| Objective To explore the clinical features of Klebsiella pneumoniae liver abscess(KPLA)and the molecular epidemiology of strains so as to provide a guidline for the clinical diagnosis and treatment of KPLA.Methods 212 strains of K.pneumoniae(KP)were isolated from inpatients in the affiliated hospital of Qingdao university between November 2013 and December 2019,and divided into two groups.Out of 212,130 KP strains recovered from patients with liver abscess were classified into LA-KP group,while 82 KP strains isolated from patients with non-liver abscess belonged to n LA-KP group.The identification and antimicrobial susceptibility testing of these strains were carried out by the Vitek-2 Compact automated system(bio Mérieux,France).The highmucoviscosity phenotypes of KP was identified by a positive string test.Capsular serotype genes(K1,K2,and K5)and virulence genes(rmp A,aerobactin,peg-344,and iuc A)were confirmed by polymerase chain reaction(PCR)amplification and sequencing assay.The clonal relatedness of LA-KP was determined by pulsed field gel electrophoresis(PFGE)and multi-site sequence typing(MLST).S1-PFGE was carried out for detecting the plasmids in all LA-KP isolates.Genomic DNA of three LA-KP isolates were extracted using bacterial genomic DNA extraction kit,and sequenced using Illumina Miseq sequencing technologies for the iuc ABCD-iut A locus sequence.The clinical data of all cases were retrospectively analyzed.The data analysis were conducted by using SPSS version 23.0.Results(1)All of 130 KPLA cases in this study were community-acquired infections.Majority of these patients were males(64.62%),and the median age of the patients was 58(51~65.25)years.Fever(93.84%)was the most common clinical manifestation.Diabetes(49.23%)was the most common underlying diseases.(2)All of the KPLA patients were investigated with CT scan.76.15% of liver abscess located in the right lobe,and 82.31% were single,and 60.00% were 5~10 cm of the dimension,and 23.85% were present of gas within the abscess.(3)Abnormal index of white blood cell count,neutrophil count,C-reactive protein,alanine aminotransferase,aspartate aminotransferase,total bilirubin,direct bilirubin,serum albumin,fasting blood glucose level was 68.46%,77.69%,100%,50.81%,34.68%,31.45%,39.66%,56.69% and 65.38%,respectively.Two strains of of LA-KP isolates were positive for Extended-Spectrum Beta-Lactamases(ESBLs),and the majority of strains were sensitive to a variety of common antibacterial drugs.(4)91.54%(119 / 130)of LA-KP group and 32.93%(27 / 82)of n LA-KP group were positive for the string test,respectively.There was a significant difference between the two groups(P<0.05).And the diagnostic accuracy was 82.08%.(5)PCR results showed that K1,K2,and K5 accounted for 66.15%(86 / 130),16.15%(21 / 130),and 2.31%(3 / 130),respectively,in LA-KP,while 19.51%(16 / 82),10.98%(9 / 82),and 1.22%(1 / 82),respectively,in n LA-KP.There were significant difference between the two groups in K1(P<0.05),K1 / K2(P<0.05),and K1 / K2 / K5(P<0.05).In addition,the accuracy of combined detection of LA-KP by three serotypes was higher than any single serotype.(6)In LA-KP,98.46%(128 / 130)of aerobactin,rmp A,and peg-344 were simultaneously positive,and the iuc A gene was detected in all strains.In n LA-KP group,the positive ratio of four virulence genes in all strains accounted for 43.90%(36 / 82),45.12%(37 / 82),43.90%(36 / 82),and 46.34%(38 / 82),respectively.There was a significant difference between the two groups in these four virulence genes(P<0.05).Meanwhile the detection accuracy was about 82%.Additionally strains with highmucoviscosity phenotypes or K1 and K2 capsular serotypes had a higher ratio of virulence gene than strains with non-highmucoviscosity phenotypes and the other capsular serotypes.(7)MLST results demonstrated that all of 130 strains in LA-KP group were divided into 27 sequence types,and ST23(47.69%,62/130)was found as a predominant sequence type,followed by ST700(16.92%,22/130).(8)PFGE profiles showed that 130 LA-KP isolates were determined to consist of 17 clusters.Group A(48.46%,63/130)and group B(17.69%,23/130)were mainly composed of K1 / ST23 and K1 / ST700 KP,respectively.(9)S1-PFGE revealed that majority of LA-KP isolates(72.31%,94/130)carried one plasmid,of which 84(64.62%)was about to 220 kb in size,whereas strains had two plasmids(16.15%,21/130)and had no plasmid(11.54%,15/130)were also observed.(10)Bioinformatics analysis showed that the sequences of iuc ABCD-iut A loci of LA-KP019(NK/ST29)and LA-KP029(K1/ST23)isolates were identical to those of previously reported plasmids p K2044 and p LVPK,while iuc D and iut A genes of LA-KP046(K1/ST700)strain were different from those of above four isolates.The 3’ fragment of the iuc D gene of LA-KP046 strain is 57 bp longer than the other four strains,and its iut A gene is only 92% sequence similarity to the other four strains.Conclusions(1)Fever is the most common clinical manifestation of KPLA.Abscess lesions are mostly single and located on the right lobe of the liver.KPLA often occurs in underlying diabetic patients.Most of the LA-KP isolates are sensitive to a variety of antibiotics,therefore narrow antibacterial spectrum drugs should be considered for the antibiotic treatment of KPLA.(2)Hypermucoviscous phenotype is expected to become an initial screening index for KPLA.Capsular serotype K1 is the most major serotype in LA-KP group.And the accuracy of combined detection of KPLA by K1,K2 and K5 serotypes is higher than any single serotype,which also has value on auxiliary diagnosis.(3)The positive ratios of aerobactin,rmp A,peg-344 and iuc A genes in LA-KP group or KP strains with hypermucoviscous phenotype or K1 and K2 capsular serotypes are higher,which indicates that the mucoviscous phenotype,capsular serotype and virulence genes are associated with the pathogenicity of KP-caused liver abscess.(4)In this study,130 LA-KP isolates are mainly composed of both group A(K1/ST23)and group B(K1/ST700).The number of ST700 KP in KPLA has shown an increasing trend in recent years,which is different from previous reports and should be highly valued.(5)Most of LA-KP only carry a plasmid with size of 220 kb.A few strains without plasmids can also be positive for virulence genes,indicating that virulence genes are on the chromosome.Therefore,virulence genes have the potential to lateral and vertical transmission.(6)The sequences of iuc D and iut A of the LA-KP046(K1 / ST700)strain are not consistent with the reference plasmids and the K1 / ST23 KP strain.Whether this difference are related to the virulence expression of the K1/ST700 type strain and the increase of this type isolates in KPLA patients in our hospital remain to be confirmed. |
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