IN VIVO BIO LU M I N E S C E N T T R A C K IN G O F E N T E R O T OX I G E N I C E S C H E R IC HI A C OLI(E TEC) IN MI CE W I T H N E W T O N FX 7AUTHORS
Gerardo E. Rodea 1, 2, Francisco X. Montiel-Infante1, Ariadnna Cruz-Córdova1,Zeus Saldaña-Ahuactzi1, Sara A. Ochoa1, Karina Espinosa-Mazariego1,Rigoberto Hernández-Castro3 and Juan Xicohtencatl-Cortes1* 2017
1.Laboratorio de Investigación en Bacteriología Intestinal, Hospital Infantil deMéxico Federico Gómez, Ciudad de México, Mexico
2.Instituto de Fisiología Celular, Universidad Nacional Autónoma de México,
Ciudad de México, Mexico
3.Departamento de Ecología de Agentes Patógenos

bacteria allows us to understand the role of ETEC during bacterial infection.
BLI permit the extent of pathogenic infection to be determined in real-time inliving animals, providing temporal and spatial information regarding labeledbacteria and their metabolic activities. Bioluminescence is an enzymatic
process by which the enzyme luciferase produces visible light in thepresence of a specific substrate, oxygen and an energy source. The
application of bioluminescence technology to study ETEC under In vivo
conditions may elucidate the behavior of this bacterium in thegastrointestinal tract in further detail.

INTRODUCTIONOBJECTIVEEnterotoxigenic Escherichia coli (ETEC) is a leading etiologic agent of
diarrhea worldwide, causing millions of diarrheic episodes andapproximately 120,000 deaths every year. Adhesion to the human intestinal
tract is one of the most important features of ETEC representing a crucial
step toward colonization. Unfortunately, the colonization dynamics andcolonic receptor interactions of this human pathogen remain largely
uncharacterized. Bioluminescence imaging (BLI) has rapidly progressed in
the field of bacterial pathogenesis to facilitate the visualization andquantitation of host-pathogen interactions in live animals. In vivo tracking ofThe aim of this study was to evaluate ETEC colonization by performing
bioluminescent tracking during In vivo mouse infection. We generated avector harboring the luc gene under the regulation of the dnaK genepromoter. Light emission and duration of light-emitting bacteria weredetermined in vitro, and bioluminescent ETEC colonization was studied
during In vivo and ex vivo mouse infection.

MATERIAL & METHODS
In vitro, In vivo, and Ex vivo Bioluminescence Assays
For in vitro agar media assays, 200 μL of luciferin (15 ng/μL) were spread on
LB agar plates before the bacteria were cultured. Strains harboring theluciferase reporter vector were cultured overnight (12 h) at 37°C on LB agar
media with luciferin. The light emission of the samples was captured for 2–
10 min using a Fusion FX imaging system (Vilber Lourmat; SU, Germany).
Three sets of 2- to 4-week-old BALB/c mice were intraperitoneallyadministered 200 μL (15 ng/μL) of luciferin prior to orogastric or
intraperitoneal inoculation at different times. Animals were anesthetized withketamine/acepromazine at a dosage of 0.3 IU (100-2.5mg/kg ratio) per g ofweight. Mouse gastrointestinal tracts were dissected following
euthanization. Complete gastrointestinal tract tissue was placed in 1x PBS
and washed, and tissues were immediately placed into the Fusion FX for exvivo imaging. Images of mice and petri dishes, are presented as pseudocolor
images indicating light intensity (red being the most intense and blue theleast intense). The colors are superimposed over grayscale reference
images. The signal is expressed as the total number of photons emitted per
second (photons/s). Images were captured and analyzed using a Fusion FX
imaging system (Vilber Lourmat; SU, Germany).

RESULTS
Figure 1. E. coli K-12 harboring pRMkluc generates bioluminescence.
(A–C) E. coli K-12 harboring pRMkluc. (D–F) E. coli K-12 harboring pBR322(negative control). Fig 1A and Fig1D: Captured under white light and fig1C:
pseudocolor representation of the petri dish. (Red = intense, blue = lessintense). In vitro bioluminescence assays were carried out to measurereporter gene activity prior to animal infection. The strains were cultured
overnight in petri dishes containing LB agar with luciferin substrate. E. coli K12 pBR322 (Figures 1D–F) did not emit bioluminescence, indicating thatneither E. coli K-12 nor the pBR322 vector carried elements that conferbioluminescence. In contrast, E. coli K-12 harboring the pRMkluc vector
emitted a bioluminescent signal (Figures 1A–C).
Figure 2. In vivo and Ex vivo Mouse Infections to Investigate ETEC
Bioluminescence Emission
Bioluminescence tracking of In vivo bacterial colonization. Light capture
began following gastric inoculation (Figure 2A), and the light signals in miceinoculated with ETEC FMU073332 harboring pRMkluc at this time point
confirmed bacterial inoculation (0 h) (Figures 2A, 3A). After inoculation, thebioluminescent signals displaced toward the small intestine. 48H aftergastric inoculation, the bioluminescent signals indicated bacterial passage
through the mouse intestine. The bacterial bioluminescent signals remainedin the mouse intestine after the 120 h post-inoculation (Figures 2A, 120 hand 3C), which corresponded to the bacterial shedding data. However, nosignals were recovered from the distal portion of the mouse intestine. E. coli
K-12 bioluminescent signals were localized in the cecum (Figures 2, 3).
Bioluminescent signals were captured at 0h, 48h and 120h post inoculation.
The bioluminescent signal is depicted as pseudocolor (red= intense, blue =less intense). Arrows indicate the bioluminescent signal. n=3.
Figure 3. Ex vivo bioluminescence imaging of bacterial passage throughthe mouse gastrointestinal tract.
After 120 h of E. coli infection, mouse gastrointestinal tracts were extracted
to perform ex vivo imaging. Intestinal tract dissection comprised theesophagus to the rectum. The bioluminescent signals emitted by ETECFMU073332 harboring pRMkluc were located in the proximal mouse ileumapproximately 6 cm from the cecum.

Figure 1.

Figure 2.

Mouse infected with ETEC FMU073332 pRMkluc0h

Figure 3.

48hTime of Infection (h)120hMouse infected with ETEC FMU073332 pRMkluc

CONCLUSION
BLI imaging suggests that passage of ETEC FMU073332 through theintestinal tract is slower than the movement of E. coli K-12(cf. topublication). The appearance of BLI signals may explain the results
observed in the colonization assay where ETEC FMU073332 is likely
retained in the intestine due to the presence of its adhesive structures andbiochemical characteristics that facilitate intestinal colonization. Ex vivo
imaging allowed us to locate the region of the intestine colonized by ETEC.
This study investigates the use of Fusion FX imaging system (VilberLourmat; SU, Germany) and enlightens the fact that the system is perfectly
suited for BLI imaging during in vitro, In vivo, and ex vivo assays.

0h

48h
Time of Infection (h)120h