Clinical Characteristics Of Klebsiella Pneumoniae And Antibiotic-Resistant Mechanism Of Hypervirulent Isolates

Widely considered an opportunistic pathogen,Klebsiella pneumoniae can be carried asymptomatically in the intestinal tract,skin and nasopharynx of healthy individuals but can also cause a range of infections in hospitalized patients,most commonly pneumonia,bloodstream,soft tissue,and urinary tract infections.Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant and hypervirulent strains.Due to widespread use of carbapenems,the global detection rate of CRKP has increased in recent years.CRKP isolates were shown to be resistant to a variety of antibiotics,and to have the ability to hydrolyze carbapenems,associated with a rising rate of treatment failure and mortality.The same is that increasing rate of hvKP infection have been reported worldwide.Although hvKP is sensitive to most commonly used antimicrobial agents,hvKP can cause life-threatening infection.In the past,the hvKP and MDR-KP located on two distinct clades on the phylogenetic tree and shared very little overlapping.But in the recent decade,the boundary between hvKP and MDR-KP blurred.HvKP with high level of drug resistance is increasingly emerging,posing a significant threat to public health.Therefore,the aim of this study was to describe the epidemiological,microbiological,and clinical characteristics of Klebsiella pneumoniae infections,and to further explore the resistance mechanism of hypervirulent isolates,that can provide a basis for clinical diagnosis and treatment.Part I.Epidemiology of carbapenem-resistant Klebsiella pneumoniae bloodstream infections in Ruijin Hospital:factors related to the morbidity and mortalityBackground:We sought to describe the epidemiological,microbiological,and clinical characteristics of CRKP-BSIs,focusing on the risk factors of the morbidity and mortality.Methods:A retrospective analysis of KP-BSI patients admitted to Ruijin hospital,between January 1,2011 and December 31,2015 was performed,and the annual percentage of patients with CRKP-BSI was determined.Risk factors related to the morbidity and mortality were analyzed using logistic regression model.Results:?1?A total of 293 incidences of KP-BSIs were identified in a 5-year period,22.2%of these?65/293?were CRKP strains,and the proportion of CRKP-BSI in ICU was 59.6%?31/52?,exceeding the national level.?2?Skin and soft tissue infection source?OR 26.63,95%CI4.8-146.8?and ICU-acquired infection?OR 5.82,95%CI 2.0-17.2?was shown to be powerful risk factors leading to the development of CRKP-BSI.?3?The crude 28-day mortality rates of KP-BSI and CRKP-BSI patients were 22.8%and 33.3%,respectively.?4?Lung as the probable source of infection?OR 4.23,95%CI 1.0-17.3?,and high SOFA score?OR 1.40,95%CI 1.2-1.6?were strong prognostic factors determining crude 28-day KP-BSI mortality.Conclusions:CRKP-BSIs are associated with high morbidity and mortality,especially in ICU.Part II.The epidemiological distribution,virulent genes and clinical characteristics of hypervirulent Klebsiella pneumonia in Ruijin HospitalBackground:To investigate the distribution,antibiotic resistance and virulent genes of the hvKP strains isolated from Ruijin Hospital and the clinical characteristics of these patients.Methods:Klebsiella pneumonia isolates were collected between September 2014and March 2016 from hospitalized patients.hvKP isolates were determined by string test.Capsular serotypes and virulent genes were detected using PCR.Clinical data of the hvKP detected patients were retrospectively studied.Results:?1?Of the 872 non-repetitive Klebsiella pneumonia isolates,the prevalence of hvKP was 14.7%,and the highest data was 44.1%from pus.Respiratory tract specimen was the most prevailing specimen of hvKP?70.3%?.?2?Majority of hvKP isolates were susceptible to main antimicrobial agents,but two carbapenem-resistant hvKP isolates capsular serotype and 32.0%for K2 capsular serotype).All hvKP isolates were fimH,wabG and ureA positive.The incidences of p-rmpA and p-rmpA2 was 91.4%?117/128?and 82.8%?106/128?,while the incidences of kfuBC and allS was 41.4%?53/128?and 27.3%?35/128?,related to K1 capsular serotype.The prevalence of capsular serotypes and virulent genes displayed no significant difference between different specimen types.?4?hvKP isolates occurred commonly in patients with diabetes mellitus?28.9%?and cancer/immunosuppression?44.5%?.60.9%?78/128?of the hvKP isolates could cause an infection,among which 47.4%?37/78?were community-acquired infection.The prevalence of capsular serotypes and virulent genes of hvKP also displayed no significant difference between infection group and non-infection group,as well as community-acquired infection group and hospital-acquired infection group.Conclusions:The prevalence of hvKP in our hospital was 14.7%,and the dominant specimen of hvKP was respiratory tract specimen,some of which were colonization bacteria.It is noteworthy that hvKP infection shows a convert from community-acquired to hospital-acquired.Part III.Antibiotic resistance mechanism of the hypervirulent Klebsiella pneumoniae Background:To investigate the antibiotic resistance mechanism of a K1-ST23 type hvKP isolate with a resistance plasmid containing bla _DHA-1 gene.Methods:We collectted the clinical information of patient with RJA166 isolate.We determined its hypervirulence by string test?virulent genes using PCR and killing assay on G.mellonella.Antibiotic resistance was confirmed through MIC and antibiotic resistance genes using PCR.The type of RJA166 was confirmed by capsular serotype?MLST and PFGE.The location of the antibiotic-resistant gene was determined by S1-PFGE and southern hybridization.The transferability of the plasmid carrying the resistant gene was confirmed by conjugation test.We used whole genome sequencing to study the genetic context around bla_DHA-1 gene.Results:?1?RJA166 was isolated from ICU.Its hypervirulence is conformed by a positive string test,a K1-ST23 type with common virulence genes and a higher G.mellonella lethality.RJA166 was resistant to penicillin??-lactam/?-lactamase inhibitor combinations?cephems except the forth?monobactams and nitrofurans,and intermediate to ertapenem,with bla_DHA-1HA-1 and bla_SHV-11 gene positive.?2?S1-PFGE and Southern hybridization clearly showed that bla_DHA-1 gene was located on a 230kb size plasmid,which was confirmed to be transferable.?3?Whole genome sequencing showed that RJA166 has three plasmids.pRJA166a,a resistant plasmid containing bla _DHA-1,was highly homologous with pKOX_R1?cover 92%;identity 99%?,and the regions surrounding the bla_DHA-1 gene were IS26-frmB-ISCR1-sul1-qacE?1-ampR-bla_DHA-1-purR-pspDCBAF-qnrB4-sdr-cinA-sapAB-IS26,which was highly homologous with pKP048 and pYDC676.The virulent plasmid pRJA166b,similar with pK2044 and pLVPK,contains a number of typical virulence genes.Bioinformatics analysis further shows that pRJA166a has a complete type IV secretion system structure that led to the transferability.Conclusions:RJA166 is the first report of K1-ST23 type hvKP isolate with a resistance plasmid containing bla _DHA-1 using whole genome sequencing.MDR-hvKP is formed by hvKP obtainned a resistant plasmid,which can spread between hvKP isolates.