Pathogenomics of neonatal meningitis causing Escherichia coli

Open Access
Author:
Wijetunge, Dona
Graduate Program:
Pathobiology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
March 03, 2014
Committee Members:
  • Subhashinie Kariyawasam, Dissertation Advisor
  • Subhashinie Kariyawasam, Committee Chair
  • Bhushan M Jayarao, Committee Member
  • Chitrita Debroy, Committee Member
  • Edward G Dudley, Committee Member
Keywords:
  • Bacterial genomics
  • E. coli
  • Neonatal meningitis
  • virulence plasmids
Abstract:
Bacterial neonatal meningitis is one of the most devastating infections in early period of human life that accounts for high mortality and morbidity among infants. Escherichia coli is the most predominant Gram-negative bacterial pathogen associated with neonatal meningitis. Evidence from previous studies has shown a significant heterogeneity of virulence properties of neonatal meningitis causing E. coli (NMEC) which makes it difficult to define the NMEC pathotype and understand its pathogenesis. In this study, we have attempted to define the NMEC pathotype and identify novel virulence traits using a pathogenomic approach. First, we have evaluated genotypic and phenotypic characteristics of NMEC (n=53) in comparison to those charactristics of fecal commensal E. coli (HFEC, n=48) in order to identify a set of genotypic and phenotypic characteristics that can be used to distinguish NMEC from HFEC. We found that a typical NMEC can be defined as K1+, sitA+ and having at least two of the three genes, vat, neuC and iucC. Then, we sequenced the genome of prototypicNMEC strain E. coli RS218 and conducted a comparative genomic analysis with reference to the other sequenced extra intestinal pathogenic E. coli genomes. Analysis of the genome revealed that RS218 chromosome is 5.087 Mb in size with an average G+C content of 50.6%. Total of 51 genomic islands (GIs) have been identified in the RS218 genome which are absent from the E. coli K12 genome. Out of these GIs, 16 GIs were common to all NMEC strains whereas two GIs were common to all ExPEC. The GIs shared in all NMEC encode for several sugar uptake pathways, an acid tolerance operon, iron uptake systems and putative adhesins and invasins, indicating potential virulence associated genes of E. coli RS218 genome that may contribute to NMEC pathogenesis. In addition to the chromosome, it contains one large plasmid, pRS218 which is 114, 231 bp in size which belongs to the IncFIB/IIA incompatibility group, and contains a genetic load region that possesses several virulence and fitness traits such as enterotoxicity, iron acquisition and copper tolerance. Based on an in vitro invasion assay that used a human brain microvascular endothelial cell line and an in vivo assay that used a neonatal rat pup model of neonatal meningitis, the pRS218-cured strain was significantly attenuated as compared to RS218 wild-type strain. We also observed that the genes (n=45) located on pRS218 were overly represented in NMEC strains compared to HFEC indicating the importance of pRS218 to NMEC pathogenesis.