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. 2011 Jan 1;2(1):34–41. doi: 10.4161/gmic.2.1.14882

Determination of spatial and temporal colonization of enteropathogenic E. coli and enterohemorrhagic E. coli in mice using bioluminescent in vivo imaging

Ki-Jong Rhee 1, Hao Cheng 1, Antoneicka Harris 1, Cara Morin 2, James B Kaper 2, Gail A Hecht 1,3,✉
PMCID: PMC3225795  PMID: 21637016

Abstract

Infectious diarrhea is a major contributor of child morbidity and mortality in developing nations. Murine models to study the pathogenesis of infectious diarrhea caused by organisms such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are not fully characterized. More emphasis has been placed on infection of mice with the murine specific pathogen Citrobacter rodentium. While these three organisms are genetically related they are not identical. Our goal was to better characterize the murine model of EPEC and EHEC infection by using bioluminescent bacteria to determine temporal and spatial colonization of these two human pathogens. EPEC and EHEC were transformed with a bacterial luciferase expression plasmid containing the constitutive OmpC promoter. C57BL/6 mice were orally inoculated with bioluminescent EPEC or EHEC and bacterial localization in the intestine was monitored ex vivo and in vivo by IVIS. At 3 days after infection, EPEC, EHEC and Citrobacter rodentium were all localized in the cecum and colon. EPEC colonization peaked at day 2–3 and was undetectable by day 7. The bioluminescent EPEC adheres to the cecum and colon of the mouse intestine. However, when EPEC infected mice were administered xylazine/ketamine for in vivo live imaging, the EPEC persisted at high densities for up to 31 days. This is the first report of a bioluminescent imaging of luciferase expressing EPEC in a mouse model.

Key words: EPEC, EHEC, IVIS, bioluminescent imaging, luciferase, animal model

Background

EPEC and EHEC contribute significantly to diarrheal disease especially in children of developing nations.1 Initially, EPEC/EHEC adhere to the host intestinal epithelium and then induce attaching and effacing (A/E) lesions which are characterized by intimate bacterial attachment to the apical membrane of the intestinal epithelial cells and effacement of the enterocyte microvilli. The genes that confer the A/E lesion phenotype are encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). These genes include the type 3 secretion system (T3SS) apparatus and other T3SS effector proteins. A/E pathogens use the T3SS to translocate bacterial effector proteins into the host epithelial cells to modulate host responses. In studies using in vitro cell cultures, Mills et al. found that translocation of EPEC T3SS effectors into the epithelial cell occurred in a hierarchical manner.2 During an in vivo infection, the expression of EPEC T3SS effector genes most likely follow a programmed expression pattern that is tailored to the timing and localization of infection. However, the temporal and spatial expression pattern of virulence genes during an in vivo infection by EPEC is unknown.

Bioluminescence imaging (BLI) has rapidly progressed in the field of bacterial pathogenesis to allow visualization and quantization of host-pathogen interactions in live animals. Bioluminescence is an enzymatic process by which the enzyme luciferase produces visible light in the presence of a specific substrate, oxygen and an energy source (ATP, FMNH2).3 The luciferase operon, luxCDABE, from the bacterium Photorhabdus luminescens encodes its own luciferase substrate. Cloning of promoters of various bacterial virulence genes upstream of the luxCDABE operon and introducing this reporter construct into a bacterial pathogen allows one to investigate the spatial and temporal expression of these virulence genes after infection of a susceptible host.4 Several enteric pathogens have been transformed with luxCDABE and their localization or gene expression examined in vivo. These include Salmonella typhimurium,5 Listeria monocytogenes,6 Yersinia enterocolitica,7,8 uropathogenic E. coli,9 EHEC10,11 Vibrio cholerae12 and Citrobacter rodentium.13 There is only one report of an EPEC strain expressing the luxCDABE operon.14 In this study, the luxCDABE was fused with a gene promoter to assess transcriptional regulation in vitro. To our knowledge there are no reports describing spatial and temporal colonization of bioluminescent EPEC in vivo.

The pathogenesis of EPEC/EHEC has been studied in various animal models. Mice have been used to study EHEC infection and more recently our laboratory reported a murine model of EPEC.15 Mice pre-treated with streptomycin are consistently susceptible to EPEC infection.15 Using this model we showed previously that EPEC colonizes the mouse large intestine, induces mild mucosal inflammation and then is eventually cleared by the host within 6–8 days.15 Most mouse research on A/E pathogens has been performed using the A/E pathogen, Citrobacter rodentium. C. rodentium is a murine specific pathogen and exhibits many characteristics of EPEC/EHEC, including the A/E phenotype. However, C. rodentium is genetically similar but not identical to EPEC. For example, C. rodentium does not possess the bundle forming pili (BFP) or flagella both of which are implicated as important virulence factors in human infections. In addition, C. rodentium induces mucosal hyperplasia which is not observed in human infections. Therefore, studying the pathogenesis of EPEC/EHEC in a mouse model may provide further insights into the mechanism of EPEC/EHEC pathogenesis in humans. In this report, we generated bioluminescent EPEC, EHEC and C. rodentium by transforming a newly developed luciferase plasmid, pCM17. We then infected mice to determine the spatial and temporal localization of EPEC.

Results

Generation of bioluminescent EPEC and EHEC.

The luciferase encoding pCM17 vector containing the OmpC promoter upstream of the luxCDABE operon was transformed into EPEC or EHEC and transformants selected by antibiotic resistance. Image of bioluminescent EPEC (Fig. 1A) and EHEC (Fig. 1B) are shown. The bioluminescence signal was proportionate to the bacterial CFU for a broad range (approximately 1 × 105 CFU to 1 × 109 CFU). The intestinal lumen of mice infected with the bioluminescent bacteria is devoid of kanamycin and therefore the pCM17 vector could be lost over the course of infection. In the streptomycin pre-treatment model of infection, EPEC is cleared by the host within 6–8 days. Therefore, we tested whether the pCM17 vector is retained in the transformants for up to 7 days. Plasmid stability was determined by the serial passaging of bacteria in LB broth in the absence of antibiotics and subsequent culturing on antibiotic-free LB agar plates. The ensuing bacterial colonies were imaged by IVIS and the percentage of bioluminescent colonies determined. The pCM17-EPEC exhibited 100% plasmid retention throughout the 7 day period. pCM17-EHEC was less stable in vitro with 95% plasmid retention at day 2 and 84% retention at day 7 (Fig. 2). These results show that both EPEC and EHEC can be transformed with pCM17 and exhibit bioluminescence with little or no loss of plasmids for up to 7 days.

Figure 1.

Figure 1

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Quantitation of bioluminescent EPEC and EHEC. LB agar plates showing EPEC (A) and EHEC (A) colonies before and after transformation with pCM17 (upper parts). pCM17 transformed EPEC (A) and EHEC (B) were serially diluted in 96-well plates and bioluminescence quantitated using IVIS and then correlated with bacterial numbers (lower parts).

Figure 2.

Figure 2

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Stability of bioluminescence in vitro. pCM17 transformed EPEC (left part) or EHEC (right part) were serially passaged at 1:100 dilution every day for 7 days in antibiotic-free LB broth and the percentage of bioluminescent colonies shown. Average of three experiments. Error bar = SEM.

To determine if pCM17 can be transformed into A/E pathogens, pCM17 was electroporated into EPEC, EHEC and the murine specific pathogen C. rodentium. We found that all three strains emitted bioluminescence when cultured on LB plates. When we infected mice with each bioluminescent strain, they all resided predominantly in the cecum and colon as described previously for EHEC and C. rodentium (Fig. 3).13 Next, we determined localization of bioluminescent EPEC immediately after oral infection. After EPEC infection for 5, 30, 60 and 180 min, the intestines were removed and imaged ex vivo (Fig. 4). We found that it took approximately 90 min for EPEC to reach the cecum/colon. By 3 hours post-infection, the EPEC localized throughout the cecum and colon.

Figure 3.

Figure 3

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Localization of bioluminescent EPEC, EHEC and C. rodentium. Streptomycin pre-treated mice were gavaged with pCM17-EPEC, -EHEC or -C. rodentium (1 × 109 CFU/200 µl) and the intestines resected at day 3. The ends of the intestines were tied and injected with air and imaged by IVIS. Representative of 5∼10 mice per group.

Figure 4.

Figure 4

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Transit of bioluminescent EPEC through the mouse intestine. Streptomycin pre-treated mice were gavaged with pCM17-EPEC (1 × 109 CFU/200 µl) and the intestines resected after 5, 30, 90 and 180 min. The ends of the intestines were tied and air injected into the intestinal lumen to maximize bioluminescence.

Optimal bioluminescence in intestinal tissue requires exposure to air.

Luciferase is an enzyme that requires the presence of oxygen to induce bioluminescence. The intestinal lumen contains a high number of facultative anaerobes which rapidly deplete the oxygen levels in the gut lumen. This would in turn diminish the bioluminescence signal. We therefore determined whether introduction of air using a syringe would enhance the bioluminescence signal. Mice were infected with pCM17-EPEC and after 3 days the entire intestine (duodenum-rectum) was excised as a single piece and immediately imaged by IVIS (Fig. 5A). EPEC was located exclusively in the cecum and colon with the highest signal being emitted from the cecum. However, when the ends of the intestine were tied and air injected into the intestinal lumen, the bioluminescence signal immediately increased approximately 5-fold (Fig. 5B). There was no detectable bioluminescence in the small intestine before or after the air injection consistent with reports that the mouse small intestine is not a major site of colonization by EPEC, EHEC or C. rodentium. These data indicate that oxygen is limited in the lumen of freshly excised intestine. Aeration increased the bioluminescence signal by 5-fold. Therefore, air was injected into the intestinal lumen prior to imaging by IVIS in all subsequent experiments.

Figure 5.

Figure 5

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Enhancement of bioluminescence with air injection. Streptomycin pre-treated mice were gavaged with pCM17-EPEC (1 × 109 CFU/200 µl) and the intestines resected after 3 days. The ends of the intestines were left alone (A, left) or tied and injected with air (A, right) and immediately imaged by IVIS. A summary of the bioluminescence quantification is shown in (B). n = 10 mice. Bar = median. p < 0.05 by Mann-Whitney analysis.

Spatial and temporal colonization of bioluminescent EPEC in the C57BL/6 mouse model. To determine the spatial and temporal colonization of EPEC in the C57BL/6 mouse model, mice were infected with pCM17-EPEC and the intestines were excised, injected with air and imaged by IVIS every day for 7 days. At day 1, EPEC colonized the cecum most heavily and progressively declined in number such that by day 7 no detectable EPEC remained in the intestinal lumen (Fig. 6A and C). The bacterial colonization as determined by stool culture showed a similar pattern but differed at day 1 post-infection (Fig. 6E). The numbers of bacteria at 1 day post-infection was variable in the stool but were consistently high in the cecum when analyzed by IVIS. To determine whether bioluminescent EPEC were adherent or free-floating in the intestinal lumen, the number of adherent EPEC was quantitated by washing vigorously the intestine with PBS and imaging by IVIS. The bioluminescence after washing was approximately 1% of the signal obtained from intact intestines suggesting that only a small portion of pCM17-EPEC is adherent (Fig. 6B and D). All EPEC colonies recovered from the stool of these mice were bioluminescent demonstrating pCM17 transformed EPEC are stable in vivo (data not shown).

Figure 6.

Figure 6

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Spatial and temporal colonization of bioluminescent EPEC. Streptomycin pre-treated mice were gavaged with pCM17-EPEC (1 × 109 CFU/200 µl) and the intestines resected at days 1–7 (Ce, cecum; Co, colon, Si, small intestine). The ends of the intestines were tied and injected with air. Representative images of intestines from infected mice (A). Red indicates stronger signal and blue indicates weaker signal. Representative images of intestines from infected mice after extensive wash with PBS (B). Total CFU per mouse as determined by the total bioluminescence emitted from the intestine using the formula from Figure 1A (C). Each dot represents one mouse. Bar, median. Total attached CFU per mouse as determined by the total bioluminescence emitted from the intestine after washing with PBS (D). Bacterial colonization (CFU/g stool) in freshly voided stool pellets (E).

We demonstrated previously that streptomycin pre-treated C57BL/6 mice infected with EPEC exhibit mild colonic inflammation and transient elevation of serum KC.15 We measured serum KC levels after EPEC infection and found that the serum KC was elevated only at 1 day post-infection (Fig. 7), similar to previous findings.

Figure 7.

Figure 7

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Serum KC levels in pCM17-EPEC infected mice. Streptomycin pre-treated mice were gavaged with pCM17-EPEC (1 × 109 CFU/200 µl) and the serum KC measured by ELISA . Each dot represents one mouse. Bar, median. p < 0.05 using the Mann-Whitney test.

In vivo BLI of pCM17-EPEC in the C57BL/6 mouse model.

A major advantage of the IVIS technology is that it allows for the serial assessment of live animals. The imaging of EPEC in a live mouse has never been conducted. Mice were infected with pCM17-EPEC and in vivo imaging of mice was conducted. In contrast to the colonization period of 7 days typically observed for EPEC infection, EPEC colonization was maintained at high levels (1 × 107 ∼ 1 × 108 CFU/mouse) for up to a month. (Fig. 8)

Figure 8.

Figure 8

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Bioluminescent imaging of pCM17-EPEC in vivo. Streptomycin pre-treated mice were gavaged with pCM17-EPEC (1 × 109 CFU/200 µl) and in vivo imaging conducted for up to 31 days. Shown are images of infected mice at day 0, 11, 21, 31 (A). Total CFU per mouse as determined by the total bioluminescence emitted from the intestine using the formula from Figure 1A. Each dot represents one mouse. n = 5. Bar, median.

Discussion

EPEC/EHEC express a multitude of genes that contribute to pathogenesis. The roles of many EPEC/EHEC genes have been investigated using bacterial infection of cultured cells. However, the study of EPEC/EHEC virulence genes in vivo has been limited. BLI provides an opportunity to visualize and quantitate bacterial gene expression in vivo. Among the A/E pathogens, both bioluminescent EHEC11 and bioluminescent C. rodentium have been generated and studied in vivo.13 However, there is no report regarding bioluminescent EPEC infection in a mouse model. Our laboratory has previously optimized an approach for studying EPEC in mice.15 Pre-treatment of C57BL/6 mice with streptomycin for one day prior to oral inoculation allows for consistent EPEC colonization which peaks at day 2–3 and clears from the host by 6–8 days. EPEC induces a mild mucosal inflammation which resolves concomitantly with decline of the bacteria. This is also reflected in the serum KC levels, which are transiently elevated at 1 day post-infection but subsequently return to normal levels. Similarly, in this study, bioluminescent EPEC infected mice exhibited the same kinetics of infection and the transient increase in serum KC levels at 1 day post-infection demonstrating that bioluminescent EPEC behaves identically to non-bioluminescent EPEC in mice.

We confirmed the observation that the luxCDABE encoded luciferase requires air (oxygen) to induce bioluminescence. In a study using bioluminescent C. rodentium, Wiles et al. found that the bioluminescent signal detected during BLI imaging was absent as soon as the mice were euthanized by cervical dislocation.17 This signal returned when the lumen of the intestines were exposed to air. This observation suggests that sufficient oxygen exists in the intestinal lumen to support bioluminescence. Oxygen, supplied by the host, is likely utilized by the luminal bacteria. We found that injection of air into the intestinal lumen was necessary to achieve maximal bioluminescence.

In the current study, we found that the cecum and colon are the main sites of colonization with EPEC initially colonizing the cecum within 3 hours after inoculation. BLI of washed tissues revealed that the highest localization was in the cecum. This may be due to attachment to the cecal patch, a lymphoid organ analogous to the appendix of other mammals. It was reported using BLI studies in C. rodentium that the cecal patch was the first site of attachment during infection.13,18 This group also showed that the rectum was another site of preferential attachment in mice.17 Although we did detect a bioluminescent signal from the rectum in some mice, it was not consistently present suggesting that this may be another difference between C. rodentium and EPEC. In order to directly test if EPEC induces attaching and effacing lesions at these sites, it would be necessary to conduct transmission electron microscopy on bacterial attachment sites. The bioluminescent EPEC generated in this study would be especially helpful in locating the intestinal regions for further evaluation.

A most unexpected result came from in vivo BLI studies of EPEC. In stark contrast to the typical 7 day infection span, EPEC persisted at high numbers in mice repeatedly anesthetized for IVIS for up to one month with colonization fluctuating between 1 × 107 ∼ 1 × 108 CFU/g stool (Fig. 8). However, pCM17 was retained in only 8% of the EPEC colonies by this point in time (data not shown). Therefore, the actual number colonizing EPEC is ten times higher. The reason for this discrepancy in colonization kinetics may lie in the in vivo versus ex vivo BLI. In the ex vivo BLI studies, infected mice are euthanized by CO2 asphyxiation followed by cervical dislocation. In the in vivo BLI studies, mice are anesthetized by intraperitoneal injection of ketamine/xylazine mixture and sedation maintained by 1% isoflurane. Several studies suggest that ketamine exhibits antiinflammatory activity both in vitro and in vivo.19–22 In a recent study, it was found that a single injection of ketamine/xylazine but not isoflurane inhalation allowed successful colonization of Vibrio cholerae in a mouse model.23 We suspect that ketamine also exerts an anti-inflammatory effect and that repeated injections may have decreased the host inflammatory response to EPEC allowing it to persist. If this is true, then inhibition of the host inflammatory response is a key hurdle to a prolonged colonization of EPEC in mice.

EPEC expresses several T3SS effector proteins with known anti-inflammatory effects which support this idea. We recently demonstrated that the EPEC T3SS effector proteins, NleH1/2, exhibit anti-inflammatory activity in vitro cell lines by dampening the NFκB activation pathway.15 Mice infected with WT-EPEC exhibited a brief increase (1 day) in serum KC levels and mild colonic inflammation whereas mice infected with ΔnleH1/2 EPEC exhibited a longer period (3 days) of elevated serum KC levels and stronger colonic inflammation. These results suggest that NleH1/2 is also anti-inflammatory in vivo. Mice infected with ΔnleH1/2 EPEC also showed a decrease in colonization persistence compared to mice infected with WT-EPEC. Plasmid complementation of the ΔnleH1/2 EPEC with NleH1 or NleH2 restored the WT-EPEC phenotype. We suggest that NleH1/2 exerts an anti-inflammatory effect that dampens the host inflammatory response to allow a higher level and longer duration of colonization. In this scenario, the ketamine may dampen the inflammatory response further providing a permanent foothold for EPEC persistence. Until the mechanisms of the anti-inflammatory effects of ketamine are elucidated, usage of ketamine may require evaluation when using IVIS to study any infectious and/or inflammatory diseases in vivo.

In this report, we demonstrate that the pCM17 vector can be used to generate bioluminescent A/E pathogens. This is the first report of a bioluminescent imaging of luciferase expressing EPEC in a mouse model. This study will set the groundwork for future studies using the pCM17 vector to investigate the spatial and temporal expression of EPEC/EHEC virulence genes in vivo.

Materials and Methods

Bacterial strains.

EPEC strain E2348/69, EHEC strain O157:H7 (Stx-negative), C. rodentium (ATCC51459) were transformed by electroporation with the plasmid pCM17 containing the luxCDABE operon driven by the OmpC promoter thus allowing for constitutive expression of luciferase.12 To ensure maintenance of pCM17 in bacteria in the absence of antibiotics, the pCM17 encodes a two plasmid partitioning loci and a hok-sok post-segregational killing mechanism.9,16 Transformants were selected on Luria-Bertani (LB) agar plates containing kanamycin (50 µg/ml) and confirmed for bioluminescence by IVIS Spectrum (Xenogen Corporation, Hopkinton, MA USA). We chose the EPEC strain E2348/69 for further evaluation based on our experience with this EPEC strain in the mouse strain C57BL/6.15

In vitro plasmid stability.

pCM17-transformed EPEC/EHEC was cultured in LB broth containing kanamycin overnight. Bacteria were serially diluted and plated on antibiotic-free LB agar plates. The plates were cultured at 37°C overnight and the bacterial colonies imaged by IVIS (day 0). The retention of pCM17 was calculated by dividing the number of luciferase positive bacterial colonies by the total number of colonies. The bacterial broth from day 0 was subcultured (1:100 dilution) into fresh antibiotic-free LB broth and cultured overnight at 37°C. The bacterial broth was serially diluted and plated onto antibiotic-free LB plates and colonies imaged by IVIS (day 1). This was repeated for up to 7 days.

Standard curve of luminescence versus transformed bacteria.

pCM17-transformed EPEC/EHEC were cultured overnight in LB broth containing kanamycin (50 µg/ml) and next day subcultured (1:100) into fresh LB broth (kanamycin, 50 µg/ml) for 3 hours. Bacteria were centrifuged, washed with PBS two times, resuspended in PBS and then serially diluted in PBS. 200 µl of each dilution was added in triplicate to an optical 96-well plate (Corning, Cat#3603). The luminescence of each well was measured by IVIS Spectrum. The bacteria numbers at each dilution was calculated by culturing the bacteria on LB agar plates. Finally, the number of bacteria per well was correlated to the bioluminescence. The sum of three independent experiments was used to calculate the standard curve.

Mouse infection.

7–8 wk old male C57BL/6 mice were purchased from Jackson Laboratories and housed under specific-pathogen free conditions. Experimental protocols were approved by the University of Illinois at Chicago Animal Care and Use Committee. Mice were given streptomycin sulfate (5 g/L) in the drinking water for 24 hr and then switched to regular water for an additional 24 hr. pCM17 transformed bacteria were cultured overnight at 37°C in LB broth containing kanamycin, then diluted in 1:10 in DMEM/F-12 media (25 mM glucose, 15 mM HEPES and 0.5% mannose) for 3 hrs at 37°C. Bacteria were centrifuged and washed with sterile PBS once and the bacterial concentration adjusted to 1 × 109 CFU/200 µl in PBS for mouse oral inoculations. Bacteria were administered via oral gavage with a 22 gauge, ball-tipped feeding needle. Mock infected mice were administered 200 µl of PBS alone.

Bacteria enumeration from fecal pellets.

Freshly collected stool from pCM17-EPEC infected mice were weighed, homogenized and serially diluted in PBS and then plated on sorbitol MacConkey agar plates containing nalidixic acid (100 µg/ml). Plates were cultured at 37°C for 48 hrs. Colonies were enumerated and represented as colony forming units (CFU) per gram stool. Random colonies were screened by PCR using espF primers for confirmation of EPEC.

Bioluminescent imaging (BLI) ex vivo and in vivo.

For ex vivo BLI, mice were euthanized at different times post infection and immediately thereafter the entire intestine from the stomach to the rectum was resected, positioned on a Petri dish and then imaged by IVIS. Then the rectum and stomach were tied off and air injected into the intestine lumen using a 27 gauge needle and syringe and then immediately imaged by IVIS. To detect adherent bacteria, the small intestine, cecum and colon tissues were opened longitudinally and washed extensively with sterile PBS before imaging with IVIS. For in vivo BLI, mice were anesthetized with an intraperitoneal injection of a ketamine (100 mg/kg)/xylazine (5 mg/kg) mixture and sedation was maintained using 1% isoflurane. Quantitation of bioluminescence was conducted by using the Living Image software (version 3.1). To remove the black fur which interferes with the bioluminescence signal, Nair Hair Remover (Church & Dwight Co., Princeton, NJ) was used at the day of infection (0 dpi) and every 4th day.

Serum KC analysis.

Blood was drawn from the heart of euthanized mice using a syringe and the blood kept in a centrifuge tube at 4°C overnight. Next day the serum was separated by centrifuging for 20 min at 2,000 g and stored at −20°C until assayed. Serum KC levels were determined by using the Quantikine Mouse CXCL1/KC ELISA kit (R&D Systems) following the manufacturer's directions. Data were analyzed using the Mann-Whitney two-tailed t-test. Differences were considered significant if p < 0.05.

Acknowledgements

This work was supported in part by the Crohn's & Colitis Foundation of America RFA 1785 (to K.J.R.), Department of Veterans Affairs Merit Award (to G.H.), NIDDK grants RO1 DK50694 (to G.H.), RO1 DK058964 (to G.H.), PO1 DK067887 (to G.H.) and NIAID grant R37 AI21657 (to J.K.). We acknowledge the assistance of Dr. Roberta Franks of the RRC TPS/IVIS at the University of Illinois at Chicago.

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