The Study Of The Clinical Characteristics,biofilm Formation And Virulence Genes Of Clinically Isolated Hypervirulent Klebsiella Pneumoniae From Respiratory Infection

PART Ⅰ Clinical characteristics Of hypervirulent klebsiella pneumoniae In patients With respiratory infectionObjective To explore the clinical characteristics of patients with hypervirulent Klebsiella pneumoniae(hvKP)in our hospital,in order to provide theoretical guidance for the clinical diagnosis and treatment of hvKP infection.Method 86 strains of Klebsiella pneumoniae isolated from inpatients with respiratory system infection in the first affiliated Hospital of Guangxi Medical University from January 2019 to January 2021 were collected.The identification and drug sensitivity test of all strains were carried out by VITEK-2 Compact system.The positive string test was defined as hypervirulent Klebsiella pneumoniae.The hypervirulent Klebsiella pneumoniae group was used as the observation group and the classica Klebsiella pneumoniae group as the control group.The clinical data of the patients were analyzed by multivariate analysis to determine the risk factors and clinical characteristics of Klebsiella pneumoniae infection.Results The string test was positive in 42 strains(48.84%)and negative in44 strains(42.5%).Among 86 strains of respiratory infection Klebsiella pneumoniae isolated in clinic,63 strains were sputum samples(72.26%)and 23 strains were lavage fluid(27.74%).Compared with the cKP control group,the hospitalization time in the hvKP observation group was significantly longer(P<0.01).The incidences of hospital acquired infection,septic shock,severe pneumonia,ICU hospitalization,mechanical ventilation,tracheal intubation,tracheotomy and fungal infection were significantly higher.(P<0.05).Compared with cKP control group,the incidences of diabetes mellitus,malignant tumor,infection within 1 week after surgery and hypoproteinemia were significantly higher in hvKP group(P<0.05).Compared with cKP,the resistance rate of hvKP to amoxicillin/clavulanic acid,ceftazidime,ceftriaxone,cefuroxime(injection),cefazolin,gentamicin,levofloxacin,piperacillin and sulfamethoxazole was significantly lower(P<0.05).In addition,22 strains(22/86,25.58%)produced extended-spectrum β-lactamases(ESBLs),the proportion of cKP strains producing ESBLs was significantly higher than that of hvKP strains(38.64%and 11.90%,P<0.01).hvKP group is prone to systemic invasive infection.Conclusion The hospitalization time,hospital acquired infection,septic shock,severe pneumonia,ICU,mechanical ventilation,tracheal intubation,tracheotomy and fungal infection in hvKP group were significantly higher than those in cKP group.Diabetes mellitus,malignant tumor,hypoproteinemia within1 week after surgery and hypoproteinemia hvKP group were significantly higher than those in cKP group.The resistance rate of hvKP to common clinical antibiotics was lower than that of cKP.In addition,the proportion of ESBLs production in hvKP group was significantly lower than that of cKP group.hvKP group is prone to systemic invasive infection.PART Ⅱ Distribution characteristics Of biofilm,capsular serotype and virulence genes of hvKP and cKP in respiratory infectionsObjective To observe the distribution characteristics of biofilm,capsule serotype and virulent gene of hcKP infection in respiratory system.Methods After hcKP group and cKP group were selected from the first part,the difference of biofilm forming ability of KP between the two groups was determined by crystal violet and canning electron microscopy,and the distribution characteristics of 6 capsular serotypes: K1,K2,K5,K20,K54 and K57 were detected by PCR,and the distribution characteristics of 9 virulence genes,aero,rmpA,rmpA2,magA,fimH,iucA,iutA,iroB and PEG-344 in the two groups were detected.qPCR was used to detect the difference of rmpA gene expression in biofilm condition between cKP group and hvKP.Results Among the 86 strains of KP,there were 60(69.77%)biofilm positive strains,including 36(85.71%)biofilm positive strains in hvKP group and 24(54.54%)biofilm positive strains in the cKP group.Scanning electron microscopy showed that the three-dimensional structure of biofilm and a small amount of extracellular matrix were observed in cKP.In the hvKP group,the three-dimensional structure of biofilm was also seen by scanning electron microscope,the extracellular matrix was sticky and dense,and Klebsiella pneumoniae gathered more in the membrane;The most common serotype was K2 in hvKPand cKP.In the hvKP group,the highest positive rates of virulent genes were rmpA,iutA,PEG-344,accounting for 78.57%、73.81%、71.43%,respectively.Compared with cKP group,the expression of rmpA,rmpA2,PEG-344,iutA and iucA genes in hvKP group was significantly higher(P<0.05).There was significant difference in the expression of rmpA gene between cKP group and hvKP group by qPCR method,and the expression of rmpA gene in hvKP group was higher than that in cKP group(P<0.05).Conclusion The clincal isolated hvKP has a stronger biofilm formation ability than cKP;K2 is the main serotype of cKP and hvKP,and the expression of K2 in hvKP group is higher than that in cKP group;rmpA,iutA,PEG-344,rmpA2 and iucA genes are all highly expressed in hvKP,and higher than those in the cKP group;Patients in the hvKP group are severely ill and have a poor clinical prognosis.At the same time,the capsular serotype K2,rmpA and PEG-344 are expressed high,K2,rmpA,PEG-344 may have a certain relationship with the severity of the disease after infection and the poor prognosis.