Functional Genomic And Transcriptomic Profiling Of Pathogenic Multidrug Resistant And Highly Virulent Escherichia Coli Of Poultry Origin

Escherichia coli(E.coli)is a Gram-negative bacterium,normally residing in the lower intestine of warm-blooded animals and humans.However,some serotypes of E.coli have developed pathogenic characters by the acquisition of virulent genetic elements and have been playing their important role in causing several diseases in animals and human.E.coli is a harmless common bacterium of poultry intestine,but with a wide range of genomic flexibility,is also causative agent of many poultry diseases,collectively called colibacillosis that is blamed for high economic loss in poultry sector worldwide.The appropriate use of antimicrobials plays a vital role in safeguarding of food producing animals and human health as well,while irrational and careless usage may cause antimicrobial resistance.Antimicrobial resistance found to be one of the worst universal risk to animal and human health.Multidrug resistance(MDR)has developed in E.coli due to excessive use of antibiotics.The presence of resistance genes has been studied not only in poultry but also in other food-producing animals and humans.Inter-transmission of MDR and virulent E.coli clones have been studied between poultry and humans,where contaminated environment and poultry products played important role.Numerous studies have been conducted to check the prevalence of pathogenic E.coli in poultry and poultry products,however limited data are available regarding their resistance and virulence associated genes expression profile.The study was designed to observe the prevalence of pathogenic poultry E.coli isolates by antibiotic susceptibility,?-lactamase screening,resistant and virulent genes detection,biofilm formation and adhesion,invasion and intracellular survivability assays in Caco-2 and Raw 264.7 cell lines along with the determination of median lethal dose in two-day old chickens.Forty-five ciprofloxacin resistant isolates of E.coli from broilers were selected from previous study.Antibiotic susceptibility test was performed by broth microdilution method and found that most of the isolates were MDR,and resistance was observed against almost all classes of antibiotics.Of them,55.5%isolates were screened as ?-lactamase positive by double-disk synergy test.For further study,five highly MDR and ?-lactamase producing isolates were selected.In selected isolates,the predominant ?-lactamase genes were,CTX-M,CTX-M-1,CTX-M3,TEM-1 and OXA,while CMY,DHA,NDM-1 and KPC genes were not found in any isolate by PCR.Furthermore,the MDR isolates were containing most of resistant and virulent genes.The detected resistant genes were,aac(3)-?,cat-A1,cml-A,sul-1,tet-A and tet-B,and the most prevalent virulent genes were pap-C,fim-C,fim-H,iuc-D,irp-2,tra-T,iro-N and iut-A.These isolates were moderate to strong biofilm producers.These isolates showed strong characteristics of the adhesion,invasion and intracellular survivability.The highest significant rate of adhesion,invasion and intracellular survival was observed for the isolate 381 among all tested isolates and reference strain E.coli ATCC 25922.Whole genome resequencing was performed using Illumina HiSeq 4000 system and found the average genome draft of MDR E.coli isolates almost 5 M bp with 50.4%GC contents.Number of in silico analyses(BLAST search,ResFinder and VirFinder)were performed to check the presence of antibiotic resistance and virulence genes in MDR poultry E.coli isolates.The SNPs loci mentioned in this data give valuable genetic markers to growing high-throughput techniques for fine-determination of genotyping of MDR and highly virulent isolates.The in silico analysis detected genes encoding resistance to ?-lactams,aminoglycosides,chloramphenicol,fosfomycin,fluoroquinolones,tetracycline,multidrug resistance(mdtE,F,G,K),resistance-nodulation-cell division(RND),multidrug resistance efflux pump(MDREP)and major facilitator superfamily transporters(MFST).Characterization of nucleotide polymorphisms on functional basis shed new bits of knowledge on the evolution,disease transmission and pathogenesis of MDR E.coli isolates.For further investigation,comparative gene expression profile of MDR E.coli 381 and the reference human strain E.coli ATCC 25922 was done using Illumina HiSeq2500 transcriptome and results were verified by RT-qPCR analyses.In isolate 381,a sum of 5141 transcripts were found and after comparison with reference strain,there were 2574 differently expressed genes(DEGs).Of total DEGs,723 were up-regulated genes and 1851 were down-regulated genes.A number of resistant encoding genes including multidrug transporters,multidrug resistance proteins,porins and autotransporters were identified.We also noticed overexpression of very important virulent genes(fimA,fimC,fimH and fiml)encoding the type-1 fimbrial proteins,curli fimbriae genes(csgC and csgF),invasin genes,(inv and ychO)toxin-encoding genes(bdm and hha)and biofilm forming regulatory genes(ycgZ,bssR and bdm).In addition,many types of stress(Ibp,Hsp,cspA,asr,usp and cstA)and metal homeostasis(zitB,mntH,dpp,bfr and fecC)controlling genes were among up-regulated genes in E.coli 381 as compared to reference strain.GO and KEGG pathway analysis results revealed that genes controlling secondary metabolism,drug transport,adhesion and invasion proteins,and mobile genetic elements were over-expressed in E.coli 381.Several genes involved in cellular and metabolic processes such as carbohydrate metabolism were responsible for stress tolerance.Gene expression profile also supported our results of in vitro and in vivo experiments.Seminal description of the transcriptomic results and other unique features of E.coli 381 confirmed that it is highly virulent and MDR strain of poultry origin.In conclusion,these findings provide evidence that most of poultry E.coli are MDR,?-lactamase producers and highly virulent,which could be a zoonotic threat.Due to emerging MDR and virulence features,novel treatment strategies are essential to control the poultry E.coli infections.Further investigations could reveal the molecular characters and correlation of resistance and virulence.This comparative study provides new avenues for further work on molecular mechanisms to prevent resistance development in bacteria and to ensure public health.The whole genome resequencing data provided higher resolution of resistance and virulence characteristics in E.coli,which can further be used for the development of prevention strategies and treatment procedures.