Molecular Epidemic Characteristics Of Carbapenem-Resistant Enterobacterales And The Adaptive Evolutionary Mechanism Of Bla_NDM-bearing Plasmid

Carbapenems are regarded as one of the most effective antibiotics for the treatment of multidrug-resistant Gram-negative bacterial infections due to the broad antibacterial spectrum and excellent antibacterial activity.Nevertheless,with the unreasonable use of these drugs,the detection rate of carbapenem-resistant Enterobacterales(CRE)in clinical settings has been increasing year by year,posing a serious threat to global human health.Although carbapenems are banned for veterinary use,the prevalence of CRE in animal sources and the surrounding environment has been reported,supporting the evidence that CRE has spread among the humans,animals,and environment,posing a threat to the common health goal proposed by "One health" concept.Consequently,this study focused on CRE isolates from clinical and animal sources in China,with the goal of elucidating the molecular epidemic characteristics through phenotypic assay,genome sequencing,and longread nanopore single-molecule analysis techniques.Subsequently,this study used three novel blaNDM-bearing plasmids as research objects,analyzing their structural features and formation mechanisms,as well as exploring the adaptive evolutionary mechanism of the fusion plasmid in Escherichia coli,to provide scientific basis for controlling the spread of CRE in different environments.1.Prevalence and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae among hospitalized patients in clinical settingsFrom March 2018 to August 2019,a total of 35 carbapenem-resistant K.pneumoniae(CRKP)strains were identified from 1413 isolates from various wards in a hospital,of which 30 strains encode KPC-2 and five strains encode NDM-1.The emergence of CRKP strains was associated with carbapenem therapy,according to binary logistic regression analysis.All CRKP strains displayed high drug resistance rate to common clinical antibiotics expect tigecycline and polymycin B,and the drug resistance spectrum of blaNDM-1 positive strains was narrower than that of blaKPC-2 positive strains.Conjugation assay revealed that carbapenem resistance genes from three blaKPC-2 positive strains and two blaNDM-positive strains could be transferred into the recipient strain.Genomic studies revealed that all strains were classified into six multilocus sequence types(MLSTs),with ST 11 being the most common(24/35).The extensive clonal transmission of ST 11 blaKPC-2 positive strains and local polyclonal transmission of blaNDM-1 positive strains were observed in this hospital.Furthermore,the blaNDM-1 gene was located on an IncX3 plasmid,a novel IncHI5-like plasmid,and an IncFIB/IncHI1B hybrid plasmid,respectively.Virulence gene screening,string testing,Galleria mellonella larvae infection testing,siderophore secretion testing,and sequence comparison jointly proved that the ST11 blaKPC-2 positive bacteria were hypervirulent.Finally,fifteen representative strains were selected to estimate the adaptability,desiccation resistance,biofilm formation,and human serum resistance,these strains have excellent environment adaptability and can survive in the hospital for a long time.2.Epidemiology and molecular characteristics of blaNDM-5 positive E.coli in a goose farmIn 2018,117 samples of anal swabs,feces,water,soil,and feed were collected from goose breeding farms and a hatchery,and 12 blNDM-5 positive E.coli strains were isolated for the first time.Eleven strains were identified from the goose breeding farms,while one strain was isolated from the hatchery.These strains had a broad drug resistance spectrum and a high resistance rate to common veterinary antibiotics.Furthermore,the blaNDM-5 gene could transfer horizontally in all strains,and their conjugation frequency was steady.Except for one strain,S1-PFGE effectively detected the conjugation of a 46 kb plasmid in the remaining strains,implying that the blaNDM-5 in these strains was located on a conjugative plasmid with a similar structure.Whole genome sequencing revealed that ten strains belong to ST48,while the remaining strains were ST3076 and ST8809,respectively,demonstrating that the clonal transmission dominated by ST48 blaNDM-5 positive E.coli in the goose farm.Yet,no clonal transmission was found between the goose farm and hatchery.In addition,blaNDM-5 was located on IncX3 plasmid with a highly comparable structure in all strains.According to these findings,the prevalence of blaNDM-5 positive E.coli in the goose farm was mainly mediated by the clonal transmission of ST48 E.coli and the horizontal transmission of the IncX3 plasmid.3.Epidemiology and molecular characteristics of blaNDM positive Enterobacterales in the pork production chain from a pig slaughterhouse to a retail marketDuring 2020 to 2021,204 samples consisting of intestinal contents,feces,etc.,as well as 40 pork samples,were collected from a pig slaughterhouse and a retail market.A total of 40 blaNDM-postive strains were discovered,including 11 Enterobacter hormaechei strains,eight Klebsiella spp.strains,and 21 E.coli strains.Significantly,11 strains,including eight strains of E.hormaechei,two strains of K.pneumoniae,and one strain of Klebsiella aerogenes,had the blaNDM-5 and tigecycline resistance gene tet(X4).The majority of the strains exhibited multidrug resistance,and the blaNDM gene in 34 strains could transfer horizontally,while the tet(X4)gene was not.Genomic analysis showed that most of the blaNDM genes were located on the IncX3 plasmid,and a novel IncX3 blaNDM positive plasmid was found as well.The blaNDM positive E.hormaechei could be divided into three clonal subgroups,with the blaNDM-5 and tet(X4)coharboring clones spreading clonally from the pig slaughterhouse to retail market.Additionally,ST48 E.coli predominated in the slaughterhouse,whereas the prevalence of E.coli in retail market was diverse.blaNDM positive K.pneumoniae and Klebsiella Michigan,on the other hand,could clonally spread across different samples,but not along the pork production chain.Finally,all blaNDM-5 and tet(X4)coharboring strains shared the same plasmid backbone,and such emerging tet(X4)bearing plasmid structure may be formed on the basis of pKAE3SP-1-like plasmids by the insertion of a tet(X4)-carrying circular intermediate and variable regions rearrangement.Based on the preceding three chapters,we discovered that,while CRE from various sources displayed distinct epidemic characteristics,there was also a potential correlation between them.In the goose farm and pig slaughterhouse,for example,a large epidemic dominated by ST48 blaNDM positive E.coli was detected.Besides,the majority of blaNDM genes were associated with the conjugative IncX3 plasmid,and a number of novel blaNDMcarrying plasmids were also discovered.4.Structure and formation mechanism of novel blaNDM-carrying plasmids in CRE strainsThis chapter explores the structural features and formation mechanisms of three types of novel blaNDM-carrying plasmids,including the blaNDM-1-carrying IncHI5-like plasmid pC39-334kb from CRKP strain in chapter 1,the fusion plasmid pL53T formed during conjugation of blaNDM-5 positive E.coli in chapter 2,and the blaNDM-5-carrying IncX3 plasmid pNTT31XS with structurally deficient in chapter 3.First,analysis of the IncHI5-like plasmid revealed that while pC39-334kb shared conserved sequences with IncHI5 plasmids,the multidrug resistance region(MDR)was distinct.The Tn125 structure carrying blaNDM-1,the region highly similar to the MDR region of plasmid pKP1814-1,and the typical type I integron complex comprised the MDR region of pC39-334kb.By retrieving all available IncHI5/IncHI5-like plasmids from NCBI database and constructing phylogenetic tree,we speculated that the generation of IncHI5-like plasmid was mediated by the mutation of the replicon of IncHI5 plasmid pNDM-1-EC12.Subsequent analysis of pNTT31XS revealed that,when compared to the typical IncX3 plasmid,pNTT31XS deleted two fragments,including an ISAba125 element and a 27kb region containing T4SS system,mobile element,and hns gene,which was mediated by the short sequence’s homologous recombination.Lastly,we discovered that the fusion plasmid pL53T was derived from the donor strains’ two daughter IncX3 and IncFII(pCoo)plasmids,and its formation was mediated by the homologous recombination of two IS26 elements during conjugation.These findings suggested that all of the novel blaNDM positive plasmids were evolved from existing plasmids via gene mutation or homologous recombination.5.Adaptive evolutionary mechanism of the blaNDM-carrying fusion plasmid in E.coliBecause the fusion plasmid is conjugative and carries high abundance of insertion sequences and drug resistant genes.Yet,the fundamental mechanism limiting plasmid dissemination is the fitness cost.As a result,the fusion plasmid pL53T was used as the research object to investigate its adaptive evolutionary mechanism in E.coli C600.C600’s fitness was reduced after acquiring pL53T,but after long-term selective pressure,the evolution of chromosome and plasmid jointly improved bacterial fitness,as evidenced by increased survival ability in vivo and in vitro pairwise competition,enhanced biofilm formation and gut colonization ability.From a plasmid aspect,a large-scale sequence loss was observed in an evolved strain.Although the evolved strain imposed a negligible fitness cost on host bacteria,its conjugation frequency was greatly reduced,and the deficiency of psiB gene was responsible for the impaired horizontal transferability.From a chromosomal perspective,all evolved strains exhibited comparable mutations in the transcriptional regulatory gene sspA(stringent starvation protein A).By constructing a sspA null mutant,transcriptome analysis,in vitro transcriptional activity assay,RT-qPCR,motility test,and scanning electron microscopy techniques,we demonstrated that the mutated sspA gene can reduce the inhibitory effect on transcription,thereby improving bacterial fitness,biofilm formation ability,and gut colonization ability by enhancing bacterial flagella synthesis.These findings collectively reveal a novel adaptive evolutionary mechanism,and expand our knowledge of how the fusion plasmid adapts to new ecological niches.In conclusion,this study studied the molecular epidemic characteristics of CRE from different sources,with particular emphasis on the clonal spread of ST 11 blaKPC-2 hypervirulent K.pneumoniae in clinical settings,the prevalence of ST48 blaNDM positive E.coli in farming environments,and the blaNDM transmission mediated by IncX3 plasmid.Meanwhile,we investigated the structure and formation mechanism of three novel blaNDMcarrying plasmids,as well as the adaptive evolutionary mechanism of the fusion plasmid in E.coli and the molecular basis responsible for the various phenotype alterations that occurred during evolution.The above research has significant scientific and practical implications for controlling the prevalence of CRE in different environments.