The Molecular Epidemiology Of Enterobacteriaceae Causing Bloodstream Infections In Shanghai And The Resistant Mechanisms Of IMP-type Carbapenemase

Bloodstream infection?BSI?is one of the most common serious clinical infections with the increasing morbidity and mortality year by year.Antibiotic resistance has become a severe healthy threat due to the globally excessive use of antibiotics.The productions of extended spectrum?-lactamases?ESBLs?,carbapenemases and plasmid-mediated AmpCs?pAmpC?are of the most important and common bacterial resistant mechanisms.Therefore,the investigation on the distributions and drug resistance of pathogens causing BSIs,the monitoring on resistant genes and molecular characteristics of resistant isolates,and the possible resistant mechanisms all have the clinical significances for prevention and treatment of nosocomial infections and drug-resistant bacteria,which should not be overlooked.We conducted this study in three university-affiliated tertiary general hospitals in Shanghai:Ruijin Hospital,Shanghai Jiao Tong University School of Medicine?HA?,Shanghai Jiao Tong University Affiliated Sixth People's Hospital?HB?and Shanghai First People's Hospital,Shanghai Jiao Tong University School of Medicine?HC?.Firstly we investigated the distributions of pathgeons causing BSIs between 2015 and 2016,and found that Enterobacteriaceae isolates were the most common?37.6%?,among which the top three followed:Escherichia coli,Klebsiella pneumoniae and Enterobacter cloacae.Based on these,we consecutively collected and isolated 697 E.coli,K.pneumoniae and E.cloacae non-duplicate bloodstream isolates in three hospitals from 2012 to 2016 and recorded the clinical data.Antimicrobial susceptibility was determined by disk diffusion accroding to CLSI.PCR was performed to detect ESBLs,carbapenemase and pAmpC genes.Multilocus sequence typing?MLST?and eBURST were employed for molecular typing and homology analysis.?1?E.coli:E.coli isolates causing BSIs were consecutively collected from three hospitals?HA to C?between 2013 and 2014.Ninety isolates randomly selected?30 per hospital?were enrolled in the study.The resistance rates for PRL,second/third-generation cephalosporins,fluoroquinolone and TE were high?>60%?.Sixty-one isolates?67.8%?produced ESBLs and no carbapenemases were found.CTX-M-15?25/61?and CTX-M-14?18/61?were the most common ESBLs.Phylogenetic group B2 predominated?43.3%?and also exhibited the highest rates of ESBLs production.ST131?20/90?was the most common sequence type?ST?and almost assigned to phylogenetic group B2?19/20?.Genetic diversity showed no evidence suggesting a spread of these antimicrobial resistant isolates between the three hospitals.?2?K.pneumoniae:K.pneumoniae isolates causing BSIs were consecutively collected from HA between 2012 and 2015.Eighty isolates?20 per year?were randomly selected and enrolled in this study.More than 50%isolates were resistant to CXM,SAM,and PRL,while carbapenems?<20%?had lower resistant rates than other antibiotics.Of all the 80 isolates,22 produced ESBLs?27.5%?,and 14 were carbapenemase producers?17.5%?.The most common ESBLs were SHV and CTX-M.Thirteen carbapenemase producers harbored KPC-2 and the other one carried NDM-5.ST11?14/80?was the most frequent ST.KPC-2presented clonal disseminations and eBURST analysis showed no homology among different STs.?3?E.cloacae:Fifty-three non-duplicate E.cloacae bloodstream isolates were consecutively collected from HA and HB between 2013 and 2016.The rates of resistance to all tested antibiotics were<40%.Eight isolates?15.1%?were ESBL producers,3?5.7%?were carbapenemase producers and 18?34.0%?were pAmpC producers.SHV?6/8,75.0%?and MIR/ACT?15/18,83.3%?predominated in ESBL and pAmpC producers respectively.Moreover,2 isolates co-carried SHV-12,IMP-26 and DHA-1.MLST distinguished the 53isolates into 51 STs with no commom STs,and multiclonality disclosed no evidence on spread of these isolates.In the part two,we also collected the above Enterobacteriaceae bloodstream isolates intermediate or resistant to carbapenems?MEM,IPM and ETP?in HA from January 2013to September 2017;a total of 65 isolates were enrolled,including 9 E.coli isolates,51 K.pneumoniae isolates and 5 E.cloacae isolates.Then we carried out the screening of bla_IMPMP among them and then through a series of experiments such as conjugation test,plasmid extraction,S1-PFGE,southern blot and long fragment PCR to characterize genotypes,genetic location and surrounding environment,and possible transmission of bla_IMP.In this study,only 3 E.cloacae isolates were detected bla _IMP,and all exhibited high resistance to cephalosporins and carbapenems.Two isolates harbouring bla_IMP-26 were ST191 and ST528respectively,while the other harbouring bla _IMP-4 was ST513.All three isolates were successfully conjugated and transferred:bla _IMP-26 was both located on conjugative IncHI2plasmids with nearly the same size?³00 kb?,while bla _IMP-4 was located on a 45 kb IncL/M plasmid.Two isolates harbouring bla_IMP-26MP-26 shared the same genetic content:Intl1-bla _IMP-26-orf II-orfIII-qacE?1-sul1,and the sourrounding environment of bla_IMP-4 was IS26-Intl1-bla_IMP-4-orf I-mobC.In summary,the resistant rates of E.coli and K.pneumonia bloodstream isolates were relatively high in Shanghai,while those of E.cloacae were low.CTX-M-producing E.coli ST131 isolates and KPC-producing K.pneumonia ST11 isolates were the most popular.Our study identified NDM-5-producing K.pneumoniae and IMP-26-producing E.cloacae in Shanghai for the first time.Therefore,the continuous monitoring on the distributions of pathogens,the changes of drug resistance and the molecular characteristics of resistance genes is still the most favorable theoretical basis for hospital infection control and clinical treatment.Additionally,bla _IMP is widespread in E.cloacae,and its resistance were mostly tranferred horizontally by plasmid,integrons or other mobile elements.IncHI2 plasmids may play a major role in the mobilization of bla_IMP-26.