| Objective:Klebsiella pneumoniae is a common clinical pathogen,which can cause multiple infections and hospital acquired infections.The most serious of infections of Klebsiella pneumoniae is bloodstream infections.With the widespread use of antibiotics,especially irrational use of drug,the emergence of strains expressing extended spectrum beta-Lactamases(ESBLs)and even carbapenem-resistant strains have brought great difficulties to treatment of clinical and control of nosocomial infection.The infections caused by multidrug-resistant strains have become an important cause of high mortality in nosocomial acquired infections worldwide.Especially in recent years,strains with high drug-resistance and high virulence have emerged.Although they have not been widely spread,the infections caused by them will be more difficult to control.In this paper,molecular microbiology technology was used to analyze Klebsiella pneumoniae isolated from bloodstream from 2010 to 2016.The purpose of this study is to understand the drug resistance,mechanism,virulence genes and phenotypes of Klebsiella pneumoniae infected by bloodstream,and to explore the epidemic trend of Klebsiella pneumoniae over the years,its molecular background,risk factors of infection and factors affecting prognosis and treatment,based on the analysis of clinical data.The results of this study will provide some reference for clinical treatment of corresponding infections.Method:1.Drug-resistant phenotype and molecular biology of Klebsiella Pneumoniae BSIs(1)Microbiological identification and drug susceptibility identification:all strains were isolated from bloodstream of the Second Affiliated Hospital of Suzhou University from 2010 to 2016,and detected by Phoenix System-100BD automated microbiology system,and their antimicrobial susceptibility was determined.(2)Confirmation test of drug resistance phenotype:ESBLs were detected by phenotype confirmation test,carbapenem inactivation method(mCIM)and modified Hodge test(MHT)were used to detect carbapenem enzyme(3)Detection of drug-resistance genes:the gene encoding ESBLs(blaCTX,blaTEM,blaSHV)and the gene encoding carbapenemase(blaKPC,b/aNDM,blaIMP,blaVIM,blaOXA-48)were amplified by Polymerase Chain Reaction(PCR),and the positive amplified products were sequenced and identified.(4)Molecular epidemiological characteristics analysis:using Pulsed Field Gel Electrophoresis(PFGE)to analyze the homology of strains.On the basis of PFGE,multilocus sequence typing(MLST)was used to molecular typing of Klebsiella pneumoniae.2.Identification and analysis of hypervirulence Klebsiella pneumoniae BSIs(1)High mucus phenotype experiment:hmvKP was detected by string-test.Positive results were defined as hmvKP groups and negative results as non-hmvKP groups.(2)Virulence gene detection and serum capsule typing:the virulence genes and serum capsule typing of the isolates were amplified by multiplex PCR,and the positive products were sent for sequencing and comparison.(3)Virulence analysis of the strains:the virulence of the related strains was tested by using the infection model of the wax borer.3.Investigation and analysis of clinical data of Klebsiella Pneumoniae BSIs with different characteristics(1)Collect informations of clinical data of Klebsiella pneumoniae BSIs.(2)Summary and analysis of related factors:according to the characteristics of strains,the factors such as gender,age,whether to use invasive equipment,different wards,primary diseases and so on were summarized,and the correlation analysis was carried out by SPSS 25.0 software.Result:1.Drug-resistant phenotype and molecular biology of Klebsiella Pneumoniae BSIs(1)A total of 153 Klebsiella pneumoniae BSIs were collected.Klebsiella pneumoniae BSIs were resistant to ampicillin in 98.7%,and the drug resistance rate of other antibiotics was lower than 50%.5.2%of the isolates developed resistance to carbapenems antibiotics.(2)It was confirmed that 33.3%of the isolates produced ESBLs and 5.2%of the isolates produced carbapenemase.(3)The positive rates of blaTEM,blaCTX and blaSHV were 15.0%,11.1%and 30.1%respectively.The positive rates of blaTEM and blaSHV were 9.8%,blaCTX and blaSHV were 7.2%.The probability of all three genes being positive was 3.9%.The positive rates of blaKPC and blaVIM genes expressing carbapenemase were 3.9%and 0.7%respectively.No other genes were detected.(4)MLST showed that the majority of Klebsiella pneumoniae was ST23(13.1%),followed by ST65(3.3%),ST65(3.3%),ST11(1.3%).ST107,ST15,ST43,ST186 and so on were all one.PFGE showed low homology of Klebsiella pneumoniae infection in blood stream except ST23 and low possibility of clonal transmission.2.Identification and analysis of hypervirulence Klebsiella pneumoniae BSIs(1)32.0%of isolates were positive in string test,which indicated that the isolates were hmvKP.(2)The results of multiple PCR showed that there were 66 hvKP isolates,rmpA(93.9%),rmpA2(77.3%),iroN(87.9%),and iutA(83.3%)were detected among all hvKP isolates.K1(19.7%)and K2(12.1%)were detected in serotypes of hvKP while K64 and K47 were not detected.The majority of hvKP were ST23(30.3%),followed by ST65(7.6%),and the rest were non-repeated STs.In 2016,a ST11-hvKP was found to carry the carbapenemase-producing resistance gene blaKPC.(3)The model experiment of the large wax borer infection showed that a total of 66 virulence related gene positive strains had the characteristics of high virulence,and the overall survival rate of the hvKP group was 0%to 70%after three days,and the overall survival rate of virulence related gene negative strains was 90%to 100%.3.Investigation and analysis of clinical data of Klebsiella Pneumoniae BSIs with different characteristicsThis study showed that the patients with hvKP were mainly in Emergency Department(n=11),ICU(n=7),Oncology Department(n=7),Digestive System Department(n=7),Hematology Department(n=6)and Endocrinology Department(n=6).Diabetes mellitus is an independent risk factor for hvKP(OR=2.123).Liver abscess(OR=3.921)and neutrophil percentage(OR=1.033)are also directly related to hvKP infection.Infection in ICU(OR=4.466),male patients(OR=2.026)and changes in mental status(OR=2.371)were independent risk factors for 30-day deterioration rate.K1 resulted in liver abscess(P=0.034).This experiment showed that the resistance rate of cKP to almost all antibiotics except imipenem,meropenem,amikacin and chloramphenicol was higher than that of hvKP,and the difference was statistically significant.Conclusion:1.Klebsiella pneumoniae BSIs are non-clonal transmission.Drug resistance,even resistance to carbapenems antibiotics is widespread.2.The prevalence of hvKP-BSIs in our hospital is high.In 2016,a strain with high virulence and high drug-resistance was found.Through animal models,this study proposes that the detection of hvKP by checking out the virulence genes is of reference value.3.This study suggests that K1 is associated with liver abscess.Changes in mental state of patients are one of the signs of poor prognosis.Male with hvKP infections may have poor prognosis and need better clinical prevention and treatment measures. |
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