iai accepted manuscript posted online 8 september 2015 infect

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Bioluminescent Imaging Reveals Novel Patterns of Colonization and Invasion in Systemic Escherichia 1

coli K1 Experimental Infection in the Neonatal Rat 2

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Luci A. Witcomb,a James W. Collins,b Alex J. McCarthy,a Gadi Frankel,b Peter W. Taylora# 5

University College London School of Pharmacy, 29-39 Brunswick Square, London, WC1N 1AX, UKa; 6

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, 7

UKb 8

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Running Head: Colonization and invasion in E. coli infection 15

#Address correspondence to Peter Taylor, [email protected] 16

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IAI Accepted Manuscript Posted Online 8 September 2015Infect. Immun. doi:10.1128/IAI.00953-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 22

Key features of Escherichia coli K1-mediated neonatal sepsis and meningitis, such as a strong age-23

dependency and development along the gut-mesentery-blood-brain course of infection, can be 24

replicated in the newborn rat. We examined temporal and spatial aspects of E. coli K1 infection 25

following initiation of gastrointestinal colonization in two-day-old (P2) rats after oral administration 26

of E. coli K1 strain A192PP and a virulent bioluminescent derivative, E. coli A192PP-lux2. A 27

combination of bacterial enumeration in the major organs, 2D bioluminescence imaging and 3D 28

diffuse light imaging tomography with integrated μCT indicated multiple sites of colonization within 29

the alimentary canal; these included the tongue, esophagus and stomach in addition to the small 30

intestine and colon. After invasion of the blood compartment, the bacteria entered the central 31

nervous system, with restricted colonization of the brain, and also invaded the major organs in line 32

with increases in the severity of symptoms of infection. Both keratinized and non-keratinized 33

surfaces of esophagi were colonized to a considerably greater extent in susceptible P2 neonates 34

compared to corresponding tissues from infection-resistant nine-day-old rat pups; the bacteria 35

appeared to damage and penetrate the non-keratinized esophageal epithelium of infection-36

susceptible P2 animals, suggesting the esophagus may represent a portal of entry for E. coli K1 into 37

the systemic circulation. Thus, multimodality imaging of experimental systemic infections in real 38

time indicate complex dynamic patterns of colonization and dissemination that provide new insights 39

into E. coli K1 infection of the neonatal rat. 40

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INTRODUCTION 48

Escherichia coli strains expressing the K1 capsule, a homopolymer of α-2,8-linked polysialic acid, are 49

a leading cause of early- and late-onset neonatal sepsis and neonatal bacterial meningitis (1, 2, 3). 50

Predisposition to these severe, often life-threatening infections is critically dependent on the vertical 51

transmission of the causative agent from mother to infant at, or soon after birth, and infection is 52

associated with following gastrointestinal (GI) colonization of the neonate (4, 5, 6); the neonatal GI 53

tract is considered sterile at birth but acquires an increasingly complex microbiota during the first 54

year of life (7). Persisting high rates of morbidity and mortality (2, 8) and the continuing emergence 55

of drug-resistant isolates (9, 10) emphasize the urgent need for new approaches to the prevention 56

and treatment of these infections. A better understanding of the etiology and pathogenesis of 57

neonatal systemic infections could provide the basis for a new generation of therapeutics and 58

prophylactics, but these infections are medical emergencies and opportunities for interventions that 59

would provide such insights are severely limited. Consequently, much of the current knowledge of 60

the underlying processes that lead to overt neonatal disease has been obtained from experimental 61

infections in small animals such as mice, rats and rabbits. 62

In some animal models, infection is initiated by parenteral administration of bacteria, 63

bypassing natural processes of colonization and dissemination and creating an artificial pathogenesis 64

scenario. Replication of the natural site of GI colonization of E. coli K1 neonatal infection in the rat, a 65

superior vehicle for such studies compared to the mouse (11), was initially employed by Moxon and 66

co-workers (12) and subsequently extended and refined (11, 13, 14). This model, initiated by gastric 67

intubation (12, 13) or feeding (11, 14) of the inoculum, produces a strongly age-dependent systemic 68

infection much as in the natural human host. For the first few days of life, newborn K1-colonized rat 69

pups are prone to develop lethal infection due to the capacity of the colonizing bacteria to 70

translocate from the lumen of the GI tract to the blood compartment (14, 15) after passage through 71

the mesenteric lymphatic system (14), from where they may establish infection in multiple organs, 72

including the brain (16). Invasion of brain tissue elicits a strong local inflammatory response induced 73


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by pro-inflammatory cytokines IL-1β, IL-6 and TNF-α (17) in similar fashion to bacterial neonatal 74

meningitis in humans (18, 19). Within a week, the pups become refractory to systemic infection, 75

even in the presence of persistent GI tract colonization (20, 21). 76

There are, however, a number of issues relating to the pathogenesis of E. coli K1 systemic 77

infection that require resolution: the basis of the strong age dependency is poorly understood, the 78

site of translocation from GI lumen to blood circulation is unknown, the mode and pattern of 79

dissemination of the circulating pathogen to peripheral organs is unclarified and there are conflicting 80

views on the route of entry into the brain, whether across the blood-brain barrier (BBB) (22, 23), the 81

blood meningeal barrier, or through the choroid plexus (1, 16), or both. Recent developments in 82

four-dimensional (4D) imaging methodologies provide an opportunity to investigate complex 83

experimental infections as real-time dynamic processes in whole animals (24, 25). In this study, we 84

shed further light on the pathogenesis of E. coli infection in neonatal rats and identify a potential 85

new site of colonization and portal of entry into the systemic circulation using a bioluminescent E. 86

coli K1 derivative combined with 2D bioluminescent imaging (2DBLI) and 3D diffuse light imaging 87

tomography with integrated µCT (DLIT- µCT). We have used this data to generate 4D movies of the 88

infection cycle to further inform on temporal aspects of disease progression. 89

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MATERIALS AND METHODS 91

Bacteria and plasmids 92

The properties of the bacteria and plasmid used in this study are detailed in Table 1. E. coli O18:K1 93

strain A192PP was derived from septicemia isolate E. coli A192 (29) by two rounds of passage 94

through neonatal rat pups, with bacterial recovery from the blood (15). The presence of K1 capsule 95

was confirmed with K1-specific bacteriophage K1E (30). Bacteria were grown in either Luria-Bertani 96

(LB) medium, M9 minimal medium (M9 salts supplemented with 1 % glucose and 0.01 M sodium 97

citrate) or Mueller Hinton (MH) broth and incubated at 37oC with agitation in an orbital incubator at 98

200 orbits min and with kanamycin (50 μg/ml) and ampicillin (100 μg/ml) as required. 99


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Generation and characterization of E. coli A192PP-lux 100

E. coli A192PP was engineered to express the bioluminescence phenotype by introduction of the lux 101

operon through mini-Tn5 mutagenesis. The method used a suicide vector that carried an 102

unpromoted lux operon (luxCDABE) from the terrestrial bacterium Photorhabdus luminescens on a 103

disarmed mini-Tn5 transposon (26). The pUTmini-Tn5luxCDABEKm2 vector was maintained in donor 104

strain E. coli S17-1 ƛ pir (27) and transferred to A192PP by conjugation, essentially as previously 105

described (28), with the exception that conjugants were selected on M9 minimal medium containing 106

kanamycin and 0.01 M sodium citrate to prevent ƛ phage lysogeny of the E. coli recipient strain (31). 107

Bioluminescent conjugants were identified with the BioRad ChemiDocTM XRS+ Imaging System (no 108

illumination, no filter, auto-exposure) and K1-positive bioluminescent colonies subjected to two 109

rounds of subculture on MH agar containing kanamycin prior to storage under glycerol at -80oC. 110

Growth kinetics (OD600) and bioluminescence of conjugants were determined in MH broth; 111

photon emission during the growth cycle (expressed as relative luminescence units) was monitored 112

with a PerkinElmer LS-55 fluorescence spectrometer. The stability of the mini-Tn5 element within 113

conjugants was determined by subculture every 24 h in MH broth in the presence and absence of 114

kanamycin; subcultures were enumerated by viability counting and bioluminescent colonies 115

identified with the ChemiDocTM XRS+ Imaging System. To determine luxCDABEKm2 insertion sites in 116

E. coli A912PP derivatives, strains were sequenced with the Illumina MiSeq platform as described 117

(32) and compared with the E. coli A192PP sequence (European Nucleotide Archive: 118

http://www.ebi.ac.uk/ena/ ; accession number PRJEB9141). The conjugant E. coli A192PP-lux2 was 119

selected for further investigation. The E. coli A192 and A192PP-lux2 genomes were assembled using 120

Velvet assembler (33) and aligned with the luxCDABE operon within the Tn-lux sequence. To confirm 121

the insertion site of the Tn-lux element, A192PP-lux2 paired sequence reads were mapped onto the 122

A192PP assembled genome. The insertion site was then confirmed by PCR using primers for the 123

amplification of A192PP-lux2-traL (forward primer 5’-TATATCGTCGGCCATGAATCC-3’; reverse primer 124

5’-AACCTCACTCCCTTTTTGCT-3’) and primers for amplification of the luxC-traL junction (forward 125


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primer 5’-CGTATCCTCCAAGCCTGAATT-3’; reverse primer 5’-TGAAGCGGTAGAAGTTGCCAA-3’), 126

producing fragments of 597 bp and 419 bp, respectively. PCR reactions (50 μl) comprised 25 μl 127

Promega Master Mix, 1 μl (10 μM) of each primer and 1 μl genomic DNA as template. Amplification 128

was carried out under the following conditions: 1 cycle at 95oC for 5 min, 35 cycles of 95oC for 1 min, 129

60oC for 1 min and 72oC for 2 min with a final extension of one cycle of 72oC for 10 min. 130

Experimental systemic infection of neonatal rats 131

Animal experiments were approved by the Ethical Committee of the UCL School of Pharmacy and 132

the UK Home Office (HO) and were conducted under HO Project Licence PPL 70/7773. The 133

procedure has been described in detail (11). In brief, two-day-old (P2) or nine-day-old (P9) Wistar rat 134

pups (Harlan UK) were fed 20 µl of mid-logarithmic phase E. coli (2-6 X 106 CFU) from an Eppendorf 135

micropipette to effect gastrointestinal (GI) colonization. No local trauma is observed as a 136

consequence of this procedure. All members of a litter, usually 12 pups, were treated as a single test 137

or control group and fed E. coli culture in identical fashion at the same time interval. GI tract 138

colonization was determined by culture of perianal swabs on MacConkey agar; bacteremia was 139

detected by MacConkey agar culture of blood taken post mortem. Disease progression was 140

determined by daily evaluation of symptoms of systemic infection and scored on a scale of rising 141

severity from 0 to 3 (11). After sacrifice, samples from the esophagus, stomach, small intestine (SI), 142

colon, blood, mesenteric lymphatic system, liver, lung, heart, kidney and spleen were excised 143

aseptically (11). Organs were washed extensively in PBS to ensure minimal contamination with 144

perfused blood. Bacteria were quantified in homogenized tissues by serial dilution culture on 145

MacConkey agar and the presence of the K1 capsule confirmed as required with bacteriophage K1E. 146

Samples from experiments involving E. coli A192PP-lux2 were cultured in the presence of 50 μg/ml 147

kanamycin. 148

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2D bioluminescence imaging (2DBLI) and 3D diffuse light imaging tomography with integrated μCT 151

(DLIT-μCT) 152

2DBLI was performed on tissues harvested from P2 and P9 rat pups colonised by A192PP-lux2; 153

tissues were collected 24, 48, and 72 h after feeding of the colonizing bacteria and the severity of 154

infection recorded. Tissues were washed in sterile PBS, placed in sterile petri dishes on black card 155

and pierced with sterile 25G needles for aeration (34). Bioluminescence was measured within 156

standardised regions of interest (ROI) and data expressed as flux (photons/s), adjusted as follows to 157

ensure all measurements had positive value: log10 [flux [p/s]+1]. Tissues from non-colonised pups 158

were used for bioluminescent background subtraction. Quantitative imaging was performed using 159

an IVIS Lumina series III (PerkinElmer). 160

For whole animal studies, both the IVIS Lumina series III (for 2DBLI) and the IVIS SpectrumCT 161

(PerkinElmer; DLIT-μCT) were employed. In DLIT-μCT experiments, the auto settings in the Living 162

Image software 4.3.1 wizard and auto-exposure settings specific for imaging bacterial luciferases 163

(maximum exposure 300 s, target count minimum 10,000) were used (25). Anesthesia (5 % 164

isofluorane, followed by maintenance under 2.5 % isofluorane) on the pre-warmed imaging platform 165

was used in both 2DBLI and DLIT-μCT experiments. Symptoms of infection were recorded 166

immediately prior to the collection of images and the pups culled for ex vivo tissue analysis; animals 167

were not subjected to repeated anesthesia. 3D animations were created using living image, as 168

previously described (25), and compiled into a movie using iMovie software (version 10.0.5). Non-169

colonised animals were used for bioluminescent background subtraction. To correlate flux 170

(photons/s) with CFU, serial dilutions in PBS from 16 h cultures of E. coli A192PP-lux were prepared 171

in 96-well black plates and wells highlighted as ROIs prior to imaging in the IVIS Lumina series III and 172

IVIS SpectrumCT. Flux within ROIs was measured and CFUs from each well determined 173

retrospectively by plating on to MH agar with kanamycin. 174

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Histology and microscopy 177

Esophageal tissues were collected and fixed in 10% neutral buffered formalin, embedded in paraffin, 178

processed, 5 μm sections obtained, mounted onto slides and stained with hematoxylin and eosin 179

(H&E). Unstained sections were prepared for immunohistochemistry: they were dewaxed in 180

HistoClear and examined for E. coli O18 lipopolysaccharide antigen as previously described (16, 35) 181

and mounted in VectaShield Mounting Medium containing 4’,6’-diamidino-2-phenylindole (DAPI) 182

stain (H-1200). Samples for scanning electron microscopy (SEM) were processed and examined as 183

previously described (36) using a JEOL JSM-5300 scanning electron microscope. 184

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RESULTS 186

Generation and characterization of E. coli A192PP-lux 187

Thirty-one bioluminescent derivatives of E. coli A192PP were obtained by mini-Tn5 mutagenesis. 188

One, designated E. coli A192PP-lux2, expressed the K1 capsule, grew in MH broth (Fig. 1A) and LB 189

minimal medium (Fig. S1) to an extent comparable to the parental A192PP strain and produced a 190

strong bioluminescent signal over the course of the growth cycle that correlated with CFU (Fig. 1B & 191

1C), indicative of constitutive expression of the lux operon (37). The strength of the bioluminescent 192

signal from A192PP-lux2 exceeded that from C. rodentium ICC180 and was maintained for seven 193

days in the absence of kanamycin (data not shown), indicative of stable genomic maintenance of the 194

lux operon. Whole genome sequencing of E. coli A192PP-lux2 revealed that the Tn5-lux element 195

inserted into traL, encoding an F-pilus assembly protein (38, 39); disruption of traL and the presence 196

of the luxC-traL junction in A192PP-lux2 was confirmed by PCR (Fig. S2); pili were not evident in SEM 197

images of either A192PP or A192PP-lux2 (Fig. S1B). The capacity of E. coli A192PP-lux2 to elicit lethal 198

infection after GI colonization in P2 and P9 neonatal rats was determined. The bioluminescent 199

derivative colonized the GI tract of P2 and P9 pups to the same extent as the parental E. coli A192PP, 200

with stable colonization occurring within 24-48 h of feeding of the bacteria (Fig. 1D); 79.17% (19/24) 201

of P2 animals developed bacteremia over the seven-day observation period and around 20% 202


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survived, whereas all receiving the parent strain were bacteremic (Fig. 1E), with no survivors (Fig. 203

1F). No P9 pups receiving either E. coli A192PP or A192PP-lux2 succumbed to bacteremia or lethal 204

infection in spite of efficient GI colonization. Thus, stable insertion of the lux transposon and 205

kanamycin resistance cassette into traL had only a minor impact on virulence; E. coli A192PP-lux2 206

and was therefore employed for all imaging experiments. 207

Distribution of E. coli A192PP-lux2 in colonized P2 and P9 rat pups 208

The available evidence indicates that, after ingestion, E. coli K1 follows the gut-mesentery-blood-CSF 209

course of infection in the neonatal rat (13, 14) but the bacteria become widely disseminated as the 210

infection progresses (16). To determine if carriage of the lux transposon and kanamycin resistance 211

genes altered the in vivo distribution of E. coli A192PP and to signpost the design of whole animal 212

and organ imaging experiments, we determined the organ tropism and organ load of E. coli A192PP 213

and A192PP-lux2 in P2 and P9 neonatal rats after initiation of colonization. A population of 214

colonizing E. coli A192PP was evident in the small intestine 24 h (the initial time point for viability 215

determination) after initiation of colonization at P2 and varied little in quantitative terms over the 216

first 72 h of colonization; there were no significant differences in numbers of colonizing bacteria 217

between E. coli A192PP and A192PP-lux2 during this period (Fig. 2A). Small numbers of bacteria 218

were present in the mesentery after 24 h (Fig. 2B) and began to appear in the blood circulation at 219

the same time point (Fig. 2C). E. coli K1 were recovered from brain tissue in a variable proportion of 220

infected P2 animals and the numbers encountered were low (Fig. 2D). There were again no 221

significant differences between E. coli A192PP and A192PP-lux2 at any given time point. Other 222

regions of the GI tract (stomach, colon) were also stably colonized by both bioluminescent strain and 223

parent strain to a similar extent, although a noticeable but insignificant decrease in A192PP-lux2 224

load present in the stomach at 24 h after initiation of colonization was observed (Fig. S3). Low to 225

moderate numbers of E. coli A192PP and A192PP-lux2 were cultured from liver, lung, heart, kidney, 226

spleen and pancreas; no significant differences in the size of the bioburden between the two strains 227

were evident when they were compared by time after initiation of colonization or by severity of 228


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symptoms of infection (Figs. S3 & S4). The bacterial load of both strains in the major organs 229

increased as the health status of the animals declined (Fig. S4). We conclude that the introduction of 230

bioluminescence- and kanamycin-associated genes into E. coli A192PP has little, if any, impact on 231

organ tropism and tissue bioburden in this widely-disseminated infection of susceptible P2 neonatal 232

rats. With resistant P9 pups, E. coli A192PP and A192PP-lux2 were recovered from the stomach, 233

small intestine and colon in numbers comparable to those found in P2 animals; only very low 234

numbers were found in the mesentery, and other tissues were free of E. coli K1 (data not shown). 235

Patterns of E. coli A192PP-lux2 infection determined by bioluminescence imaging 236

2DBLI of live P2 rats revealed a significant increase in total bioluminescence between 24 h and 48 h 237

after feeding of E. coli A192PP-lux2 (P< 0.05), coincident with the onset of signs of infection. 238

Representative images are shown in Fig. 3. Colonizing bacteria were not uniformly distributed along 239

the small intestine. Typically, there was rapid dissemination of the pathogen from the site of 240

colonization between these two time points and an apparent reduction in both flux and degree of 241

dissemination at 72 h. However, in individual animals the infection progresses at different rates (16) 242

with a proportion succumbing to infection within three days after seeding of E. coli K1, and survivors 243

at 72 h are likely to be animals in which infection progressed at a relatively low rate compared to 244

non-survivors. The apparent peak of infection at 48 h was reflected in photon emission from excised 245

major organs with the exception of the GI tract tissues, indicative of stable GI colonization (Fig. 3). 246

We therefore examined the data relative to the severity of symptoms of infection (Fig. 4). 247

In live P2 animals the E. coli K1 burden increased significantly as symptoms of infection 248

became evident, indicating that although the total burden increased with time it correlated more 249

closely with the severity of infection. The degree of colonization of the stomach, small intestine and 250

mesentery increased with disease severity and invasion of the central nervous system occurred only 251

in animals displaying severe symptoms of infection. Association of E. coli K1 with liver, spleen, 252

pancreas, heart and kidney were similarly associated with the late stages of infection. In contrast, 253

the distribution of E. coli A192PP-lux2 in colonized P9 pups was restricted to the GI tract (Fig. S5). 254


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The bioburden in live P9 animals did not increase significantly between 24 h and72 h and few 255

bacteria were visualized in the mesentery. Niches rich in colonizing bacteria were evident in the 256

small intestine, colon and mesentery in both P2 and P9 pups. Overall, the distribution of E. coli K1 257

determined by 2DBLI was consistent with data obtained by viability determination (Figs. 2 & S4). 258

DLIT-μCT was used to investigate the relationship between the development of symptoms of 259

disease and the whole-animal distribution of E. coli A192PP-lux2 in P2 animals over a 72 h period 260

following initiation of colonization (Video file 1). As anesthesia modified the progression of disease 261

and increased the risk of rejection by the mother, longitudinal monitoring of individual pups was not 262

performed. As determined earlier in the study, the rate of disease progression differed significantly 263

between individual animals and clearer representations of disease development were obtained 264

when comparisons were made on the basis of severity of symptoms rather than on time from 265

colonization. Multiple organ involvement can be seen in cases of severe infection (score of three or 266

more) and it is noteworthy that infection of the CNS was restricted to the surface of the brain, 267

lending support to the view that the choroid plexus rather than the brain microvascular endothelium 268

represents the portal of entry into the CNS in experimental E. coli K1 meningitis. The authenticity of 269

the DLIT-μCT reconstruction was confirmed by 2DBLI of the corresponding pups and their organs 270

(Fig. S6). DLIT-μCT revealed that the initial bolus of E. coli K1 entered the colon within 3 h of oral 271

application of the inoculum, seeding the entirety of the alimentary tract including the oral cavity, 272

esophagus, stomach and small intestine (Video file 2; Fig. S7). Frequent colonisation of the oral 273

cavity was observed, which presented as photon emission from the head region (Video file 3), and 274

was evident over the entire 72 h observation period. Stable oral cavity colonization was confirmed 275

by 2DBLI of excised elements of this region and revealed an intense colonization of tongue tissue 276

(Fig. 5). During later stages of disease progression, photon emission from multiple lymph nodes in 277

the face, neck, back and joints was observed (Video file 4; Fig. 6), demonstrating extensive 278

dissemination of E. coli K1 within the lymphatic system. 279

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Age-dependent colonization and invasion of the esophagus 281

2DBLI indicated that the esophagus could represent a novel site for entry and dissemination in 282

systemic E. coli K1 experimental infection (Figs. S7 & 7A). Photon emission from the head region did 283

not originate from the trachea (data not shown). Determination of photon emission by the 284

bioluminescent derivative (Fig. 7B) and CFU of E. coli A192PP and A192PP-lux2 (Fig. 7C) in excised 285

esophagi showed P2 rats to be significantly more susceptible to colonization at this site than P9 286

animals. No significant quantitative differences in colonization capacity between the two strains 287

were apparent (Fig. 7C) and no difference between E. coli A192PP and A192PP-lux2 with respect to 288

severity of symptoms was found (data not shown). H&E staining of the esophagus revealed the 289

presence of a keratinised epithelium at P9; this layer was absent or only partially developed in the 290

esophagus of susceptible P2 neonatal rats (Fig. 8A), began to appear 2-3 days post-partum (Fig. S8) 291

and developed progressively during the early neonatal period. Esophageal sections from rats 292

colonized with E. coli A192PP at P2 were probed for the presence of the O18 antigen; bacterial 293

attachment to keratinized and non-keratinized esophageal surfaces was evident at 24, 48 and 72 h 294

following bacterial feeding (Fig. 8B); 018 antigen was not detected in tissues from non-colonized 295

animals. Bacteria were also observed attached to sloughing keratin strands present in the lumen of 296

the esophagus. Additionally, at 48 h peripheral invasion and damage to regions of non-keratinized 297

esophageal epithelium by E. coli K1 was evident (Fig. 8B) and further evidence of invasion of non-298

keratinized regions was also observed by SEM (Fig. 8C). 299

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DISCUSSION 301

The isolation of E. coli K1 from the cerebrospinal fluid of sick newborn infants frequently coincides 302

with the presence of the bacteria in the feces of both infant and mother (4, 6, 40), providing a strong 303

indication that the maternal GI tract represents the primary reservoir of infection for these neonatal 304

pathogens, which are associated with the infant gut prior to systemic invasion. Oral administration 305

of E. coli K1 to susceptible neonatal rats clearly shows that colonization of the GI tract precedes 306


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invasion (13, 14, 15). The site of translocation and the molecular processes involved have not been 307

defined with any precision although evidence is emerging that colonization of the small intestine is 308

essential for systemic infection (21). In the current study, DLIT-μCT imaging showed that the 309

colonizing E. coli K1 bolus reached the colon within three hours of administration and 2DBLI revealed 310

regions of intense colonization in both the small intestine and colon of P2 and P9 pups and, 311

unexpectedly, that bacteria were seeded along the entire length of the alimentary tract. Thus, the 312

oral cavity, esophagus and stomach of P2 animals were usually heavily colonized and may represent 313

additional reservoirs of infection in this model. Small numbers of E. coli K1 were found in the 314

mesenteric lymphatic system at an early stage of the infection cycle, supporting evidence (14) that 315

invasion of the blood circulation occurs by this route. The presence of E. coli K1 bacteria in some of 316

the major organs, particularly in pups with symptoms of systemic infection, may represent end-stage 317

invasion as immune defenses are compromised. However, with highly perfused organs the bacterial 318

viability counts are very likely to include bacteria present in blood as well as in tissue; the organs of 319

newborn rats are fragile and will not withstand ex vivo perfusion for blood removal (PW Taylor; 320

unpublished observation). 321

Colonization of the GI tract by E. coli K1 at P2 dysregulates the maturation of the mucus 322

barrier which is poorly formed in newborn pups; mucosal barrier function at this age is insufficient to 323

prevent translocation of E. coli K1 from gut lumen to blood circulation (21). An integral barrier has 324

formed by P9 (21), preventing systemic invasion and accounting for the restricted distribution of the 325

bacteria following colonization of P9 animals observed in the current study. We also found an 326

unexpected age-dependent, stable colonization of the esophagus and obtained evidence that, in P2 327

pups, E. coli K1 may invade non-keratinized esophageal tissue, raising the possibility that the 328

esophagus represents an additional locus of translocation to the blood. Thus, photon emission from 329

this site was significantly increased in susceptible P2 rats compared to resistant P9 animals over the 330

three days following initiation of colonization and the strong age-dependency confirmed by 331

enumeration of viable E. coli K1 within esophageal tissue. 332


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H&E staining revealed the age-dependent development of a keratinized esophageal layer, 333

appearing as early as two days postpartum; age-related keratinization has also been documented in 334

the mouse but appears to proceed more slowly in comparison to the rat as determined in the 335

current study (41). Esophageal keratinization provides protection against the consumption of coarse 336

foods (42), although the human esophageal epithelium is not normally keratinized to any extent (43, 337

44) and, like the rodent esophagus, does not develop a viscoelastic protective mucus barrier (45). E. 338

coli K1 attached to all regions of the P2 esophagus, including keratinized and non-keratinized 339

surfaces. As binding is independent of the keratin layer, the presence of keratin cannot account for 340

differences in esophageal binding between P2 and P9 pups. However, invading E. coli K1 were found 341

in non-keratinised regions of the P2 esophagus, suggesting that the incomplete keratinization of this 342

site predisposes the underlying epithelium to invasion and may enable the bacteria to persist; this 343

contention is supported by SEM imaging of P2 esophageal tissue. Full-term human neonates exhibit 344

reduced esophageal motility and luminal clearance compared to adults which is further decreased in 345

preterm neonates (46, 47, 48), suggesting that the neonatal esophagus may be vulnerable to 346

colonization and invasion by neuropathogens in the human host. Thus, reduced esophageal 347

peristalsis, an exposed epithelial layer lacking keratin and a mucosal barrier may increase the risk of 348

pathogen attachment, overgrowth and invasion at this site. 349

We noted colonization of the oral cavity, in particular the tongue. There have been few 350

studies of sites of E. coli K1 colonization upstream of the GI tract but Guerina and colleagues (49) 351

recorded colonization of the oral cavity in virtually all neonatal rats examined. In another study, 352

colonization of the oropharynx and bloodstream invasion occurred in neonatal rats fed E. coli K1; 353

pilus-deficient mutants were unable to maintain colonization of this site but continued to colonize 354

the GI tract and cause bacteremia (50), suggesting that oropharyngeal colonization was not essential 355

for development of sepsis and meningitis. In support, we found that the Tn5-lux construct inserted 356

into traL of E. coli A912PP; this gene is involved in F-pilus assembly (38) but the virulence of the 357

transposant E. coli A192PP-lux2 was to a great extent retained. Further studies should be 358


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implemented to determine if colonization of sites other than the GI tract predispose towards 359

systemic infection and whether colonization of the distal tongue can be used for diagnostic 360

swabbing of neonates to detect K1 colonization prior to the onset of sepsis. Although a strong 361

correlation between systemic E. coli K1 infection and GI tract colonization has been frequently 362

reported in clinical studies of neonatal infection (3, 4, 6), there are indications that in some cases of 363

bacterial meningitis, aspiration of the infecting dose into the lungs and other regions of the 364

respiratory tract may provide opportunities for an alternative portal of entry into the systemic 365

circulation (51). This is unlikely to be a significant factor in the development of infection in the 366

neonatal rat model, as E. coli A192PP appears in the lung at the later time points and only in pups 367

displaying severe symptoms of infection. 368

Soon after initiation of colonization, susceptible pups develop bacteremia before 369

succumbing to overwhelming infection involving the major organs. We found E. coli K1 in the major 370

groups of lymph nodes, suggesting that disseminated infection may arise due to failure of the 371

lymphatic system to control localized foci of bacteria. E. coli K1 is strongly associated with sepsis and 372

meningitis (1, 2, 3) and we found significant associations between severity of infection and recovery 373

of viable bacteria from brain tissue. In a previous study (16), we visualized E. coli K1 in brain sections 374

using a modified Gram stain and found bacteria associated only with the surfaces of this organ. The 375

current study lends further support to this pattern of distribution: DLIT-μCT imaging (video 1) 376

indicated that E. coli A192PP-lux2 cells were restricted to superficial layers on the surface of the 377

brain, much as in the human condition (52) and supports previous observations (16) that E. coli K1 378

gains access to the CNS through the choroid plexus, a component of the blood-CSF barrier. Transit 379

from the blood circulation through this epithelial barrier to the CSF would present the invading 380

bacteria with the opportunity to adhere to the most superficial layer of the brain, the 381

leptomeninges, but restrict access to cortical tissue. However, it is widely accepted that E. coli K1 382

invade the CNS by traversal of the endothelium (BBB) (23, 53), even though the evidence supporting 383

this route of entry is modest (22). As the mammalian brain accounts for only 2% of body mass yet 384


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receives 20% of the cardiac output, it is a highly vascularized organ; the human brain contains an 385

estimated one hundred billion vessels, one for each neuron (54). If circulating neuropathogens 386

penetrated the BBB to any extent at multiple site sites within this vascular network, one would 387

expect to encounter the bacteria throughout the brain in both naturally-occurring and experimental 388

E. coli K1 meningitis but this is not the case. The choroid plexus provides a much lower resistance to 389

transport into the CNS than the BBB (55) and is strong candidate for portal of entry. Resolution of 390

the important issues of colonization and tissue penetration may open up new avenues for resolving 391

lethal infections of the CNS. 392

393

ACKNOWLEDGMENTS 394

This study was supported by project grant GN2075 from Action Medical Research. Further support 395

was provided by the National Institute for Health Research University College London Hospitals 396

Biomedical Research Centre. We thank Dr Richard Stabler, London School of Hygiene and Tropical 397

Medicine, for generating the sequence reads. We thank Prof Thilo M. Fuchs, Technische Universitat 398

Munchen, for kindly providing the Tn-lux sequence. 399

400

Accession numbers 401

Nucleotide sequence of the A192PP-lux2 genome has been deposited in the European Nucleotide 402

Archive (http://www.ebi.ac.uk/ena/; accession number PRJEB9940). 403

404

405

406

407

408

409

410


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REFERENCES 411

1. Tunkel AR, Scheld WM. 1993. Pathogenesis and pathophysiology of bacterial meningitis. 412

Clin Microbiol Rev 6:118-136. 413

2. Bonacorsi S, Bingen E. 2005. Molecular epidemiology of Escherichia coli causing neonatal 414

meningitis. Int J Med Microbiol 295:373-381. 415

3. Simonsen KA, Anderson-Berry AL, Delair SF, Davies HD. 2014. Early-onset neonatal sepsis. 416

Clin Microbiol Rev 27:21-47. 417

4. Sarff LD, McCracken GH, Schiffer MS, Glode MP, Robbins JB, Ørskov I, Ørskov F. 1975. 418

Epidemiology of Escherichia coli K1 in healthy and diseased newborns. Lancet 1:1099-1104. 419

5. Schiffer MS, Oliveira E, Glode MP, McCracken G., Sarff LM, Robbins JB. 1976. A review: 420

relation between invasiveness and the K1 capsular polysaccharide of Escherichia coli. 421

Pediatr Res 10:82-87. 422

6. Obata-Yasuoka M, Ba-Thein W, Tsukamoto T, Yoshikawa H, Hayashi H. 2002. Vaginal 423

Escherichia coli share common virulence factor profile, serotypes and phylogeny with other 424

extraintestinal E. coli. Microbiology 148:2745-2752. 425

7. Palmer C, Bik EM, DiGuilio DB, Relman DA, Brown PO. 2007. Development of the human 426

infant intestinal microbiota. PLoS Biol 5:1556-1573. 427

8. Shah BA, Padbury JF. 2014. Neonatal sepsis: an old problem with new insights. Virulence 428

5:170-178. 429

9. Bizzarro MJ, Dembry LM, Baltimore RS, Gallagher PG. 2008. Changing patterns in neonatal 430

Escherichia coli sepsis and ampicillin resistance in the era of intrapartum antibiotic 431

prophylaxis. Pediatrics 121:689-696. 432

10. Brouwer MC, Tunkel AR, van de Beek D. 2010. Epidemiology, diagnosis, and antimicrobial 433

treatment of acute bacterial meningitis. Clin Microbiol Rev 23:467-492. 434

11. Dalgakiran F, Witcomb LA, McCarthy AJ, Birchenough GM, Taylor PW. 2014. Non-invasive 435

model of neuropathogenic Escherichia coli infection in the neonatal rat. J Vis Exp 436


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

18

92:e52018. 437

12. Moxon ER, Glode MP, Sutton A, Robbins JB. 1977. The infant rat as a model of bacterial 438

meningitis. J Infect Dis 136:S186-S190. 439

13. Glode MP, Sutton A, Moxon ER, Robbins JB. 1977. Pathogenesis of neonatal Escherichia 440

coli meningitis: induction of bacteremia and meningitis in infant rats fed E. coli K1. Infect 441

Immun 16:75-80. 442

14. Pluschke G, Mercer A, Kusećek B, Pohl A, Achtman M. 1983. Induction of bacteremia in 443

newborn rats by Escherichia coli K1 is correlated with only certain O (lipopolysaccharide) 444

antigen types. Infect Immun 39:599-608. 445

15. Mushtaq N, Redpath MB, Luzio JP, Taylor PW. 2004. Prevention and cure of systemic 446

Escherichia coli K1 infection by modification of the bacterial phenotype. Antimicrob Agents 447

Chemother 48:1503-1508. 448

16. Zelmer A, Bowen M, Jokilammi A, Finne J, Luzio JP, Taylor PW. 2008. Differential 449

expression of the polysialyl capsule during blood-to-brain transit of neuropathogenic 450

Escherichia coli K1. Microbiology 154:2522-2532. 451

17. Zelmer A, Martin M, Gundogdu O, Birchenough G, Lever R, Wren BW, Luzio JP, Taylor PW. 452

2010. Administration of capsule-selective endosialidase E minimizes changes in organ gene 453

expression induced by experimental systemic infection with Escherichia coli K1. 454

Microbiology 156:2205-2215. 455

18. Täuber MG, Moser B. 1999. Cytokines and chemokines in meningeal inflammation: biology 456

and clinical implications. Clin Infect Dis 28:1-12. 457

19. Polin RA, Harris MC. 2001. Neonatal bacterial meningitis. Semin Neonatol 6:157-172. 458

20. Mushtaq N, Redpath MB, Luzio JP, Taylor PW. 2005. Treatment of experimental 459

Escherichia coli infection with recombinant bacteriophage-derived capsule depolymerase. J 460

Antimicrob Chemother 56:160-165. 461

21. Birchenough GMH, Johannson MEV, Stabler RA, Dalgakiran F, Hansson GC, Wren BW, 462


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

19

Luzio JP, Taylor PW. 2013. Altered innate defenses in the neonatal gastrointestinal tract in 463

response to colonization by neuropathogenic Escherichia coli. Infect Immun 81:3264-3275. 464

22. Kim KS, Itabashi H, Gemski P, Sadoff J, Warren RL, Cross AS. 1992. The K1 capsule is the 465

critical determinant in the development of Escherichia coli meningitis in the rat. J Clin Invest 466

90:897-905. 467

23. Kim KS. 2010. Acute bacterial meningitis in infants and children. Lancet Infect Dis 10:32-42. 468

24. Hutchens M, Luker GD. 2007. Applications of bioluminescence imaging to the study of 469

infectious diseases. Cell Microbiol 9:2315-2322. 470

25. Collins JW, Meganck JA, Kuo C, Francis KP, Frankel G. 2013. 4D multimodality imaging of 471

Citrobacter rodentium infections in mice. J Vis Exp 78:e50450. 472

26. Winson MK, Swift S, Hill PJ, Sims CM, Griesmayr G, Bycroft BW, Williams P, Stewart GS. 473

1998. Engineering the luxCDABE genes from Photorhabdus luminescens to provide a 474

bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 475

constructs. FEMS Microbiol Lett 163:193-202. 476

27. de Lorenzo V, Herrero M, Jakubzik U, Timmis KN. 1990. Mini-Tn5 transposon derivatives 477

for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in 478

gram-negative eubacteria. J Bacteriol 172:6568-6572. 479

28. Wiles S, Clare S, Harker J, Huett A, Young D, Dougan G, Frankel G. 2004. Organ specificity, 480

colonization and clearance dynamics in vivo following oral challenges with the murine 481

pathogen Citrobacter rodentium. Cell Microbiol 6:963-972. 482

29. Achtman M, Mercer A, Kusećek B, Pohl A, Heuzenroeder M, Aaronson W, Sutton A, Silver 483

RP. 1983. Six widespread bacterial clones among Escherichia coli K1 isolates. Infect Immun 484

39:315-335. 485

30. Gross RJ, Cheasty T, Rowe B. 1977. Isolation of bacteriophages specific for the K1 486

polysaccharide antigen of Escherichia coli. J Clin Microbiol 6:548-550. 487

31. Karlyshev AV, Oyston PC, Williams K, Clark GC, Titball RW, Winzeler EA, Wren BW. 2001. 488


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

20

Application of high-density array-based signature-tagged mutagenesis to discover novel 489

Yersinia virulence-associated genes. Infect Immun 69:7810-7819. 490

32. McCarthy AJ, Martin P, Cloup E, Stabler RA, Oswald E, Taylor PW. 2015. The genotoxin 491

colibactin is a determinant of virulence in Escherichia coli K1 experimental neonatal 492

systemic infection. Infect Immun 83:0000-0000. 493

33. Zerbino DR. 2010. Using the Velvet de novo assembler for short-read sequencing 494

technologies. Curr Protoc Bioinformatics Chapter 11: Unit 11.5. 495

http://dx.doi.org/10.1002/0471250953.bi1105s31. PubMed. 496

34. Rhee KJ, Cheng H, Harris A, Morin C, Kaper JB, Hecht G. 2011. Determination of spatial and 497

temporal colonization of enteropathogenic E. coli and enterohemorrhagic E. coli in mice 498

using bioluminescent in vivo imaging. Gut Microbes 2:34-41. 499

35. Girard F, Dziva F, van Diemen P, Phillips AD, Stevens MP, Frankel G. 2007. Adherence of 500

enterohemorrhagic Escherichia coli O157, O26, and O111 strains to bovine intestinal 501

explants ex vivo. Appl Environ Microbiol 73:3084-3090. 502

36. Collins JW, Akin AR, Kosta A, Zhang N, Tangney M, Francis KP, Frankel G. 2012. Pre-503

treatment with Bifidobacterium breve UCC2003 modulates Citrobacter rodentium-induced 504

colonic inflammation and organ specificity. Microbiology 158:2826-2834. 505

37. Xu X, Miller SA, Baysal-Gurel F, Gartemann KH, Eichenlaub R, Rajashekara G. 2010. 506

Bioluminescence imaging of Clavibacter michiganensis subsp. michiganensis infection of 507

tomato seeds and plants. Appl Environ Microbiol 76:3978-3988. 508

38. Achtman M, Willetts N, Clark AJ. 1971. Beginning a genetic analysis of conjugational 509

transfer determined by the F factor in Escherichia coli by isolation and characterization of 510

transfer-deficient mutants. J Bacteriol 106:529-538. 511

39. Frost LS, Paranchych W, Willetts NS. 1984. DNA sequence of the F traALE region that 512

includes the gene for F pilin. J Bacteriol 160:395-401. 513

40. Schiffer MS, Oliveira E, Glode MP, McCracken GH, Sarff LM, Robbins JB. 1976. A review: 514


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

21

relation between invasiveness and the K1 capsular polysaccharide of Escherichia coli. 515

Pediatr Res 10:82-87. 516

41. Duan H, Gao F, Li S, Nagata T. 1993. Postnatal development and aging of esophageal 517

epithelium in mouse: a light and electron microscopic radioautographic study. Cell Mol Biol 518

39:309-316. 519

42. Craddock VM. 1993. Biology of the Esophagus, p 5-14. In Craddock VM (ed), Cancer of the 520

Esophagus: Approaches to the Etiology. Cambridge University Press, Cambridge UK. 521

43. Al Yassin TM, Toner PG. 1977. Fine structure of squamous epithelium and submucosal 522

glands of human oesophagus. J Anat 123:705-721. 523

44. Orlando RC. 2006. Esophageal mucosal defense mechanisms. GI Motility online 524

doi:10.1038/gimo15. 525

45. Dixon J, Strugala V, Griffin SM, Welfare MR, Dettmar PW, Allen A, Pearson JP. 2001. 526

Esophageal mucin: an adherent mucus gel barrier is absent in the normal esophagus but 527

present in columnar-lined Barrett's esophagus. Am J Gastroenterol 96:2575-2583. 528

46. Gryboski JD. 1965. The swallowing mechanism of the neonate. I. Esophageal and gastric 529

motility. Pediatrics 35:445-452. 530

47. Jadcherla SR, Duong HQ, Hofmann C, Hoffmann R, Shaker R. 2005. Characteristics of upper 531

oesophageal sphincter and oesophageal body during maturation in healthy human 532

neonates compared with adults. Neurogastroenterol Motil 17:663-670. 533

48. Stoll BJ, Hansen NI, Sánchez PJ, Faix RG, Poindexter BB, Van Meurs KP, Bizzarro MJ, 534

Goldberg RN, Frantz ID, Hale EC, Shankaran S, Kennedy K, Carlo WA, Watterberg KL, Bell 535

EF, Walsh MC, Schibler K, Laptook AR, Shane AL, Schrag SJ, Das A, Higgins RD. 2011. Early 536

onset neonatal sepsis: the burden of group B streptococcal and E. coli disease continues. 537

Pediatrics 127:817-826. 538

49. Guerina NG, Kessler TW, Guerina VJ, Neutra MR, Clegg HW, Langermann S, Scannapieco 539

FA, Goldmann DA. 1983. The role of pili and capsule in the pathogenesis of neonatal 540


http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

22

infection with Escherichia coli K1. J Infect Dis 148:395-405. 541

50. Bloch CA, Orndorff PE. 1990. Impaired colonization by and full invasiveness of Escherichia 542

coli K1 bearing a site-directed mutation in the type 1 pilin gene. Infect Immun 58:275-278. 543

51. Trivedi K, Tang CM, Exley RM. 2011. Mechanisms of meningococcal colonisation. Trends 544

Microbiol 19:456-463. 545

52. Siegel JD, McCracken GH. 1981. Sepsis neonatorum. N Engl J Med 304:642-647. 546

53. Nassif X, Bourdoulous S, Eugène E, Couraud PO. 2002. How do extracellular pathogens 547

cross the blood-brain barrier? Trends Microbiol 10:227-232. 548

54. Quaegebeur A, Lange C, Carmeliet P. 2011. The neurovascular link in health and disease: 549

molecular mechanisms and therapeutic implications. Neuron 71:406-424. 550

55. Moody DM. 2006. The blood-brain barrier and blood-cerebral spinal fluid barrier. Semin 551

Cardiothorac Vasc Anesth 10:128-131. 552

553

554

555

556

557

558

559

560

561

562

563

564

565

566


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Table 1. Citrobacter rodentium and Escherichia coli strains and plasmid used in this study 567

Description Ref

Bacteria C. rodentium ICC180

Luminescent derivative of C. rodentium, nalR kmR

28

E. coli S17-1 ƛ pir Pir + maintenance and donor strain, ampR

kmR

27

E. coli A192PP

Serially passaged (A192) strain

15

E. coli A192PP-lux2 Plasmid pUTmini-Tn5luxCDABEKm2

Luminescent A192PP derivative, kmR

Suicide vector, with unpromoted luxCDABE transposon, ampR kmR

This study 26

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583


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LEGENDS 584

585

FIG. 1 Characterization of E. coli K1 bioluminescent derivative A192PP-lux2. (A), Growth of E. coli 586

A192PP and A192PP-lux2 in MH broth at 37°C (200 orbits min); ±SD, n=5. (B), Photon emission by E. 587

coli A192PP-lux2 in relative luminescence units correlates with growth phase and shows 588

constitutive expression of the lux operon. (C), Relationship of photon emission to CFU (Spearman’s 589

rank test (two-tailed), n=3; P< 0.001). Colonization (D), accumulated bacteremia (E), and survival (F) 590

of E. coli A192PP and A192PP-lux2 in P2 and P9 neonatal rat pups. 591

592

FIG. 2 Colonization of the small intestine (A), and dissemination to the mesentery (B), blood (C) and 593

brain (D) of E. coli A192PP and A192PP-lux2 after oral application of bacteria (2-6 X 106 CFU) to P2 594

neonatal rat pups. Fewer data points at later time intervals reflect decreases in survival over time. 595

596

FIG. 3 Progression of systemic infection after oral application of E. coli A192PP-lux2 (2-6 X 106 CFU) 597

to P2 neonatal rat pups determined by 2D bioluminescence imaging (2DBLI) of live rats and excised 598

organs. Bioluminescence values were determined as log10 [flux [p/s]+1]; p, number of photons. 599

Images were collected from live animals at the time points indicated; they were then sacrificed, 600

organs collected and images obtained immediately. Representative images of whole animals and 601

excised organs are shown. Fewer data points at later time intervals reflect decreases in survival 602

over time. Data represent means ±SD (one-way ANOVA with Tukey’s multiple comparison test; *, P< 603

0.05). 604

605

FIG. 4 Relationship between the severity of disease and distribution of E. coli A192PP-lux2 in live 606

rats and excised organs after oral application of bacteria (2-6 X 106 CFU) to P2 neonatal rat pups 607

determined by 2D bioluminescence imaging (2DBLI). Bioluminescence values were determined as 608

log10 [flux [p/s]+1]; p, number of photons. Images were collected from live animals at the time 609


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points indicated; they were then sacrificed, organs collected and images obtained immediately. 610

Representative images of whole animals and excised organs are shown. Fewer data points at later 611

time intervals reflect decreases in survival over time. The disease severity of each animal was 612

monitored 4 - 5 times daily using a seven-point scoring system (11) based on color of the skin, 613

agility (righting reflex), response to gentle pressure on the abdomen, visibility of stomach/milk line, 614

temperature, weight gain and behavior when placed in cage. Animals were culled immediately 615

when three or more of symptoms became evident and the animal recorded as dead. Data represent 616

means ±SD (one-way ANOVA with Tukey’s multiple comparison test; *, P< 0.05; **, P< 0.01; ***, P< 617

0.001). 618

619

FIG. 5 Colonization of the oral cavity of P2 neonatal rats by E. coli A192PP-lux2. (A), 2DBLI 72 h after 620

feeding of bacteria (2-6 X 106 CFU) shows photon emission from the head region. (B), 2DBLI and 621

DLIT-μCT reveals intense oral cavity colonization with foci associated with tongue tissue. Images 622

were collected from live animals; they were then sacrificed, organs collected and images obtained 623

immediately. Representative images of whole animals and excised organs are shown. 624

625

FIG. 6 Dissemination of E. coli A192PP-lux2 to regional lymph nodes in live animals and excised 626

nodes 72 h after oral application of 2-6 X 106 CFU bacteria, revealed by 2DBLI and DLIT-μCT. Arrows 627

indicate excised tissues. Images were collected from live animals; they were then sacrificed, organs 628

collected and images obtained immediately. Representative images of whole animals and excised 629

organs are shown; different animals were used to generate each image. 630

631

FIG. 7 Age-dependent colonization of the esophagus by E. coli A192PP and A192PP-lux2. (A), 2DBLI 632

images of colonized P2 rats, showing oral cavity and esophageal involvement. (B), Photon emission 633

from esophageal tract tissue excised from colonized P2 and P9 rat pups; ***, P< 0.001 (Student’s t-634

test). (C), Enumeration of colonizing bacteria from esophageal samples of P2 and P9 rat pups; **, P< 635


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0.01; ***, P< 0.001 (Student’s t-test). 636

637

FIG. 8 Imaging of the age-dependency of esophageal colonization. (A), Age-dependent 638

keratinization of the esophagus in P2 and P9 neonatal rats. Scale bars: 25 μm. (B), 639

Immunohistochemical detection of E. coli O18 antigen in esophageal samples excised from two P2 640

animals 48 h after feeding of E. coli A192PP, damage and peripheral invasion of non-keratinised 641

sites can be seen. Scale bars: 25 μm. The O18 antigen was expressed by both strains to a 642

comparable extent. (C), SEM images of esophagi from pups fed E. coli A192PP at P2; images 643

obtained 24 h later reveal evidence of peripheral epithelial invasion. 644


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iai accepted manuscript posted online 8 september 2015 infect

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