Altered innate defenses in the neonatal gastrointestinal tract in response to colonization by neuropathogenic Escherichia coli

Two-day-old (P2), but not nine-day-old (P9), rat pups are susceptible to systemic infection following gastrointestinal colonization by Escherichia coli K1. Age dependency reflects the capacity of colonizing K1 to translocate from gastrointestinal (GI) tract to blood. A complex GI microbiota developed by P2, showed little variation over P2-P9 and did not prevent stable K1 colonization. Substantial developmental expression was observed over P2-P9, including up-regulation of genes encoding components of the small intestinal (α-defensins Defa24 and Defa-rs1) and colonic (trefoil factor Tff2) mucus barrier. K1 colonization modulated expression of these peptides: developmental expression of Tff2 was dysregulated in P2 tissues and was accompanied by a decrease in mucin Muc2. Conversely, α-defensin genes were up-regulated in P9 tissues. We propose that incomplete development of the mucus barrier during early neonatal life and the capacity of colonizing K1 to interfere with mucus barrier maturation provide opportunities for neuropathogen translocation into the bloodstream

Pathoadaptive mutations of Escherichia coli K1 in experimental neonatal systemic infection

Although Escherichia coli K1 strains are benign commensals in adults, their acquisition at birth by the newborn may result in life-threatening systemic infections, most commonly sepsis and meningitis. Key features of these infections, including stable gastrointestinal (GI) colonization and age-dependent invasion of the bloodstream, can be replicated in the neonatal rat. We previously increased the capacity of a septicemia isolate of E. coli K1 to elicit systemic infection following colonization of the small intestine by serial passage through two-day-old (P2) rat pups. The passaged strain, A192PP (belonging to sequence type 95), induces lethal infection in all pups fed 2–6 x 106 CFU. Here we use whole-genome sequencing to identify mutations responsible for the threefold increase in lethality between the initial clinical isolate and the passaged derivative. Only four single nucleotide polymorphisms (SNPs), in genes (gloB, yjgV, tdcE) or promoters (thrA) involved in metabolic functions, were found: no changes were detected in genes encoding virulence determinants associated with the invasive potential of E. coli K1. The passaged strain differed in carbon source utilization in comparison to the clinical isolate, most notably its inability to metabolize glucose for growth. Deletion of each of the four genes from the E. coli A192PP chromosome altered the proteome, reduced the number of colonizing bacteria in the small intestine and increased the number of P2 survivors. This work indicates that changes in metabolic potential lead to increased colonization of the neonatal GI tract, increasing the potential for translocation across the GI epithelium into the systemic circulation

Age-dependent host factors and the site of gastrointestinal translocation in Escherichia coli K1 neonatal systemic infection

Escherichia coli K1 is a major cause of neonatal bacterial meningitis (NBM). Mortality and morbidity of NBM remains significant, requiring new treatments to alleviate the disease burden. Our understanding of many aspects of disease pathogenesis has been acquired through the use of a robust neonatal rat model of E. coli K1 systemic infection. Translocation of E. coli K1 from the GI tract into the blood compartment is a key step in the disease progression that is poorly understood. Knowledge of the key mechanisms underlying the age-dependent susceptibility of the neonates to E. coli K1 could identify novel therapeutic targets. Using the neonatal rat model, this study aimed to analyse 1) the spatial distribution of E. coli K1 in the GI tract in an attempt to identify the site of translocation, 2) the role of Paneth cells in E. coli K1 pathogenesis and 3) the possibility of endogenous Trefoil factor 2 (Tff2) feeding as a prophylactic therapy. Evidence was found suggesting that the middle small intestine of susceptible animals may be the site where E. coli K1 translocates into the bloodstream by a transcellular route. Susceptible animals were unable to control E. coli K1 numbers at the site of translocation. Two thirds of the resistant animals became susceptible to E. coli K1 infection when their antimicrobial peptide (α-defensins) producing Paneth cells were selectively ablated with a chemical reagent, dithizone. Daily feeding of recombinant Tff2 (a protein that promotes mucin layer maturation) to susceptible animals reduced the incidence of bacteraemia and increased survival by approximately 30%. These results indicate that the E. coli K1 systemic infection is a multi-factorial process. Enhancement of the GI tract barrier in two-day-old animals at the site of translocation with recombinant Tff2 or α-defensins represents a potential strategy for the prophylaxis of neonatal E. coli K1 infection

Postnatal development of the small intestinal mucosa drives age-dependent, regio-selective susceptibility to Escherichia coli K1 infection

The strong age dependency of neonatal systemic infection with Escherichia coli K1 can be replicated in the neonatal rat. Gastrointestinal (GI) colonization of two-day-old (P2) rats leads to invasion of the blood within 48 h of initiation of colonization; pups become progressively less susceptible to infection over the P2-P9 period. We show that, in animals colonized at P2 but not at P9, E. coli K1 bacteria gain access to the enterocyte surface in the mid-region of the small intestine and translocate through the epithelial cell monolayer by an intracellular pathway to the submucosa. In this region of the GI tract, the protective mucus barrier is poorly developed but matures to full thickness over P2-P9, coincident with the development of resistance to invasion. At P9, E. coli K1 bacteria are physically separated from villi by the mucus layer and their numbers controlled by mucus-embedded antimicrobial peptides, preventing invasion of host tissues