Bioluminescent Imaging Reveals Novel Patterns of Colonization and Invasion in Systemic Escherichia coli K1 Experimental Infection in the Neonatal Rat Luci A. Witcomb,a James W. Collins,b Alex J. McCarthy,a Gadi Frankel,b Peter W. Taylora University College London School of Pharmacy, London, United Kingdoma; MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdomb

Key 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.

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scherichia coli strains expressing the K1 capsule, a homopolymer of ␣-2,8-linked polysialic acid, are a leading cause of earlyand late-onset neonatal sepsis and neonatal bacterial meningitis (1–3). Predisposition to these severe, often life-threatening infections is critically dependent on the vertical transmission of the causative agent from mother to infant at or soon after birth, and infection is associated with the ensuing gastrointestinal (GI) colonization of the neonate (4–6); the neonatal GI tract is considered sterile at birth but acquires an increasingly complex microbiota during the first year of life (7). Persisting high rates of morbidity and mortality (2, 8) and the continuing emergence of drug-resistant isolates (9, 10) emphasize the urgent need for new approaches to the prevention and treatment of these infections. A better understanding of the etiology and pathogenesis of neonatal systemic infections could provide the basis for a new generation of therapeutics and prophylactics, but these infections are medical emergencies, and opportunities for interventions that would provide such insights are severely limited. Consequently, much of the current knowledge of the underlying processes that lead to overt neonatal disease has been obtained from experimental infections in small animals such as mice, rats, and rabbits. In some animal models, infection is initiated by parenteral administration of bacteria, bypassing natural processes of colonization and dissemination and creating an artificial pathogenesis scenario. Replication of the natural site of GI colonization of E. coli K1 neonatal infection in the rat, a superior vehicle for such studies compared to the mouse (11), was employed initially by Moxon and coworkers (12) and subsequently extended and refined (11, 13, 14). This model, initiated by gastric intubation (12, 13) or feeding (11, 14) of the inoculum, produces a strongly age-dependent systemic infection much like that in the natural human host.

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For the first few days of life, newborn K1-colonized rat pups are prone to develop lethal infection due to the capacity of the colonizing bacteria to translocate from the lumen of the GI tract to the blood compartment (14, 15) after passage through the mesenteric lymphatic system (14), from where they may establish infection in multiple organs, including the brain (16). The invasion of brain tissue elicits a strong local inflammatory response induced by proinflammatory cytokines interleukin-1␤ (IL-1␤), IL-6, and tumor necrosis factor alpha (TNF-␣) (17) in a fashion similar to that of bacterial neonatal meningitis in humans (18, 19). Within a week, the pups become refractory to systemic infection, even in the presence of persistent GI tract colonization (20, 21). There are, however, a number of issues relating to the pathogenesis of E. coli K1 systemic infection that require resolution: the basis of the strong age dependency is poorly understood, the site of translocation from GI lumen to blood circulation is unknown, the mode and pattern of dissemination of the circulating pathogen to

Received 23 July 2015 Returned for modification 17 August 2015 Accepted 3 September 2015 Accepted manuscript posted online 8 September 2015 Citation Witcomb LA, Collins JW, McCarthy AJ, Frankel G, Taylor PW. 2015. Bioluminescent imaging reveals novel patterns of colonization and invasion in systemic Escherichia coli K1 experimental infection in the neonatal rat. Infect Immun 83:4528 – 4540. doi:10.1128/IAI.00953-15. Editor: A. J. Bäumler Address correspondence to Peter W. Taylor, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.00953-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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TABLE 1 Citrobacter rodentium and Escherichia coli strains and plasmid used in this study Bacterial strain or plasmid

Description

Reference or source

Strains C. rodentium ICC180 E. coli S17-1 ␭pir E. coli A192PP E. coli A192PP-lux2

Luminescent derivative of C. rodentium; Nalr Kmr Pir⫹ maintenance and donor strain; Ampr Kmr Serially passaged (A192) strain Luminescent A192PP derivative; Kmr

28 27 15 This study

Plasmid pUTmini-Tn5luxCDABEKm2

Suicide vector, with unpromoted luxCDABE transposon; Ampr Kmr

26

peripheral organs is unclarified, and there are conflicting views on the route of entry into the brain, whether across the blood-brain barrier (BBB) (22, 23), the blood meningeal barrier, through the choroid plexus (1, 16), or both. Recent developments in fourdimensional (4D) imaging methodologies provide an opportunity to investigate complex experimental infections as real-time dynamic processes in whole animals (24, 25). In this study, we shed further light on the pathogenesis of E. coli infection in neonatal rats and identify a potential new site of colonization and portal of entry into the systemic circulation using a bioluminescent E. coli K1 derivative combined with 2D bioluminescent imaging (2DBLI) and 3D diffuse light imaging tomography with integrated micro-computed tomography (DLIT-␮CT). We have used these data to generate 4D movies of the infection cycle to further inform on temporal aspects of disease progression. MATERIALS AND METHODS Bacteria and plasmids. The properties of the bacteria and plasmid used in this study are detailed in Table 1. E. coli O18:K1 strain A192PP was derived from septicemia isolate E. coli A192 (26) by two rounds of passage through neonatal rat pups, with bacterial recovery from the blood (15). The presence of K1 capsule was confirmed with K1-specific bacteriophage K1E (27). Bacteria were grown in either Luria-Bertani (LB) medium, M9 minimal medium (M9 salts supplemented with 1% glucose and 0.01 M sodium citrate), or Mueller-Hinton (MH) broth and incubated at 37°C with agitation in an orbital incubator at 200 orbits per min and with kanamycin (50 ␮g/ml) and ampicillin (100 ␮g/ml) as required. Generation and characterization of E. coli A192PP-lux. E. coli A192PP was engineered to express the bioluminescence phenotype by introduction of the lux operon through mini-Tn5 mutagenesis. The method used a suicide vector that carried an unpromoted lux operon (luxCDABE) from the terrestrial bacterium Photorhabdus luminescens on a disarmed mini-Tn5 transposon (28). The pUTmini-Tn5luxCDABEKm2 vector was maintained in donor strain E. coli S17-1 ␭pir (29) and transferred to A192PP by conjugation, essentially as previously described (30), with the exception that conjugants were selected on M9 minimal medium containing kanamycin and 0.01 M sodium citrate to prevent ␭ phage lysogeny of the E. coli recipient strain (31). Bioluminescent conjugants were identified with the Bio-Rad ChemiDoc XRS⫹ imaging system (no illumination, no filter, autoexposure), and K1-positive bioluminescent colonies were subjected to two rounds of subculture on MH agar containing kanamycin prior to storage under glycerol at ⫺80°C. Growth kinetics (optical density at 600 nm [OD600]) and bioluminescence of conjugants were determined in MH broth; photon emission during the growth cycle (expressed as relative luminescence units) was monitored with a PerkinElmer LS-55 fluorescence spectrometer. The stability of the mini-Tn5 element within conjugants was determined by subculture every 24 h in MH broth in the presence and absence of kanamycin; subcultures were enumerated by viability counting, and bioluminescent colonies were identified with the ChemiDoc XRS⫹ imaging system. To determine luxCDABEKm2 insertion sites in E. coli A912PP derivatives,

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strains were sequenced with the Illumina MiSeq platform as described previously (32) and compared with the E. coli A192PP sequence (European Nucleotide Archive [http://www.ebi.ac.uk/ena/] accession number PRJEB9141). The conjugant E. coli A192PP-lux2 was selected for further investigation. The E. coli A192 and A192PP-lux2 genomes were assembled using Velvet assembler (33) and aligned with the luxCDABE operon within the Tn-lux sequence. To confirm the insertion site of the Tn-lux element, A192PP-lux2 paired sequence reads were mapped onto the A192PP assembled genome. The insertion site then was confirmed by PCR using primers for the amplification of A192PP-lux2-traL (forward primer, 5=-TATATCGTCGGCCATGAATCC-3=; reverse primer, 5=-AAC CTCACTCCCTTTTTGCT-3=) and primers for the amplification of the luxC-traL junction (forward primer, 5=-CGTATCCTCCAAGCCTGAAT T-3=; reverse primer, 5=-TGAAGCGGTAGAAGTTGCCAA-3=), producing fragments of 597 bp and 419 bp, respectively. PCRs (50 ␮l) contained 25 ␮l Promega master mix, 1 ␮l (10 ␮M) of each primer, and 1 ␮l genomic DNA as the template. Amplification was carried out under the following conditions: 1 cycle at 95°C for 5 min; 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 2 min; and a final extension of one cycle of 72°C for 10 min. Experimental systemic infection of neonatal rats. Animal experiments were approved by the Ethical Committee of the UCL School of Pharmacy and the UK Home Office (HO) and were conducted under HO Project License PPL 70/7773. The procedure has been described in detail (11). In brief, 2-day-old (P2) or 9-day-old (P9) Wistar rat pups (Harlan UK) were fed 20 ␮l of mid-logarithmic-phase E. coli (2 ⫻ 106 to 6 ⫻ 106 CFU) from an Eppendorf micropipette to effect gastrointestinal (GI) colonization. No local trauma is observed as a consequence of this procedure. All members of a litter, usually 12 pups, were treated as a single test or control group and fed E. coli culture in identical fashion at the same time interval. GI tract colonization was determined by culture of perianal swabs on MacConkey agar; bacteremia was detected by MacConkey agar culture of blood taken postmortem. Disease progression was determined by daily evaluation of symptoms of systemic infection and scored on a scale of rising severity from 0 to 3 (11). After sacrifice, samples from the esophagus, stomach, small intestine (SI), colon, blood, mesenteric lymphatic system, liver, lung, heart, kidney, and spleen were excised aseptically (11). Organs were washed extensively in phosphate-buffered saline (PBS) to ensure minimal contamination with perfused blood. Bacteria were quantified in homogenized tissues by serial dilution culture on MacConkey agar and the presence of the K1 capsule confirmed as required with bacteriophage K1E. Samples from experiments involving E. coli A192PP-lux2 were cultured in the presence of 50 ␮g/ml kanamycin. 2DBLI and DLIT-␮CT. 2DBLI was performed on tissues harvested from P2 and P9 rat pups colonized by A192PP-lux2; tissues were collected 24, 48, and 72 h after feeding of the colonizing bacteria, and the severity of infection was recorded. Tissues were washed in sterile PBS, placed in sterile petri dishes on a black card, and pierced with sterile 25-guage needles for aeration (34). Bioluminescence was measured within standardized regions of interest (ROI), and data are expressed as flux (photons per second), adjusted using the following formula to ensure all measurements had positive value: log10(flux ⫹1). Tissues from noncolonized pups were

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used for bioluminescent background subtraction. Quantitative imaging was performed using an IVIS Lumina series III (PerkinElmer). For whole-animal studies, both the IVIS Lumina series III (for 2DBLI) and the IVIS SpectrumCT (for DLIT-␮CT; PerkinElmer) were employed. In DLIT-␮CT experiments, the automatic settings in the Living Image software 4.3.1 wizard and autoexposure settings specific for imaging bacterial luciferases (maximum exposure, 300 s; target count minimum, 10,000) were used (25). Anesthesia (5% isoflurane, followed by maintenance under 2.5% isoflurane) on the prewarmed imaging platform was used in both 2DBLI and DLIT-␮CT experiments. Symptoms of infection were recorded immediately prior to the collection of images and the pups culled for ex vivo tissue analysis; animals were not subjected to repeated anesthesia. 3D animations were created using Living Image, as previously described (25), and compiled into a movie using iMovie software (version 10.0.5). Noncolonized animals were used for bioluminescent background subtraction. To correlate flux (photons per second) with CFU, serial dilutions in PBS from 16-h cultures of E. coli A192PP-lux were prepared in 96-well black plates, and wells were highlighted as ROIs prior to imaging in the IVIS Lumina series III and IVIS SpectrumCT. Flux within ROIs was measured and CFU from each well determined retrospectively by plating on MH agar with kanamycin. Histology and microscopy. Esophageal tissues were collected and fixed in 10% neutral buffered formalin, embedded in paraffin, and processed, and 5-␮m sections were obtained, mounted onto slides, and stained with hematoxylin and eosin (H&E). Unstained sections were prepared for immunohistochemistry: they were dewaxed in HistoClear and examined for E. coli O18 lipopolysaccharide antigen as previously described (16, 35) and mounted in VectaShield mounting medium containing 4=,6=-diamidino-2-phenylindole (DAPI) stain (H-1200). Samples for scanning electron microscopy (SEM) were processed and examined as previously described (36) using a JEOL JSM-5300 scanning electron microscope. Nucleotide sequence accession number. Nucleotide sequence of the A192PP-lux2 genome has been deposited in the European Nucleotide Archive (http://www.ebi.ac.uk/ena/) under accession number PRJEB9940.

RESULTS

Generation and characterization of E. coli A192PP-lux. Thirtyone bioluminescent derivatives of E. coli A192PP were obtained by mini-Tn5 mutagenesis. One, designated E. coli A192PP-lux2, expressed the K1 capsule, grew in MH broth (Fig. 1A) and LB minimal medium (see Fig. S1 in the supplemental material) to an extent comparable to that of the parental A192PP strain, and produced a strong bioluminescent signal over the course of the growth cycle that correlated with CFU (Fig. 1B and C), indicative of constitutive expression of the lux operon (37). The strength of the bioluminescent signal from A192PP-lux2 exceeded that from C. rodentium ICC180 and was maintained for 7 days in the absence of kanamycin (data not shown), indicative of stable genomic maintenance of the lux operon. Whole-genome sequencing of E. coli A192PP-lux2 revealed that the Tn5-lux element inserted into traL, encoding an F-pilus assembly protein (38, 39). The disruption of traL and the presence of the luxC-traL junction in A192PPlux2 was confirmed by PCR (see Fig. S2); pili were not evident in SEM images of either A192PP or A192PP-lux2 (see Fig. S1B). The capacity of E. coli A192PP-lux2 to elicit lethal infection after GI colonization in P2 and P9 neonatal rats was determined. The bioluminescent derivative colonized the GI tract of P2 and P9 pups to the same extent as the parental E. coli A192PP, with stable colonization occurring within 24 and 48 h of feeding of the bacteria (Fig. 1D); 79.17% (19/24) of P2 animals developed bacteremia over the 7-day observation period and around 20% survived, whereas all

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receiving the parent strain were bacteremic (Fig. 1E), with no survivors (Fig. 1F). No P9 pups receiving either E. coli A192PP or A192PP-lux2 succumbed to bacteremia or lethal infection in spite of efficient GI colonization. Thus, the stable insertion of the lux transposon and kanamycin resistance cassette into traL had only a minor impact on virulence. Therefore, E. coli A192PP-lux2 was employed for all imaging experiments. Distribution of E. coli A192PP-lux2 in colonized P2 and P9 rat pups. The available evidence indicates that, after ingestion, E. coli K1 follows the gut-mesentery-blood-cerebrospinal fluid (CSF) course of infection in the neonatal rat (13, 14), but the bacteria become widely disseminated as the infection progresses (16). To determine if carriage of the lux transposon and kanamycin resistance genes altered the in vivo distribution of E. coli A192PP and to signpost the design of whole-animal and organ imaging experiments, we determined the organ tropism and organ load of E. coli A192PP and A192PP-lux2 in P2 and P9 neonatal rats after the initiation of colonization. A population of colonizing E. coli A192PP was evident in the small intestine 24 h (the initial time point for viability determination) after initiation of colonization at P2 and varied little in quantitative terms over the first 72 h of colonization; there were no significant differences in numbers of colonizing bacteria between E. coli A192PP and A192PP-lux2 during this period (Fig. 2A). Small numbers of bacteria were present in the mesentery after 24 h (Fig. 2B) and began to appear in the blood circulation at the same time point (Fig. 2C). E. coli K1 were recovered from brain tissue in a variable proportion of infected P2 animals, and the numbers encountered were low (Fig. 2D). There again were no significant differences between E. coli A192PP and A192PP-lux2 at any given time point. Other regions of the GI tract (stomach and colon) also were stably colonized by both bioluminescent strain and parent strain to a similar extent, although a noticeable but insignificant decrease in A192PP-lux2 load present in the stomach at 24 h after initiation of colonization was observed (see Fig. S3 in the supplemental material). Low to moderate numbers of E. coli A192PP and A192PP-lux2 were cultured from liver, lung, heart, kidney, spleen, and pancreas; no significant differences in the size of the bioburden between the two strains were evident when they were compared by time after initiation of colonization or by severity of symptoms of infection (see Fig. S3 and S4). The bacterial load of both strains in the major organs increased as the health status of the animals declined (see Fig. S4). We conclude that the introduction of bioluminescence- and kanamycin-associated genes into E. coli A192PP has little, if any, impact on organ tropism and tissue bioburden in this widely disseminated infection of susceptible P2 neonatal rats. With resistant P9 pups, E. coli A192PP and A192PP-lux2 were recovered from the stomach, small intestine, and colon in numbers comparable to those found in P2 animals; only very low numbers were found in the mesentery, and other tissues were free of E. coli K1 (data not shown). Patterns of E. coli A192PP-lux2 infection determined by bioluminescence imaging. 2DBLI of live P2 rats revealed a significant increase in total bioluminescence between 24 h and 48 h after feeding of E. coli A192PP-lux2 (P ⬍ 0.05), coincident with the onset of signs of infection. Representative images are shown in Fig. 3. Colonizing bacteria were not uniformly distributed along the small intestine. Typically, there was rapid dissemination of the pathogen from the site of colonization between these two time points and an apparent reduction in both flux and degree of dis-

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FIG 1 Characterization of E. coli K1 bioluminescent derivative A192PP-lux2. (A) Growth of E. coli A192PP and A192PP-lux2 in MH broth at 37°C (200 orbits per min); results are given as means ⫾ standard deviations (SD), n ⫽ 5. (B) Photon emission by E. coli A192PP-lux2, in relative luminescence units (RLU), correlates with growth phase and shows constitutive expression of the lux operon. (C) Relationship of photon emission to CFU (Spearman’s rank test [two-tailed]; n ⫽ 3; P ⬍ 0.001). Colonization (D), accumulated bacteremia (E), and survival (F) of E. coli A192PP and A192PP-lux2 in P2 and P9 neonatal rat pups are shown.

semination at 72 h. However, in individual animals the infection progresses at different rates (16), with a proportion succumbing to infection within 3 days after seeding of E. coli K1, and survivors at 72 h are likely to be animals in which infection progressed at a relatively low rate compared to that of nonsurvivors. The apparent peak of infection at 48 h was reflected in photon emission from excised major organs with the exception of the GI tract tissues, indicative of stable GI colonization (Fig. 3).

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Therefore, we examined the data relative to the severity of symptoms of infection (Fig. 4). In live P2 animals, the E. coli K1 burden increased significantly as symptoms of infection became evident, indicating that although the total burden increased with time, it correlated more closely with the severity of infection. The degree of colonization of the stomach, small intestine, and mesentery increased with disease severity, and invasion of the central nervous system occurred only

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FIG 2 Colonization of the small intestine (A) and dissemination to the mesentery (B), blood (C), and brain (D) of E. coli A192PP and A192PP-lux2 after oral application of bacteria (2 to 6 ⫻ 106 CFU) to P2 neonatal rat pups. Fewer data points at later time intervals reflect decreases in survival over time.

in animals displaying severe symptoms of infection. The association of E. coli K1 with liver, spleen, pancreas, heart, and kidney were similarly associated with late stages of infection. In contrast, the distribution of E. coli A192PP-lux2 in colonized P9 pups was restricted to the GI tract (see Fig. S5 in the supplemental material). The bioburden in live P9 animals did not increase significantly between 24 h and 72 h, and few bacteria were visualized in the mesentery. Niches rich in colonizing bacteria were evident in the small intestine, colon, and mesentery in both P2 and P9 pups. Overall, the distribution of E. coli K1 determined by 2DBLI was consistent with data obtained by viability determination (Fig. 2; also see Fig. S4). DLIT-␮CT was used to investigate the relationship between the development of symptoms of disease and the whole-animal distribution of E. coli A192PP-lux2 in P2 animals over a 72-h period following the initiation of colonization (see Video S1 in the supplemental material). As anesthesia modified the progression of disease and increased the risk of rejection by the mother, longitudinal monitoring of individual pups was not performed. As determined earlier in the study, the rate of disease progression differed significantly between individual animals, and clearer representations of disease development were obtained when comparisons were made on the basis of severity of symptoms rather than on time from colonization. The involvement of multiple organs can be seen in cases of severe infection (score of three or more), and it is noteworthy that the infection of the central nervous system (CNS) was restricted to the surface of the brain, lending support to the view that the choroid plexus rather than the brain microvascular endothelium represents the portal of entry into the CNS in experimental E. coli K1 meningitis. The authenticity of the DLIT-

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␮CT reconstruction was confirmed by 2DBLI of the corresponding pups and their organs (see Fig. S6). DLIT-␮CT revealed that the initial bolus of E. coli K1 entered the colon within 3 h of oral application of the inoculum, seeding the entirety of the alimentary tract, including the oral cavity, esophagus, stomach, and small intestine (see Video S2 and Fig. S7). Frequent colonization of the oral cavity was observed, which presented as photon emission from the head region (see Video S3), and was evident over the entire 72-h observation period. Stable oral cavity colonization was confirmed by 2DBLI of excised elements of this region and revealed an intense colonization of tongue tissue (Fig. 5). During later stages of disease progression, photon emission from multiple lymph nodes in the face, neck, back, and joints was observed (Fig. 6; also see Video S4), demonstrating extensive dissemination of E. coli K1 within the lymphatic system. Age-dependent colonization and invasion of the esophagus. 2DBLI indicated that the esophagus could represent a novel site for entry and dissemination in systemic E. coli K1 experimental infection (Fig. 7A; also see Fig. S7 in the supplemental material). Photon emission from the head region did not originate from the trachea (data not shown). The determination of photon emission by the bioluminescent derivative (Fig. 7B) and CFU of E. coli A192PP and A192PP-lux2 (Fig. 7C) in excised esophagi showed P2 rats to be significantly more susceptible to colonization at this site than P9 animals. No significant quantitative differences in colonization capacity between the two strains were apparent (Fig. 7C), and no difference between E. coli A192PP and A192PP-lux2 with respect to the severity of symptoms was found (data not shown). H&E staining of the esophagus revealed the presence of a keratinized epithelium at P9; this layer was absent from or only

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FIG 3 Progression of systemic infection after oral application of E. coli A192PP-lux2 (2 to 6 ⫻ 106 CFU) to P2 neonatal rat pups determined by 2D biolumi-

nescence imaging (2DBLI) of live rats and excised organs. Bioluminescence values were determined as log10(flux ⫹1), where flux is measured in photons per second. Images were collected from live animals at the time points indicated; they then were sacrificed, organs were collected, and images were obtained immediately. Representative images of whole animals and excised organs are shown. Fewer data points at later time intervals reflect decreases in survival over time. Data represent means ⫾ SD (one-way analysis of variance [ANOVA] with Tukey’s multiple-comparison test; *, P ⬍ 0.05).

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018 antigen was not detected in tissues from noncolonized animals. Bacteria also were observed attached to sloughing keratin strands present in the lumen of the esophagus. Additionally, at 48 h, peripheral invasion and damage to regions of nonkeratinized esophageal epithelium by E. coli K1 was evident (Fig. 8B), and further evidence of invasion of nonkeratinized regions also was observed by SEM (Fig. 8C). DISCUSSION

FIG 5 Colonization of the oral cavity of P2 neonatal rats by E. coli A192PPlux2. (A) 2DBLI 72 h after feeding of bacteria (2 ⫻ 106 CFU to 6 ⫻ 106 CFU) shows photon emission from the head region. (B) 2DBLI and DLIT-␮CT reveal intense oral cavity colonization with foci associated with tongue tissue. Images were collected from live animals; they then were sacrificed, organs were collected, and images were obtained immediately. Representative images of whole animals and excised organs are shown.

partially developed in the esophagus of susceptible P2 neonatal rats (Fig. 8A). It began to appear 2 to 3 days postpartum (see Fig. S8) and developed progressively during the early neonatal period. Esophageal sections from rats colonized with E. coli A192PP at P2 were probed for the presence of the O18 antigen; bacterial attachment to keratinized and nonkeratinized esophageal surfaces was evident at 24, 48, and 72 h following bacterial feeding (Fig. 8B);

The isolation of E. coli K1 from the cerebrospinal fluid of sick newborn infants frequently coincides with the presence of the bacteria in the feces of both infant and mother (4–6), providing a strong indication that the maternal GI tract represents the primary reservoir of infection for these neonatal pathogens, which are associated with the infant gut prior to systemic invasion. Oral administration of E. coli K1 to susceptible neonatal rats clearly shows that colonization of the GI tract precedes invasion (13–15). The site of translocation and the molecular processes involved have not been defined with any precision, although evidence is emerging that the colonization of the small intestine is essential for systemic infection (21). In the current study, DLIT-␮CT imaging showed that the colonizing E. coli K1 bolus reached the colon within 3 h of administration, and 2DBLI revealed regions of intense colonization in both the small intestine and colon of P2 and P9 pups and, unexpectedly, that bacteria were seeded along the entire length of the alimentary tract. Thus, the oral cavity, esophagus, and stomach of P2 animals were usually heavily colonized and may represent additional reservoirs of infection in this model. Small numbers of E. coli K1 were found in the mesenteric lymphatic system at an early stage of the infection cycle, supporting evidence (14) that invasion of blood circulation occurs by this route. The presence of E. coli K1 bacteria in some of the major organs, particularly in pups with symptoms of systemic infection, may represent end-stage invasion as immune defenses are compromised. However, with highly perfused organs the bacterial viability counts are very likely to include bacteria present in blood as well as in tissue; the organs of newborn rats are fragile and will not withstand ex vivo perfusion for blood removal (P. W. Taylor, unpublished observations). Colonization of the GI tract by E. coli K1 at P2 dysregulates the maturation of the mucus barrier, which is poorly formed in newborn pups; mucosal barrier function at this age is insufficient to prevent translocation of E. coli K1 from gut lumen to blood circulation (21). An integral barrier has formed by P9 (21), preventing systemic invasion and accounting for the restricted distribution of the bacteria following colonization of P9 animals observed in the current study. We also found an unexpected age-dependent, stable colonization of the esophagus and obtained evidence that, in P2 pups, E. coli K1 may invade nonkeratinized esophageal tissue, raising the possibility that the esophagus represents an additional locus of translocation to the blood. Thus, photon emission from this

FIG 4 Relationship between the severity of disease and distribution of E. coli A192PP-lux2 in live rats and excised organs after oral application of bacteria (2 ⫻ 106 CFU to 6 ⫻ 106 CFU) to P2 neonatal rat pups determined by 2D bioluminescence imaging (2DBLI). Bioluminescence values were determined as log10(flux ⫹1), where flux is measured in photons per second. Images were collected from live animals at the time points indicated. They then were sacrificed, organs collected, and images obtained immediately. Representative images of whole animals and excised organs are shown. Fewer data points at later time intervals reflect decreases in survival over time. The disease severity of each animal was monitored 4 to 5 times daily using a seven-point scoring system (11) based on color of the skin, agility (righting reflex), response to gentle pressure on the abdomen, visibility of stomach/milk line, temperature, weight gain, and behavior when placed in a cage. Animals were culled immediately when three or more symptoms became evident, and the animals were recorded as dead. Data represent means ⫾ SD (one-way ANOVA with Tukey’s multiple-comparison test; *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).

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FIG 6 Dissemination of E. coli A192PP-lux2 to regional lymph nodes in live animals and excised nodes 72 h after oral application of 2 ⫻ 106 to 6 ⫻ 106 CFU bacteria, revealed by 2DBLI and DLIT-␮CT. Arrows indicate excised tissues. Images were collected from live animals; they were then sacrificed, and organs were collected and images obtained immediately. Representative images of whole animals and excised organs are shown; different animals were used to generate each image.

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FIG 7 Age-dependent colonization of the esophagus by E. coli A192PP and A192PP-lux2. (A) 2DBLI images of colonized P2 rats, showing oral cavity and esophageal involvement. (B) Photon emission from esophageal tract tissue excised from colonized P2 and P9 rat pups. ***, P ⬍ 0.001 (Student’s t test). (C) Enumeration of colonizing bacteria from esophageal samples of P2 and P9 rat pups. **, P ⬍ 0.01; ***, P ⬍ 0.001 (Student’s t test).

site was significantly increased in susceptible P2 rats compared to that from resistant P9 animals over the 3 days following initiation of colonization and the strong age dependency confirmed by enumeration of viable E. coli K1 within esophageal tissue. H&E staining revealed the age-dependent development of a keratinized esophageal layer, appearing as early as 2 days postpartum; age-related keratinization also has been documented in the mouse but appears to proceed more slowly compared to that of the rat as determined in the current study (40). Esophageal keratinization provides protection against the consumption of coarse foods (41), although the human esophageal epithelium is not normally keratinized to any extent (42, 43) and, like the rodent esophagus, does not develop a viscoelastic protective mucus barrier (44). E. coli K1 attached to all regions of the P2 esophagus, including keratinized and nonkeratinized surfaces. As binding is inde-

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pendent of the keratin layer, the presence of keratin cannot account for differences in esophageal binding between P2 and P9 pups. However, invading E. coli K1 cells were found in nonkeratinized regions of the P2 esophagus, suggesting that the incomplete keratinization of this site predisposes the underlying epithelium to invasion and may enable the bacteria to persist; this contention is supported by SEM imaging of P2 esophageal tissue. Full-term human neonates exhibit reduced esophageal motility and luminal clearance compared to that of adults, which is further decreased in preterm neonates (45–47), suggesting that the neonatal esophagus is vulnerable to colonization and invasion by neuropathogens in the human host. Thus, reduced esophageal peristalsis, an exposed epithelial layer lacking keratin, and a mucosal barrier may increase the risk of pathogen attachment, overgrowth, and invasion at this site.

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FIG 8 Imaging of the age dependency of esophageal colonization. (A) Age-dependent keratinization of the esophagus in P2 and P9 neonatal rats. Scale bars, 25

␮m. (B) Immunohistochemical detection of E. coli O18 antigen in esophageal samples excised from two P2 animals 48 h after feeding of E. coli A192PP. Damage and peripheral invasion of nonkeratinized sites can be seen. Scale bars, 25 ␮m. The O18 antigen was expressed by both strains to a comparable extent. (C) SEM images of esophagi from pups fed E. coli A192PP at P2; images obtained 24 h later reveal evidence of peripheral epithelial invasion.

We noted colonization of the oral cavity, in particular the tongue. There have been few studies of sites of E. coli K1 colonization upstream of the GI tract, but Guerina and colleagues (48) recorded the colonization of the oral cavity in virtually all neonatal rats examined. In another study, colonization of the oropharynx and bloodstream invasion occurred in neonatal rats fed E. coli K1; pilus-deficient mutants were unable to maintain colonization of this site but continued to colonize the GI tract and cause bactere-

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mia (49), suggesting that oropharyngeal colonization was not essential for the development of sepsis and meningitis. In support of this, we found that the Tn5-lux construct inserted into traL of E. coli A912PP; this gene is involved in F-pilus assembly (38), but the virulence of the transposant E. coli A192PP-lux2 was, to a great extent, retained. Further studies should be implemented to determine if colonization of sites other than the GI tract predispose toward systemic infection and whether colonization of the distal

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Colonization and Invasion in E. coli Infection

tongue can be used for diagnostic swabbing of neonates to detect K1 colonization prior to the onset of sepsis. Although a strong correlation between systemic E. coli K1 infection and GI tract colonization has been reported frequently in clinical studies of neonatal infection (3, 4, 6), there are indications that in some cases of bacterial meningitis, the aspiration of the infecting dose into the lungs and other regions of the respiratory tract provides opportunities for an alternative portal of entry into the systemic circulation (50). This is unlikely to be a significant factor in the development of infection in the neonatal rat model, as E. coli A192PP appears in the lung at later time points and only in pups displaying severe symptoms of infection. Soon after the initiation of colonization, susceptible pups develop bacteremia before succumbing to overwhelming infection involving the major organs. We found E. coli K1 in the major groups of lymph nodes, suggesting that disseminated infection arises due to failure of the lymphatic system to control localized foci of bacteria. E. coli K1 is strongly associated with sepsis and meningitis (1–3), and we found significant associations between the severity of infection and the recovery of viable bacteria from brain tissue. In a previous study (16), we visualized E. coli K1 in brain sections using a modified Gram stain and found bacteria associated only with the surfaces of this organ. The current study lends further support to this pattern of distribution: DLIT-␮CT imaging (see Video S1 in the supplemental material) indicated that E. coli A192PP-lux2 cells were restricted to superficial layers on the surface of the brain, much as in the human condition (51), and supports previous observations (16) that E. coli K1 gains access to the CNS through the choroid plexus, a component of the blood-CSF barrier. Transit from the blood circulation through this epithelial barrier to the CSF would present the invading bacteria with the opportunity to adhere to the most superficial layer of the brain, the leptomeninges, but restrict access to cortical tissue. However, it is widely accepted that E. coli K1 invades the CNS by traversal of the endothelium (BBB) (23, 52), even though the evidence supporting this route of entry is modest (22). As the mammalian brain accounts for only 2% of body mass yet receives 20% of the cardiac output, it is a highly vascularized organ; the human brain contains an estimated one hundred billion vessels, one for each neuron (53). If circulating neuropathogens penetrated the BBB to any extent at multiple sites within this vascular network, one would expect to encounter the bacteria throughout the brain in both naturally occurring and experimental E. coli K1 meningitis, but this is not the case. The choroid plexus provides a much lower resistance to transport into the CNS than the BBB (54) and is a strong candidate for portal of entry. The resolution of the important issues of colonization and tissue penetration may open new avenues for resolving lethal infections of the CNS. ACKNOWLEDGMENTS This study was supported by project grant GN2075 from Action Medical Research. Further support was provided by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. We thank Richard Stabler, London School of Hygiene and Tropical Medicine, for generating the sequence reads. We thank Thilo M. Fuchs, Technische Universitat Munchen, for kindly providing the Tn-lux sequence.

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