1. E.coli Strain KVP104 Does Not Induce Apoptosis In IEC-6
Cells
Jonathan Nkangabwa 1, Brandon Bell 2, BA2, Anatoly Grishin 3, PhD2, Henri Ford 4, MD2, MHA2
George Washington University,1 Children’s Hospital Los Angeles,2 Division of Pediatric Surgery, Los Angeles, CA
Introduction:
Necrotizing Enterocolitis (NEC) is a deadly
inflammatory disorder of the gut that
predominantly affects preterm infants. It is
commonly believed that pathogenic
bacteria, such as Cronobacter muytjensii,
may colonize the intestines of a premature
infant, which can harm the gut epithelial
barrier. These bacteria activate an
inflammatory signaling cascade that leads to
enterocyte apoptosis, breach of the
epithelial barrier and further invasion by
harmful microbes. Studies have shown that
administration of beneficial bacteria
(probiotics) may counteract the effects of
pathogens and can significantly lower the
incidence and severity of NEC. Previous
work in our lab identified E. coli strain
KVP104 as a potential probiotic that
decreases the incidence of NEC in a
neonatal rat model.
Hypothesis:
E. coli KVP104 is a probiotic strain that does
not induce enterocyte apoptosis.
Methods:
IEC-6 rat intestinal epithelioid cells were
grown in Dulbecco-Modified Eagle Medium
(DMEM) in 4-well coated chamber slides.
Liquid cultures of C. muytjensii and E. coli
KVP104 were grown overnight in Luria
Broth. A total 107 cfu of each bacterium in
DMEM was added to chambers for 5 hours.
After treatment, the cells were fixed with
paraformaldehyde and apoptosis was
measured using the Terminal deoxy-Uridine
Nick End Labeling (TUNEL) assay.
Results:
Photomicrographs from the three treatment
groups show differences in apoptosis
induction by E. coli KVP104 and C.
muytjensii. Cells incubated with DMEM
alone demonstrated low levels of apoptosis
(Figure A).
Cells treated with C. muytjensii (Figure
C) demonstrated significantly higher
level of apoptosis. Treatment with E. coli
KVP104 (Figure B) did not significantly
increase the level of IEC-6 apoptosis
when compared to DMEM alone.
Quantitative data are shown in Figure D.
A) Cells treated with DMEM alone; B) Cells
treated with E. coli KVP104; C) Cells treated
with C. muytjensii
A
Conclusion:
Our findings indicate that E. coli KVP104
does not induce enterocyte apoptosis
and thus may be a beneficial first
colonizer of the gut. Further, KVP104
may be a useful therapeutic adjunct to
prevent the development of NEC in
neonates. Future directions include
determining if E. coli KVP104 can
prevent apoptosis caused by C.
muytjensii and other pathogenic
bacteria.
References:
Hunter, Catherine J. et al. “Enterobacter Sakazakii: An
Emerging Pathogen in Infants and Neonates.” Surgical
Infections 9.5 (2008): 533–539. PMC. Web. 5 Aug. 2015.
Hunter, Catherine J. et al. “Lactobacillus Bulgaricus Prevents
Intestinal Epithelial Cell Injury Caused by Enterobacter
Sakazakii-Induced Nitric Oxide Both In Vitro and in the
Newborn Rat Model of Necrotizing Enterocolitis .”Infection and
Immunity 77.3 (2009): 1031–1043. PMC. Web. 5 Aug. 2015.
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120
Control KVP104 C. muytjensii
TUNELCells/DAPICells
KVP104 does not induce
Enterocyte Apoptosis
C
B
DAPI TUNEL
Figure D