Bioluminescent Imaging Reveals Novel Patterns of Colonization and Invasion in Systemic Escherichia coli K1 Experimental Infection in the Neonatal Rat

Abstract

bKey features of Escherichia coli K1-mediated neonatal sepsis and meningitis, such as a strong age dependency and development along the gut-mesentery-blood-brain course of infection, can be replicated in the newborn rat. We examined temporal and spatial aspects of E. coli K1 infection following initiation of gastrointestinal colonization in 2-day-old (P2) rats after oral administration of E. coli K1 strain A192PP and a virulent bioluminescent derivative, E. coli A192PP-lux2. A combination of bacterial enumeration in the major organs, two-dimensional bioluminescence imaging, and three-dimensional diffuse light imaging tomography with integrated micro-computed tomography indicated multiple sites of colonization within the alimentary canal; these included the tongue, esophagus, and stomach in addition to the small intestine and colon. After invasion of the blood compartment, the bacteria entered the central nervous system, with restricted colonization of the brain, and also invaded the major organs, in line with increases in the severity of symptoms of infection. Both keratinized and nonkeratinized surfaces of esophagi were colonized to a considerably greater extent in susceptible P2 neonates than in corresponding tissues from infection-resistant 9-day-old rat pups; the bacteria appeared to damage and penetrate the nonkeratinized esophageal epithelium of infection-susceptible P2 animals, suggesting the esophagus represents a portal of entry for E. coli K1 into the systemic circulation. Thus, multimodality imaging of experimental systemic infections in real time indicates complex dynamic patterns of colonization and dissemination that provide new insights into the E. coli K1 infection of the neonatal rat.

Non-invasive three-dimensional imaging of Escherichia coli K1 infection using diffuse light imaging tomography combined with micro-computed tomography

“…3), which showed only a small reduction in virulence compared to the parent [12]. Thus, threedimensional DLIT-μCT, in combination with enumeration by culture of organ bioburden and twodimensional bioluminescent imaging, was employed to illustrate temporal and spatial aspects of systemic infection in P2 rat pups following oral administration of E. coli A192PP-lux2 [12]. Bacteria translocated from the lumen of the GI tract to the blood compartment and entered the central nervous system with colonisation of the brain restricted to the meningeal surfaces.…”

“…We investigated over thirty bioluminescent derivatives of E. coli A192PP and identified only one suitable lux strain for detailed in vivo investigations, E. coli A192PP-lux2 (Fig. 3), which showed only a small reduction in virulence compared to the parent [12]. Thus, threedimensional DLIT-μCT, in combination with enumeration by culture of organ bioburden and twodimensional bioluminescent imaging, was employed to illustrate temporal and spatial aspects of systemic infection in P2 rat pups following oral administration of E. coli A192PP-lux2 [12].…”

“…Clones belonging to serotype O18 tend to cause the most severe systemic neonatal infections in rodents [9]. In order to undertake extensive characterization of the basis of systemic infection due to E. coli K1, we selected the O18:K1 septicaemia isolate A192 [28], reported as colonizing new born Wistar rats with an incidence of 100%, inducing bacteraemia in 35% with survival of 75% [12]. This strain, like many other E. coli K1 strains, belongs to sequence type 95 [29].…”

“…2), but not to GI colonization [30,31]. For DLIT-μCT imaging, we engineered E. coli A192PP for expression of the bioluminescence phenotype by introduction of the lux operon through mini-Tn5 mutagenesis [12]. It is imperative to ensure that bioluminescent conjugants produce a strong bioluminescent signal over the course of the growth cycle in the absence of the selective antibiotic to confirm constitutive and stable expression of the lux operon [32], display a rate of growth in nutrientreplete medium comparable to that of the parent strain and retain a comparable level of virulence in the animal model.…”

“…Importantly, key features of E. coli K1 systemic infections, including the age dependency, can be replicated in the neonatal rat by oral administration of bacteria, a procedure that frequently initiates stable colonization of the GI tract [8,9]. Rat pups colonized in the first few days postpartum develop lethal systemic infection as the colonizing E. coli K1 translocate from the lumen of the GI tract to the blood circulation to cause a persistent bacteraemia [9,10]; they then elicit infection in multiple organs, including the brain [11,12]. Bacteria appear to enter the cerebrospinal compartment predominantly at the choroid plexus and penetrate superficial brain tissue [11], where they give rise to localized inflammation through cytokine-induced pathways [13].…”