Bioluminescent Imaging Reveals Novel Patterns of Colonization andInvasion in Systemic Escherichia coli K1 Experimental Infection in theNeonatal 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 developmentalong the gut-mesentery-blood-brain course of infection, can be replicated in the newborn rat. We examined temporal and spa-tial aspects of E. coli K1 infection following initiation of gastrointestinal colonization in 2-day-old (P2) rats after oral adminis-tration of E. coli K1 strain A192PP and a virulent bioluminescent derivative, E. coli A192PP-lux2. A combination of bacterialenumeration in the major organs, two-dimensional bioluminescence imaging, and three-dimensional diffuse light imaging to-mography 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 com-partment, the bacteria entered the central nervous system, with restricted colonization of the brain, and also invaded the majororgans, in line with increases in the severity of symptoms of infection. Both keratinized and nonkeratinized surfaces of esophagiwere colonized to a considerably greater extent in susceptible P2 neonates than in corresponding tissues from infection-resistant9-day-old rat pups; the bacteria appeared to damage and penetrate the nonkeratinized esophageal epithelium of infection-sus-ceptible P2 animals, suggesting the esophagus represents a portal of entry for E. coli K1 into the systemic circulation. Thus, mul-timodality imaging of experimental systemic infections in real time indicates complex dynamic patterns of colonization and dis-semination that provide new insights into the E. coli K1 infection of the neonatal rat.

Escherichia coli strains expressing the K1 capsule, a homopoly-mer of �-2,8-linked polysialic acid, are a leading cause of early-

and late-onset neonatal sepsis and neonatal bacterial meningitis(1–3). Predisposition to these severe, often life-threatening infec-tions is critically dependent on the vertical transmission of thecausative agent from mother to infant at or soon after birth, andinfection is associated with the ensuing gastrointestinal (GI) col-onization of the neonate (4–6); the neonatal GI tract is consideredsterile at birth but acquires an increasingly complex microbiotaduring the first year of life (7). Persisting high rates of morbidityand mortality (2, 8) and the continuing emergence of drug-resis-tant isolates (9, 10) emphasize the urgent need for new approachesto the prevention and treatment of these infections. A better un-derstanding of the etiology and pathogenesis of neonatal systemicinfections could provide the basis for a new generation of thera-peutics and prophylactics, but these infections are medical emer-gencies, and opportunities for interventions that would providesuch insights are severely limited. Consequently, much of the cur-rent knowledge of the underlying processes that lead to overt neo-natal disease has been obtained from experimental infections insmall animals such as mice, rats, and rabbits.

In some animal models, infection is initiated by parenteral ad-ministration of bacteria, bypassing natural processes of coloniza-tion and dissemination and creating an artificial pathogenesis sce-nario. Replication of the natural site of GI colonization of E. coliK1 neonatal infection in the rat, a superior vehicle for such studiescompared to the mouse (11), was employed initially by Moxonand coworkers (12) and subsequently extended and refined (11,13, 14). This model, initiated by gastric intubation (12, 13) orfeeding (11, 14) of the inoculum, produces a strongly age-depen-dent systemic infection much like that in the natural human host.

For the first few days of life, newborn K1-colonized rat pups areprone to develop lethal infection due to the capacity of the colo-nizing bacteria to translocate from the lumen of the GI tract to theblood compartment (14, 15) after passage through the mesentericlymphatic system (14), from where they may establish infection inmultiple organs, including the brain (16). The invasion of braintissue elicits a strong local inflammatory response induced by pro-inflammatory cytokines interleukin-1� (IL-1�), IL-6, and tumornecrosis factor alpha (TNF-�) (17) in a fashion similar to that ofbacterial neonatal meningitis in humans (18, 19). Within a week,the pups become refractory to systemic infection, even in the pres-ence of persistent GI tract colonization (20, 21).

There are, however, a number of issues relating to the patho-genesis of E. coli K1 systemic infection that require resolution: thebasis of the strong age dependency is poorly understood, the site oftranslocation from GI lumen to blood circulation is unknown, themode and pattern of dissemination of the circulating pathogen to

Received 23 July 2015 Returned for modification 17 August 2015Accepted 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 insystemic 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, peter.taylor@ucl.ac.uk.

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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peripheral organs is unclarified, and there are conflicting views onthe route of entry into the brain, whether across the blood-brainbarrier (BBB) (22, 23), the blood meningeal barrier, through thechoroid plexus (1, 16), or both. Recent developments in four-dimensional (4D) imaging methodologies provide an opportu-nity to investigate complex experimental infections as real-timedynamic processes in whole animals (24, 25). In this study, weshed further light on the pathogenesis of E. coli infection in neo-natal rats and identify a potential new site of colonization andportal of entry into the systemic circulation using a biolumines-cent E. coli K1 derivative combined with 2D bioluminescent im-aging (2DBLI) and 3D diffuse light imaging tomography with in-tegrated micro-computed tomography (DLIT-�CT). We haveused these data to generate 4D movies of the infection cycle tofurther inform on temporal aspects of disease progression.

MATERIALS AND METHODSBacteria and plasmids. The properties of the bacteria and plasmid used inthis study are detailed in Table 1. E. coli O18:K1 strain A192PP was derivedfrom septicemia isolate E. coli A192 (26) by two rounds of passage throughneonatal rat pups, with bacterial recovery from the blood (15). The pres-ence of K1 capsule was confirmed with K1-specific bacteriophage K1E(27). Bacteria were grown in either Luria-Bertani (LB) medium, M9 min-imal medium (M9 salts supplemented with 1% glucose and 0.01 M so-dium citrate), or Mueller-Hinton (MH) broth and incubated at 37°C withagitation 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. coliA192PP was engineered to express the bioluminescence phenotype byintroduction of the lux operon through mini-Tn5 mutagenesis. Themethod used a suicide vector that carried an unpromoted lux operon(luxCDABE) from the terrestrial bacterium Photorhabdus luminescens ona disarmed mini-Tn5 transposon (28). The pUTmini-Tn5luxCDABEKm2vector was maintained in donor strain E. coli S17-1 �pir (29) and trans-ferred to A192PP by conjugation, essentially as previously described (30),with the exception that conjugants were selected on M9 minimal mediumcontaining kanamycin and 0.01 M sodium citrate to prevent � phagelysogeny of the E. coli recipient strain (31). Bioluminescent conjugantswere identified with the Bio-Rad ChemiDoc XRS� imaging system (noillumination, no filter, autoexposure), and K1-positive bioluminescentcolonies were subjected to two rounds of subculture on MH agar contain-ing kanamycin prior to storage under glycerol at �80°C.

Growth kinetics (optical density at 600 nm [OD600]) and biolumines-cence of conjugants were determined in MH broth; photon emission dur-ing the growth cycle (expressed as relative luminescence units) was mon-itored with a PerkinElmer LS-55 fluorescence spectrometer. The stabilityof the mini-Tn5 element within conjugants was determined by subcultureevery 24 h in MH broth in the presence and absence of kanamycin; sub-cultures were enumerated by viability counting, and bioluminescent col-onies were identified with the ChemiDoc XRS� imaging system. To de-termine luxCDABEKm2 insertion sites in E. coli A912PP derivatives,

strains were sequenced with the Illumina MiSeq platform as describedpreviously (32) and compared with the E. coli A192PP sequence (Euro-pean Nucleotide Archive [http://www.ebi.ac.uk/ena/] accession numberPRJEB9141). The conjugant E. coli A192PP-lux2 was selected for furtherinvestigation. The E. coli A192 and A192PP-lux2 genomes were assembledusing Velvet assembler (33) and aligned with the luxCDABE operonwithin the Tn-lux sequence. To confirm the insertion site of the Tn-luxelement, A192PP-lux2 paired sequence reads were mapped onto theA192PP assembled genome. The insertion site then was confirmed byPCR using primers for the amplification of A192PP-lux2-traL (forwardprimer, 5=-TATATCGTCGGCCATGAATCC-3=; reverse primer, 5=-AACCTCACTCCCTTTTTGCT-3=) and primers for the amplification of theluxC-traL junction (forward primer, 5=-CGTATCCTCCAAGCCTGAATT-3=; reverse primer, 5=-TGAAGCGGTAGAAGTTGCCAA-3=), produc-ing fragments of 597 bp and 419 bp, respectively. PCRs (50 �l) contained25 �l Promega master mix, 1 �l (10 �M) of each primer, and 1 �l genomicDNA as the template. Amplification was carried out under the followingconditions: 1 cycle at 95°C for 5 min; 35 cycles of 95°C for 1 min, 60°C for1 min, and 72°C for 2 min; and a final extension of one cycle of 72°C for10 min.

Experimental systemic infection of neonatal rats. Animal experi-ments were approved by the Ethical Committee of the UCL School ofPharmacy and the UK Home Office (HO) and were conducted under HOProject 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 (HarlanUK) were fed 20 �l of mid-logarithmic-phase E. coli (2 � 106 to 6 � 106

CFU) from an Eppendorf micropipette to effect gastrointestinal (GI) col-onization. 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 orcontrol group and fed E. coli culture in identical fashion at the same timeinterval. GI tract colonization was determined by culture of perianalswabs on MacConkey agar; bacteremia was detected by MacConkey agarculture of blood taken postmortem. Disease progression was determinedby daily evaluation of symptoms of systemic infection and scored on ascale of rising severity from 0 to 3 (11). After sacrifice, samples from theesophagus, stomach, small intestine (SI), colon, blood, mesenteric lym-phatic system, liver, lung, heart, kidney, and spleen were excised asepti-cally (11). Organs were washed extensively in phosphate-buffered saline(PBS) to ensure minimal contamination with perfused blood. Bacteriawere quantified in homogenized tissues by serial dilution culture on Mac-Conkey agar and the presence of the K1 capsule confirmed as requiredwith bacteriophage K1E. Samples from experiments involving E. coliA192PP-lux2 were cultured in the presence of 50 �g/ml kanamycin.

2DBLI and DLIT-�CT. 2DBLI was performed on tissues harvestedfrom P2 and P9 rat pups colonized by A192PP-lux2; tissues were collected24, 48, and 72 h after feeding of the colonizing bacteria, and the severity ofinfection was recorded. Tissues were washed in sterile PBS, placed in ster-ile petri dishes on a black card, and pierced with sterile 25-guage needlesfor aeration (34). Bioluminescence was measured within standardizedregions of interest (ROI), and data are expressed as flux (photons persecond), adjusted using the following formula to ensure all measurementshad positive value: log10(flux �1). Tissues from noncolonized pups were

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

Bacterial strain or plasmid Description Reference or source

StrainsC. rodentium ICC180 Luminescent derivative of C. rodentium; Nalr Kmr 28E. coli S17-1 �pir Pir� maintenance and donor strain; Ampr Kmr 27E. coli A192PP Serially passaged (A192) strain 15E. coli A192PP-lux2 Luminescent A192PP derivative; Kmr This study

PlasmidpUTmini-Tn5luxCDABEKm2 Suicide vector, with unpromoted luxCDABE transposon; Ampr Kmr 26

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used for bioluminescent background subtraction. Quantitative imagingwas 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 Imagesoftware 4.3.1 wizard and autoexposure settings specific for imaging bac-terial luciferases (maximum exposure, 300 s; target count minimum,10,000) were used (25). Anesthesia (5% isoflurane, followed by mainte-nance under 2.5% isoflurane) on the prewarmed imaging platform wasused in both 2DBLI and DLIT-�CT experiments. Symptoms of infectionwere recorded immediately prior to the collection of images and the pupsculled for ex vivo tissue analysis; animals were not subjected to repeatedanesthesia. 3D animations were created using Living Image, as previouslydescribed (25), and compiled into a movie using iMovie software (version10.0.5). Noncolonized animals were used for bioluminescent backgroundsubtraction. To correlate flux (photons per second) with CFU, serial di-lutions in PBS from 16-h cultures of E. coli A192PP-lux were prepared in96-well black plates, and wells were highlighted as ROIs prior to imagingin the IVIS Lumina series III and IVIS SpectrumCT. Flux within ROIs wasmeasured and CFU from each well determined retrospectively by platingon MH agar with kanamycin.

Histology and microscopy. Esophageal tissues were collected andfixed in 10% neutral buffered formalin, embedded in paraffin, and pro-cessed, and 5-�m sections were obtained, mounted onto slides, andstained with hematoxylin and eosin (H&E). Unstained sections were pre-pared for immunohistochemistry: they were dewaxed in HistoClear andexamined for E. coli O18 lipopolysaccharide antigen as previously de-scribed (16, 35) and mounted in VectaShield mounting medium contain-ing 4=,6=-diamidino-2-phenylindole (DAPI) stain (H-1200). Samples forscanning electron microscopy (SEM) were processed and examined aspreviously described (36) using a JEOL JSM-5300 scanning electron mi-croscope.

Nucleotide sequence accession number. Nucleotide sequence ofthe A192PP-lux2 genome has been deposited in the European Nucle-otide Archive (http://www.ebi.ac.uk/ena/) under accession numberPRJEB9940.

RESULTSGeneration and characterization of E. coli A192PP-lux. Thirty-one bioluminescent derivatives of E. coli A192PP were obtained bymini-Tn5 mutagenesis. One, designated E. coli A192PP-lux2, ex-pressed the K1 capsule, grew in MH broth (Fig. 1A) and LB min-imal medium (see Fig. S1 in the supplemental material) to anextent comparable to that of the parental A192PP strain, and pro-duced a strong bioluminescent signal over the course of thegrowth cycle that correlated with CFU (Fig. 1B and C), indicativeof constitutive expression of the lux operon (37). The strength ofthe bioluminescent signal from A192PP-lux2 exceeded that fromC. rodentium ICC180 and was maintained for 7 days in the absenceof kanamycin (data not shown), indicative of stable genomicmaintenance of the lux operon. Whole-genome sequencing of E.coli A192PP-lux2 revealed that the Tn5-lux element inserted intotraL, encoding an F-pilus assembly protein (38, 39). The disrup-tion of traL and the presence of the luxC-traL junction in A192PP-lux2 was confirmed by PCR (see Fig. S2); pili were not evident inSEM images of either A192PP or A192PP-lux2 (see Fig. S1B). Thecapacity of E. coli A192PP-lux2 to elicit lethal infection after GIcolonization in P2 and P9 neonatal rats was determined. The bio-luminescent derivative colonized the GI tract of P2 and P9 pups tothe same extent as the parental E. coli A192PP, with stable coloni-zation occurring within 24 and 48 h of feeding of the bacteria (Fig.1D); 79.17% (19/24) of P2 animals developed bacteremia over the7-day observation period and around 20% survived, whereas all

receiving the parent strain were bacteremic (Fig. 1E), with no sur-vivors (Fig. 1F). No P9 pups receiving either E. coli A192PP orA192PP-lux2 succumbed to bacteremia or lethal infection in spiteof efficient GI colonization. Thus, the stable insertion of the luxtransposon and kanamycin resistance cassette into traL had only aminor impact on virulence. Therefore, E. coli A192PP-lux2 wasemployed for all imaging experiments.

Distribution of E. coli A192PP-lux2 in colonized P2 and P9rat 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 thebacteria become widely disseminated as the infection progresses(16). To determine if carriage of the lux transposon and kanamy-cin resistance genes altered the in vivo distribution of E. coliA192PP and to signpost the design of whole-animal and organimaging experiments, we determined the organ tropism and or-gan load of E. coli A192PP and A192PP-lux2 in P2 and P9 neonatalrats after the initiation of colonization. A population of colonizingE. coli A192PP was evident in the small intestine 24 h (the initialtime point for viability determination) after initiation of coloni-zation at P2 and varied little in quantitative terms over the first 72h of colonization; there were no significant differences in numbersof colonizing bacteria between E. coli A192PP and A192PP-lux2during this period (Fig. 2A). Small numbers of bacteria were pres-ent in the mesentery after 24 h (Fig. 2B) and began to appear in theblood circulation at the same time point (Fig. 2C). E. coli K1 wererecovered from brain tissue in a variable proportion of infected P2animals, and the numbers encountered were low (Fig. 2D). Thereagain were no significant differences between E. coli A192PP andA192PP-lux2 at any given time point. Other regions of the GI tract(stomach and colon) also were stably colonized by both biolumi-nescent strain and parent strain to a similar extent, although anoticeable but insignificant decrease in A192PP-lux2 load presentin the stomach at 24 h after initiation of colonization was observed(see Fig. S3 in the supplemental material). Low to moderate num-bers of E. coli A192PP and A192PP-lux2 were cultured from liver,lung, heart, kidney, spleen, and pancreas; no significant differ-ences in the size of the bioburden between the two strains wereevident when they were compared by time after initiation of col-onization or by severity of symptoms of infection (see Fig. S3 andS4). The bacterial load of both strains in the major organs in-creased as the health status of the animals declined (see Fig. S4).We conclude that the introduction of bioluminescence- and ka-namycin-associated genes into E. coli A192PP has little, if any,impact on organ tropism and tissue bioburden in this widely dis-seminated infection of susceptible P2 neonatal rats. With resistantP9 pups, E. coli A192PP and A192PP-lux2 were recovered fromthe stomach, small intestine, and colon in numbers comparable tothose found in P2 animals; only very low numbers were found inthe mesentery, and other tissues were free of E. coli K1 (data notshown).

Patterns of E. coli A192PP-lux2 infection determined by bio-luminescence imaging. 2DBLI of live P2 rats revealed a significantincrease in total bioluminescence between 24 h and 48 h afterfeeding of E. coli A192PP-lux2 (P 0.05), coincident with theonset of signs of infection. Representative images are shown in Fig.3. Colonizing bacteria were not uniformly distributed along thesmall intestine. Typically, there was rapid dissemination of thepathogen from the site of colonization between these two timepoints and an apparent reduction in both flux and degree of dis-

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semination at 72 h. However, in individual animals the infectionprogresses at different rates (16), with a proportion succumbingto infection within 3 days after seeding of E. coli K1, and survivorsat 72 h are likely to be animals in which infection progressed at arelatively low rate compared to that of nonsurvivors. The ap-parent peak of infection at 48 h was reflected in photon emis-sion from excised major organs with the exception of the GItract tissues, indicative of stable GI colonization (Fig. 3).

Therefore, we examined the data relative to the severity ofsymptoms of infection (Fig. 4).

In live P2 animals, the E. coli K1 burden increased significantlyas symptoms of infection became evident, indicating that al-though the total burden increased with time, it correlated moreclosely with the severity of infection. The degree of colonization ofthe stomach, small intestine, and mesentery increased with diseaseseverity, and invasion of the central nervous system occurred only

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 orbitsper 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 ratpups are shown.

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in animals displaying severe symptoms of infection. The associa-tion of E. coli K1 with liver, spleen, pancreas, heart, and kidneywere similarly associated with late stages of infection. In contrast,the distribution of E. coli A192PP-lux2 in colonized P9 pups wasrestricted to the GI tract (see Fig. S5 in the supplemental material).The bioburden in live P9 animals did not increase significantlybetween 24 h and 72 h, and few bacteria were visualized in themesentery. Niches rich in colonizing bacteria were evident in thesmall intestine, colon, and mesentery in both P2 and P9 pups.Overall, the distribution of E. coli K1 determined by 2DBLI wasconsistent with data obtained by viability determination (Fig. 2;also see Fig. S4).

DLIT-�CT was used to investigate the relationship betweenthe development of symptoms of disease and the whole-animaldistribution of E. coli A192PP-lux2 in P2 animals over a 72-hperiod following the initiation of colonization (see Video S1 in thesupplemental material). As anesthesia modified the progression ofdisease and increased the risk of rejection by the mother, longitu-dinal monitoring of individual pups was not performed. As deter-mined earlier in the study, the rate of disease progression differedsignificantly between individual animals, and clearer representa-tions of disease development were obtained when comparisonswere made on the basis of severity of symptoms rather than ontime from colonization. The involvement of multiple organs canbe seen in cases of severe infection (score of three or more), and itis noteworthy that the infection of the central nervous system(CNS) was restricted to the surface of the brain, lending support tothe view that the choroid plexus rather than the brain microvas-cular endothelium represents the portal of entry into the CNS inexperimental E. coli K1 meningitis. The authenticity of the DLIT-

�CT reconstruction was confirmed by 2DBLI of the correspond-ing pups and their organs (see Fig. S6). DLIT-�CT revealed thatthe initial bolus of E. coli K1 entered the colon within 3 h of oralapplication of the inoculum, seeding the entirety of the alimentarytract, including the oral cavity, esophagus, stomach, and smallintestine (see Video S2 and Fig. S7). Frequent colonization of theoral cavity was observed, which presented as photon emissionfrom the head region (see Video S3), and was evident over theentire 72-h observation period. Stable oral cavity colonization wasconfirmed by 2DBLI of excised elements of this region and re-vealed an intense colonization of tongue tissue (Fig. 5). Duringlater stages of disease progression, photon emission from multiplelymph 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 sitefor entry and dissemination in systemic E. coli K1 experimentalinfection (Fig. 7A; also see Fig. S7 in the supplemental material).Photon emission from the head region did not originate from thetrachea (data not shown). The determination of photon emissionby the bioluminescent derivative (Fig. 7B) and CFU of E. coliA192PP and A192PP-lux2 (Fig. 7C) in excised esophagi showedP2 rats to be significantly more susceptible to colonization at thissite than P9 animals. No significant quantitative differences incolonization capacity between the two strains were apparent (Fig.7C), and no difference between E. coli A192PP and A192PP-lux2with respect to the severity of symptoms was found (data notshown). H&E staining of the esophagus revealed the presence of akeratinized epithelium at P9; this layer was absent from or only

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 oralapplication 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.

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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 persecond. Images were collected from live animals at the time points indicated; they then were sacrificed, organs were collected, and images were obtainedimmediately. Representative images of whole animals and excised organs are shown. Fewer data points at later time intervals reflect decreases in survival overtime. Data represent means SD (one-way analysis of variance [ANOVA] with Tukey’s multiple-comparison test; *, P 0.05).

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partially developed in the esophagus of susceptible P2 neonatalrats (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 P2were probed for the presence of the O18 antigen; bacterial attach-ment to keratinized and nonkeratinized esophageal surfaces wasevident at 24, 48, and 72 h following bacterial feeding (Fig. 8B);

018 antigen was not detected in tissues from noncolonized ani-mals. Bacteria also were observed attached to sloughing keratinstrands present in the lumen of the esophagus. Additionally, at 48h, peripheral invasion and damage to regions of nonkeratinizedesophageal epithelium by E. coli K1 was evident (Fig. 8B), andfurther evidence of invasion of nonkeratinized regions also wasobserved by SEM (Fig. 8C).

DISCUSSION

The isolation of E. coli K1 from the cerebrospinal fluid of sicknewborn infants frequently coincides with the presence of thebacteria in the feces of both infant and mother (4–6), providing astrong indication that the maternal GI tract represents the pri-mary reservoir of infection for these neonatal pathogens, whichare associated with the infant gut prior to systemic invasion. Oraladministration of E. coli K1 to susceptible neonatal rats clearlyshows that colonization of the GI tract precedes invasion (13–15).The site of translocation and the molecular processes involvedhave not been defined with any precision, although evidence isemerging that the colonization of the small intestine is essentialfor systemic infection (21). In the current study, DLIT-�CT im-aging showed that the colonizing E. coli K1 bolus reached thecolon within 3 h of administration, and 2DBLI revealed regions ofintense colonization in both the small intestine and colon of P2and P9 pups and, unexpectedly, that bacteria were seeded alongthe entire length of the alimentary tract. Thus, the oral cavity,esophagus, and stomach of P2 animals were usually heavily colo-nized and may represent additional reservoirs of infection in thismodel. Small numbers of E. coli K1 were found in the mesentericlymphatic system at an early stage of the infection cycle, support-ing evidence (14) that invasion of blood circulation occurs by thisroute. The presence of E. coli K1 bacteria in some of the majororgans, particularly in pups with symptoms of systemic infection,may represent end-stage invasion as immune defenses are com-promised. However, with highly perfused organs the bacterial vi-ability counts are very likely to include bacteria present in blood aswell as in tissue; the organs of newborn rats are fragile and will notwithstand ex vivo perfusion for blood removal (P. W. Taylor, un-published observations).

Colonization of the GI tract by E. coli K1 at P2 dysregulates thematuration of the mucus barrier, which is poorly formed in new-born pups; mucosal barrier function at this age is insufficient toprevent translocation of E. coli K1 from gut lumen to blood circu-lation (21). An integral barrier has formed by P9 (21), preventingsystemic invasion and accounting for the restricted distribution ofthe bacteria following colonization of P9 animals observed in thecurrent study. We also found an unexpected age-dependent, sta-ble colonization of the esophagus and obtained evidence that, inP2 pups, E. coli K1 may invade nonkeratinized esophageal tissue,raising the possibility that the esophagus represents an additional lo-cus 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 aslog10(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 timeintervals 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) basedon color of the skin, agility (righting reflex), response to gentle pressure on the abdomen, visibility of stomach/milk line, temperature, weight gain, and behaviorwhen placed in a cage. Animals were culled immediately when three or more symptoms became evident, and the animals were recorded as dead. Data representmeans SD (one-way ANOVA with Tukey’s multiple-comparison test; *, P 0.05; **, P 0.01; ***, P 0.001).

FIG 5 Colonization of the oral cavity of P2 neonatal rats by E. coli A192PP-lux2. (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-�CTreveal intense oral cavity colonization with foci associated with tongue tissue.Images were collected from live animals; they then were sacrificed, organs werecollected, and images were obtained immediately. Representative images ofwhole animals and excised organs are shown.

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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 CFUbacteria, revealed by 2DBLI and DLIT-�CT. Arrows indicate excised tissues. Images were collected from live animals; they were then sacrificed, and organs werecollected and images obtained immediately. Representative images of whole animals and excised organs are shown; different animals were used to generate eachimage.

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site was significantly increased in susceptible P2 rats compared to thatfrom resistant P9 animals over the 3 days following initiation of col-onization and the strong age dependency confirmed by enumerationof viable E. coli K1 within esophageal tissue.

H&E staining revealed the age-dependent development of akeratinized esophageal layer, appearing as early as 2 days postpar-tum; age-related keratinization also has been documented in themouse but appears to proceed more slowly compared to that ofthe rat as determined in the current study (40). Esophageal kera-tinization provides protection against the consumption of coarsefoods (41), although the human esophageal epithelium is not nor-mally keratinized to any extent (42, 43) and, like the rodent esoph-agus, does not develop a viscoelastic protective mucus barrier(44). E. coli K1 attached to all regions of the P2 esophagus, includ-ing keratinized and nonkeratinized surfaces. As binding is inde-

pendent of the keratin layer, the presence of keratin cannot ac-count for differences in esophageal binding between P2 and P9pups. However, invading E. coli K1 cells were found in nonkera-tinized regions of the P2 esophagus, suggesting that the incom-plete keratinization of this site predisposes the underlying epithe-lium to invasion and may enable the bacteria to persist; thiscontention is supported by SEM imaging of P2 esophageal tissue.Full-term human neonates exhibit reduced esophageal motilityand luminal clearance compared to that of adults, which is furtherdecreased in preterm neonates (45–47), suggesting that the neo-natal esophagus is vulnerable to colonization and invasion by neu-ropathogens in the human host. Thus, reduced esophageal peri-stalsis, an exposed epithelial layer lacking keratin, and a mucosalbarrier may increase the risk of pathogen attachment, overgrowth,and invasion at this site.

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 andesophageal 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).

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

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

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. Damageand 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) SEMimages of esophagi from pups fed E. coli A192PP at P2; images obtained 24 h later reveal evidence of peripheral epithelial invasion.

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tongue can be used for diagnostic swabbing of neonates to detectK1 colonization prior to the onset of sepsis. Although a strongcorrelation between systemic E. coli K1 infection and GI tract col-onization has been reported frequently in clinical studies of neo-natal infection (3, 4, 6), there are indications that in some cases ofbacterial meningitis, the aspiration of the infecting dose into thelungs and other regions of the respiratory tract provides opportu-nities for an alternative portal of entry into the systemic circula-tion (50). This is unlikely to be a significant factor in the develop-ment of infection in the neonatal rat model, as E. coli A192PPappears in the lung at later time points and only in pups displayingsevere symptoms of infection.

Soon after the initiation of colonization, susceptible pups de-velop bacteremia before succumbing to overwhelming infectioninvolving the major organs. We found E. coli K1 in the majorgroups of lymph nodes, suggesting that disseminated infectionarises due to failure of the lymphatic system to control localizedfoci of bacteria. E. coli K1 is strongly associated with sepsis andmeningitis (1–3), and we found significant associations betweenthe severity of infection and the recovery of viable bacteria frombrain tissue. In a previous study (16), we visualized E. coli K1 inbrain sections using a modified Gram stain and found bacteriaassociated only with the surfaces of this organ. The current studylends further support to this pattern of distribution: DLIT-�CTimaging (see Video S1 in the supplemental material) indicatedthat E. coli A192PP-lux2 cells were restricted to superficial layerson the surface of the brain, much as in the human condition (51),and supports previous observations (16) that E. coli K1 gains ac-cess to the CNS through the choroid plexus, a component of theblood-CSF barrier. Transit from the blood circulation throughthis epithelial barrier to the CSF would present the invading bac-teria with the opportunity to adhere to the most superficial layer ofthe brain, the leptomeninges, but restrict access to cortical tissue.However, it is widely accepted that E. coli K1 invades the CNS bytraversal of the endothelium (BBB) (23, 52), even though the ev-idence supporting this route of entry is modest (22). As the mam-malian brain accounts for only 2% of body mass yet receives 20%of the cardiac output, it is a highly vascularized organ; the humanbrain contains an estimated one hundred billion vessels, one foreach neuron (53). If circulating neuropathogens penetrated theBBB to any extent at multiple sites within this vascular network,one would expect to encounter the bacteria throughout the brainin both naturally occurring and experimental E. coli K1 meningi-tis, but this is not the case. The choroid plexus provides a muchlower resistance to transport into the CNS than the BBB (54) andis a strong candidate for portal of entry. The resolution of theimportant issues of colonization and tissue penetration may opennew avenues for resolving lethal infections of the CNS.

ACKNOWLEDGMENTS

This study was supported by project grant GN2075 from Action MedicalResearch. Further support was provided by the National Institute forHealth Research University College London Hospitals Biomedical Re-search Centre.

We thank Richard Stabler, London School of Hygiene and TropicalMedicine, for generating the sequence reads. We thank Thilo M. Fuchs,Technische Universitat Munchen, for kindly providing the Tn-lux se-quence.

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