Mechanisms Mediating In-hospital Transmission Of Carbapenem-resistant Klebsiella Pneumoniae Belonging To Sequence Type 11

Objectives:To characterize molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae(CRKP)in West China Hospital by their antimicrobial resistance determinants,virulence genes and sequence types(ST)based on the whole genome sequences analysis.To identify characteristics of the transmission of ST11 type CRKP.To examine clonal relatedness of ST11 CRKP isolates based on single nucleotide polymorphisms(SNP).To study mechanisms mediating the successful transmission of the predominant clone using a set of experiments informed by comparative genomics analysis.Method:1.CRKP isolates recovered from clinical samples and rectal swab isolates at ICU of West China Hospital,Sichuan University,between March 27 th and August 31 st in 2017 were collected.After species identification of these strains by sequencing the gyr B gene,the resistant phenotypes for 12 commonly-used antimicrobial agents were tested using the broth microdilution method of the Clinical and Laboratory Standards Institute(CLSI).All of the CRKP isolates were sequenced using the Illumina Hi Seq X10 platform,and the generated raw reads were trimmed by BBMap v35.85,assembled by Shovill v1.1.0 and annotated by Prokka v1.14.5.Antimicrobial resistance genes,virulence genes and plasmid information were then identified using the Abricate v0.9.2 program.Multilocus sequence typing(MLST)was performed using the MLST database.Clinical data were also analyzed.2.SNPs were called by Snippy v4.5.2 after removing recombination detected by Gubbins v2.4.1.Pairwise SNP distance matrix was generated by Snp-dists v0.6.3.A maximum-likelihood phylogenetic tree including all isolates was inferred by RAx ML v8.2.12.ST11 CRKP isolates were assigned to clones based on the SNPs and the phylogenetic tree.By including the collection date of individual isolates,the maximum-likelihood tree was further dated using Bact Dating v1.0.Genes and SNP specific to the predominant clone were identified by Scoary v1.6.16 and Roary v3.13.0 or Snippy v4.5.2.The three-dimension(3D)model of proteins with amino acid substitutions were predicted by the homology modelling web tool Swiss-model and the threading server I-TASSER.3.Fimbriae morphology of the representative strain of the predominant clone and that of the subdominant clone was observed through transmission electron microscope.Cell motility was examined on semi-solid agar.Serum resistance assay was performed by co-incubation with serum donated by health volunteers.Biofilm formation capacity was measured in sterile polyvinyl chloride(PVC)microtiter panel using crystal violet staining and glacial acetic acid dissolution.The Galleria mellonella larva infection model was used to assess the virulence of representative strains.Growth in iron-adjusted nutrient-restricted media was tested.In vitro competition assays between the two representative strains were performed incubated in iron-adjusted media.In vivo competition assay was tested using antimicrobial-treated mice inoculated with bacterial suspensions via oral gavage.Competition advantage was then measured by cell viable counts collected from mice fecal particles.4.Transcriptome sequencing was performed for the strain that showed obvious competition advantage in mice from the co-inoculated mixture.HTSeq v0.6.1 was used to count the number of reads mapped to each gene.Differential expression of two groups was analyzed using the DESeq R package v1.18.0.Gene ontology(GO)enrichment analysis of differentially expressed genes was performed by the GOseq R package.KOBAS software was used to examine the statistical enrichment of differential expression genes in KEGG pathways.RNA used for quantitative reverse transcription polymerase chain reaction(q RT-PCR)was prepared using the RNAiso Plus kit.q RT-PCR was then performed to eut B(encoding ethanolamine ammonia-lyase heavy chain)and eut R gene(encoding HTH-type transcriptional regulator)expression levels.The capacity of the two representative strains to grow with ethanolamine as the sole carbon or nitrogen source was tested on modified M9 medium supplemented with the corresponding substances.Competition assays were performed between the two representative strains co-incubated in modified M9 media with ethanolamine as the sole nitrogen source.The concentration of vitamin B12 in the mice fecal particle after the mice were mouth-fed with different bacterial suspensions for day 1,day 3 and day 7 respectively,were measured by ELISA.Vitamin B12 in the medium incubating different cultures was determined using the above method.Results:1.A total of 174 CRKP isolates were obtained with 153 from rectal swabs and 21 from clinical specimens.All isolates were multi-drug resistance.Most of the isolates carried carbapenemase gene bla KPC-2.One low-level colistin-resistant strain was also found.All KPC-producing CRKP isolates were susceptible to ceftazidime-avibactam,although was ineffective against CRKP producing class B carbapenemase NDM.ST11 was the dominant ST type of CRKP in this study.The major capsular K-antigen type was KL64,and the major lipopolysaccharide O-antigen type was O2v1.As for virulence factors,all isolates carried type 3 fimbriae coding genes and yersiniabactin associated with iron uptake.2.ST11 CRKP in this study could be assigned to seven distinct clones,among which there was a predominant clone comprising most ST11 isolates.A time-calibrated phylogenetic analysis was inferred base on all ST11 CRKP isolates in this study,suggesting that the emerging time of the most recent common ancestor was irrelevant to the formation of predominant clone.An average of 10.8 antimicrobial resistance(AMR)genes were identified from strains belonging to the predominant clone,whereas fewer than the 15.8 on average in those from subdominant clone,the clone comprising the second largest number of isolates.The comparative genomics analysis demonstrated that the predominant clone lacked an approximate 10.5-kb gene fragment,which contained 10 consecutive protein-coding genes including fim D,yad V,mlt F,fab G,ghr A,clo R and btu D and three genes with unknown function.Most of those genes encoding proteins for fimbriae synthesis process,while others were associated with redox and energy metabolism.This fragment was replaced by an ISKpn26 in the predominant clone,implying the defective fragment emerged before divergence between the predominant clone and the subdominant clone.In addition,the predominant clone had 5 specific non-synonymous SNPs and 1 nucleotide deletion,all of which involved in the biofilm synthesis.3.The predominant and subdominant clones displayed no significant difference in fimbriae morphology and function,as well as the ability of serum resistance ability.The capacity of biofilm formation on PVC surface was weaker in predominant clone compared with the other clone,indicating that the dissemination advantage was no relevant to biofilm formation.In addition,the predominant clone exhibited lower virulence than the subdominant clone.The representative strain of the predominant clone had a slightly competitive advantage over the other when co-incubated in the iron-defective medium.Obvious competition advantage fitness was also observed for the representative strain of the predominant clone over that of the subdominant clone in fecal particles from mice received mouth-fed commensal bacterial suspension.4.Transcriptome sequencing of the representative strains in the mono-and co-cultures from mice fecal particles was performed.Differential gene expression analysis of the representative strain of the predominant clone compared to the monoculture control in mice gut revealed four significantly upregulated gene clusters,including type 2 secretion system,vitamin B12 anaerobic synthesis pathway,ethanolamine utilization and 1,2-propanediol utilization pathway.Relative fold change in eut B and eut R confirmed the upregulation at the m RNA level.Both of the tested CRKP strains could use ethanolamine as the sole nitrogen source for growth.While co-cultured in the nutrient-restricting condition with ethanolamine as the sole nitrogen source,the representative strain of the predominant clone showed a slight competition fitness to the other one.Compared with that in mice without antimicrobial treatment,the vitamin B12 concentration in feces of mice with microflora disturbed by antimicrobials increased after gavage of the bacterial suspension.The concentration of vitamin B12 in the mice fecal particle after the mice were mouth-fed with the predominant bacterial suspensions on day 3 was increased compare to those mouth-fed with the subdominant bacterial suspensions,suggesting that the predominant clone had stronger capability to synthesize vitamin B12.Conclusions:CRKP collected from West China Hospital showed high levels resistance to carbapenems,and most of them carry bla KPC-2.ST11 was the dominant ST type spread in the hospital.A predominant clone comprising most ST11 CRKP isolates was identified.The emerging time of the most recent common ancestor of each clone was irrelevant to the emergence of predominant clone.The predominant clone had competition advantage mainly due to the increased vitamin B12 anaerobic synthesis and enhanced ethanolamine utilization in co-culturation.The capacity of utilizing ethanolamine as a nitrogen source conferred a growth advantage to the representative strain of the predominant clone when co-cultured with that of the subdominant clone.Lower virulence might contribute to the long-term carriage of the predominant clone in colonized patients,thereby leading to more successful dissemination.The stable gut microbe community was critical to gut colonization resistance.The prevention and management of hospital infection should be combined with the antimicrobial stewardship(AMS).Our findings suggested that the ability of enteric pathogens to utilize substrates provided an advantage during colonization,which was important for rationally designing future strategies for prevention of gut colonization and decolonization.