| Objective: To analyse the epidemiological situation of Klebsiella pneumoniae(KP)in the region by using Multilocus sequence typing(MLST)technique and to investigate whether there are differences in the biofilm formation ability of different ST types of KP.The effect of azithromycin combined with levofloxacin on the biofilm of different ST types of Klebsiella pneumoniae was investigated,aiming to provide new ideas for the prevention and treatment of Klebsiella pneumoniae infections with biofilm.Methods: 1.Collect 9 strains of Klebsiella pneumoniae from the sensitive group(wild group),19 strains of extended-spectrum β-lactamases(ESBLs)-producing Klebsiella pneumoniae,and 37 strains of Carbapenem resistant Klebsiella pneumoniae(CRKP)isolated from inpatient specimens at Yijishan Hospital Affiliated to Wannan Medical College from August 2019 to November 2021.All strains were identified by MALDI-TOF-MS mass spectrometry.Perform MLST homology analysis on each strain.2.In vitro biofilm models of different ST types of Klebsiella pneumoniae were constructed,and the optical density at 590 nm(OD590)was measured by crystalline violet staining to detect the growth trend of biofilms of different ST types of Klebsiella pneumoniae,and to compare the differences in biofilm formation ability between different ST types of Klebsiella pneumoniae.3.The minimal inhibitory concentration(MIC)of azithromycin and levofloxacin against Klebsiella pneumoniae was determined using a micro broth dilution method.The Fractional inhibitory concentration index(FICI)was calculated to determine the combined effect and select the optimal combined concentration.Explore the inhibitory and clearance effects of azithromycin combined with levofloxacin on biofilms of Klebsiella pneumoniae of different ST types.4.Use confocal laser scanning microscopy(CLSM)to observe the structural changes of Klebsiella pneumoniae biofilm before and after the action of antibiotics.Results:1.MLST typing showed that the sensitive group of Klebsiella pneumoniae strains belonged to 8 different sequence types: ST86,ST727,ST29,ST111,ST1049,ST421,ST592,and ST268;The Klebsiella pneumoniae strains in the ESBLs group belong to 14 different sequence types,including ST15,ST37,ST11,ST23,ST25,ST1109,ST893,ST485,ST1798,ST133,ST967,ST264,ST726,and ST273,with ST15 accounting for26.30% and ST37 accounting for 10.50%;The Klebsiella pneumoniae strains in the CRKP group belong to 9 different sequence types: ST11,ST15,ST656,ST1805,ST147,ST4587,ST219,ST5636,and ST34.Among them,ST11 accounts for 48.70%,ST15 accounts for 27.00%,and ST656 accounts for 5.40%.Our hospital’s carbapenem resistant Klebsiella pneumoniae is mainly characterized by ST11 and ST15.Among the37 CRKPs,13 strains were mainly from neurosurgery,including 5 strains of ST11,5strains of ST15,and 8 strains of ICU.Among them,7 strains of ST11 and 1 strain of ST15 were the main epidemic strains.2.ESBLs group ST15 type,CRKP group ST11 type,and CRKP group ST15 type Klebsiella pneumoniae all formed biofilms and reached maturity on the 5th day,followed by biofilm dispersion.Compare the biofilm production of the ST15(ESBLs)group and ST15(CRKP)group,as well as the ST11(CRKP)and ST15(CRKP)groups on the fifth day of biofilm maturation.The ST15(ESBLs)group has stronger biofilm production ability than the ST15(CRKP)group,and the ST11(CRKP)group has stronger biofilm formation ability than the ST15(CRKP)group,and the differences are statistically significant(P<0.01).3.MIC of antibacterial drugs: ST11 type CRKP has a MIC of 512 ug/ml for azithromycin and 128 μg/ml for levofloxacin;The MIC of azithromycin and levofloxacin in ST15 type CRKP is 512 μg/ml and 256 μg/ml respectively;The MIC of azithromycin and levofloxacin in the ST15 type ESBLs group was 512 μg/ml and 4μg/ml,respectively.Partial inhibitory concentration index(FICI): The FICI of the ST11 type CRKP group was 0.53,the FICI of the ST15 type CRKP group was 0.51,and the FICI of the ST15 type ESBLs group was 0.53,indicating that the combined effect of azithromycin and levofloxacin on different ST type Klebsiella pneumoniae was additive.4.The 2×MIC azithromycin group,1×MIC levofloxacin group and 2×MIC Azithromycin+1×MIC levofloxacin group had inhibitory effects on the biofilm formation of three KP strains.On the 5th day,the ST11(CRKP)group had an inhibitory rate of biofilm formation on 2×MIC azithromycin group 84.00%,1×MIC levofloxacin group 46.83% and combination drug group 85.13%;The inhibition rate of biofilm formation in the ST15(CRKP)group is 2×MIC azithromycin group 78.07%,66.20% in the 1×MIC levofloxacin group and 83.93% in the combination drug group;The inhibition rate of biofilm formation in the ST15(ESBLs)group is 2×MIC azithromycin group 75.73%,1×MIC levofloxacin group 51.10% and combination drug group 89.93%.Within the selected experimental concentration range,the 2×MIC MIC azithromycin group and the combination drug group showed significant inhibitory effects on the biofilm formation of the three strains.5.After the biofilm was matured,the clearance rates of Klebsiella pneumoniae biofilm in each group were 42.70% in the ST11(CRKP)azithromycin group,12.50% in the levofloxacin group,and 44.79% in the combination drug group,respectively;35.85% in the ST15(CRKP)azithromycin group,20.38% in the levofloxacin group,and 36.23%in the combination drug group;ST15(ESBLs)azithromycin group 29.17%,levofloxacin group 16.67%,and combination drug group 35.12%.In the biofilm clearance test,the clearance effect of the combination drug concentration >2×MIC azithromycin>1×MIC levofloxacin,but the highest clearance rate in the combination drug group was only 44.79%.Conclusion: The carbapenemase resistant Klebsiella pneumoniae in our hospital are mainly ST11 and ST15.There may be clonal transmission in neurosurgery and ICU wards,so we should strengthen the prevention and control of hospital infection.There are differences in biofilm formation ability among different ST types of Klebsiella pneumoniae.The ST15(ESBLs)type has stronger biofilm formation ability than the ST15(CRKP)type,while the ST11(CRKP)type has stronger biofilm formation ability than the ST15(CRKP)type.Azithromycin and its combination therapy have good inhibitory effects on the biofilms of Klebsiella pneumoniae of different ST types.The combination therapy has a certain clearance effect on the biofilms of Klebsiella pneumoniae of different ST types,but the clearance rate is not high.Therefore,it is recommended that early combination therapy be used to treat biofilm infections in clinical practice. |
PDF Full Text Request