine（P<0.05）.2.The biofilm formation ability of 85 strains of Klebsiella pneumoniae varied,with76 strains（89.41%）able to form biofilms,among which 22 strains（25.88%）had strong positive biofilms.21 strains（24.71%）were moderately positive.33 strains（38.82%）were weakly positive.Only 9 strains（10.59%）could not form biofilm.The biofilm formation of ESBLs-producing strains were significantly higher than that of non-ESBLs-producing strains（χ²=13.052,P=0.003）.There was a significant difference in biofilm formation between MDR and non-MDR groups（χ²=8.279,P=0.041）.3.Crystal violet staining showed that Klebsiella pneumoniae began to form biofilm on the first day,and then the amount of biofilm increased gradually.The OD₅₇₀of the biofilm increased rapidly from the second day to the third day,and reached maturity on the fifth day,the absorbance increased from 0.213±0.018 to 0.793±0.041.From the fifth day to the sixth day,the absorbance decreased to 0.681±0.046.The results were analyzed and found that the amount of the biofilm of Klebsiella pneumoniae was related to the culture time（χ²=35.116,P<0.05）.The detection of biofilm by ordinary light microscopy and crystal violet staining had a good consistency.After one day of culture,Klebsiella pneumoniae began to adhere to and scattered on the surface of the cover glass.The bacteria gradually gathered together after two days of culture.On the fifth day of culture,the biofilm became mature,the structure was tight,and the gap between the bacteria was small.On the sixth day of culture,the biofilm spread and fell off,and the gap between the bacteria increased.4.Among the 85 strains of Klebsiella pneumoniae in this study,the detection rates of efflux pump genes acr B,Kde A,Kpn E and regulatory genes rob A,sox S,and rar A were higher than 95%,while the detection rates of Kex D,oqx A,oqx B,and acr R were lower,and the detection rate of Kex D was the lowest（83.53%）.Statistical analysis showed that except for efflux pump gene rar A（χ²=4.954,P=0.106）,The biofilm formation ability of Klebsiella pneumoniae was different from that of Kex D,Kde A,Kpn E,acr A,acr B,oqx A,oqx B,tol C,ket M,rob A,ram A,mar A,sox S and acr R（P<0.05）.5.The expression levels of four efflux pump genes in 15 strains of Klebsiella pneumoniae in different conditions were measured.The relative transcription levels of acr B,oqx B,rar A and ram A in the biofilm were up-regulated compared with those in the plankcells（P<0.01）.6.When the concentration of CCCP was 0.625μg/ml-5μg/ml,it could promote the biofilm of Klebsiella pneumoniae to a certain extent.However,at the concentration of10μg/ml,CCCP could inhibit biofilm formation,and the inhibition was more significant at the concentration of 20μg/ml.Conclusion1.The drug resistance of Klebsiella pneumoniae isolated from our hospital is relatively serious.2.The biofilm formation ability of MDR is significantly increased.3.The formation of biofilm is a dynamic process.4.The biofilm formation of Klebsiella pneumoniae is associated with efflux pump genes.5.Compared with planktonic state,the expression of acr B,oqx B,rar A and ram A in biofilm state increases.6.At lower concentration,CCCP could promote the formation of biofilm.While at higher concentration,CCCP could inhibit the formation of biofilm.