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RESEARCH POSTER PRESENTATION DESIGN © 2012
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Salmonella is a food borne illness resulting in gastroenteritis in adults and children
with increased mortality rates in newborns and children primarily, but also cause
disease in adults. Historically, consumption of human milk promotes the growth of
probiotics, sometimes suppressing gastrointestinal colonization by pathogens including
Salmonella. Human milk is a complex mixture of oligosaccharides, milk fat globules,
and whey cream, all of which have been reported to have antimicrobial and/or
prebiotic properties in humans and animals. In this study it was hypothesized that the
antimicrobial effects of different human milk components will be recapitulated during
Salmonella infection in vitro using differentiated colonic epithelial cells (Caco-2).
Modified gentamicin protection assays were done in the presence of diet components
to elucidate the effects of each component on Salmonella infectivity. Results
demonstrated that while human milk oligosaccharides and milk fat globules were
unable to significantly alter S. Typhimurium association with Caco-2 cells, priming the
bacterial cells with whey cream resulted in a 300% increase in bacterial adhesion and
200% increase in S. Typhimurium invasion. Given that whey cream exists in a liquid
state at 37°C, further experiments were conducted to characterize the effects of a
variety of lipids on Salmonella infectivity of Caco-2 cells. Lipids derived from fish, krill,
and plants were assayed along with additional Salmonella strains to examine the
breadth of this effect. Results from these experiments illustrate the consequence of
differences in lipid composition on Salmonella infectivity and the impact of strain
variation on sensitivity to different oils. Priming S. Typhimurium LT2 with each lipid
increased the capacity to adhere to and invade Caco-2 cells. Conversely, the more
pathogenic S. Typhimurium 14028S strain showed no significant change in infectivity
while S. Enteritidis was most susceptible to lipid priming, resulting in an 80% decrease
in overall association to Caco-2 cells. This study illustrates the independent effects of
different oils on Salmonella, emphasizing the effects of diet changes on pathogen
susceptibility, all of which increased pathogen association in vitro.
ABSTRACT
With the increase in drug resistant pathogens due to overuse of antibiotics,
dietary supplements especially in the maintenance of food animals are becoming the
primary alternate to combat foodborne diseases. This practice has long been in place in
the preference for breastfeeding over milk formula for human infants. In addition to the
antibodies provided in breast milk, breast milk components are also shown to be effective
in promoting the colonization of the neonatal gastrointestinal tract by beneficial microbes
such as Bifidobacterium sp1. Additionally breast milk components are thought to inhibit
colonization by pathogens such as Salmonella2.
Salmonella enterica is a human foodborne pathogen that is of concern in
children in developing countries and in the food animal and meat processing industry here
in the United States. Human infection leading to self limiting gastroenteritis facilitates
Salmonella transmission to subsequent hosts, including humans, and carriers such as
poultry and livestock. Dietary supplements have been shown to be effective in reducing
pathogen load in the gastrointestinal tract3. Preliminary experiments on Salmonella
infection of Caco-2 in the presence of different human milk components indicate whey
cream as the most effective milk component at reducing Salmonella association with Caco-
2 although the mechanism behind this observation remains to be elucidated.
INTRODUCTION
MATERIALS and METHODS
Figure 1. Effects of distinct milk components on Salmonella association with
Caco-2.
RESULTS
CONCLUSIONS
REFERENCES
ACKNOWLEDGEMENTS
This study was made possible by intellectual and technical support from Nguyet Kong. We
are grateful to Dr. Bart Weimer for his guidance in the development of this study. This work
was supported by the California Dairy Center.
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California at Davis
Poyin Chen, Nguyet Kong, Bart C. Weimer
Inhibitory Effects of Lipids on Salmonella Association with
the Gastrointestinal Barrier
Component Lipid type Chemical structures
Whey
cream
Milk fat
Milk fat membrane
Borage oil
Palmitic acid
Gamma linoleic acid
Linoleic acid
Oleic acid
Fish oil Eicosapentaenoic acid
Decosahexanaeoic acid
Krill oil Eicosapentanoic acid
Decosahexaneoic acid
Table 1. Lipids screened in host association assay
a. Effect of human milk fat globules on Salmonella –Caco2 association
b. Effect of whey cream on Salmonella-Caco2 association
c. Effect of human milk oligosaccharides on Salmonella-Caco2 association
Figure 2. Differential effects of lipids on Salmonella association with Caco-2.
ASSAY WORKFLOW
RESULTS
a. Effect of differentially sourced oils on S. Enteritidis total infection of Caco-2.
b. Effects of differentially sourced oils on S. Typhimurium strain LT2 infection of Caco-2.
c. Effects of differentially sourced oils on S. Typhimurium strain 14028S infection of Caco2
Bart Weimer, Ph. D. (bcweimer@ucdavis.edu)
1089 Veterinary Medicine Drive, VM3B , Room 4016
Davis, California, 95616
http://weimermicrolab.wix.com/thelab
(530) 752-6426
CONTACT
Incubate Caco-2 with lipid for 15 minutes
Infect Caco-2 with Salmonella for 60 minutes
Remove unassociated Salmonella and treat samples with gentramicin
Wash and lyse all cells in the sample for quantitation via qPCR
Control
1% HMO
Control
1% HMO
-35
-25
-15
-5
5
15
25
35
100
300
500
700
900
1100
1300
Adhesion
Invasion
SE ST
cfu/Caco2
Control
1% whey cream
Control
1% whey cream
Control
1% whey cream
-100
-50
0
50
100
200
400
600
800
1000
Adhesion
Invasion
ST SE SH
cfu/Caco2
Control
1% HMFG
Control
1% HMFG
Control
1% HMFG
-200
-100
0
100
200
300
400
Adhesion
Invasion
SE ST SH
cfu/Caco2
Linoleic acid
Eicosapentaenoic acid
Docosahexaenoic acid
Palmitic acid
Oleic acid
Gamma linoleic acid
1. Martin, R., et al., Isolation of bifidobacteria from breast milk and assessment of the
bifidobacterial population by PCR-denaturing gradient gel electrophoresis and
quantitative real-time PCR. Appl Environ Microbiol, 2009. 75(4): p. 965-9.
2. Morrow, A.L., et al., Human-milk glycans that inhibit pathogen binding protect breast-
feeding infants against infectious diarrhea. J Nutr, 2005. 135(5): p. 1304-7.
3. Fernandez, F., M. Hinton, and B. Van Gils, Dietary mannan-oligosaccharides and their
effect on chicken caecal microflora in relation to Salmonella Enteritidis colonization.
Avian Pathol, 2002. 31(1): p. 49-58.
Of the three milk components, whey cream displayed optimal efficacy in reducing S.
Typhimurium association with Caco-2.
Variability in fish and krill oil results highlights not only the importance of fatty acid
composition, but also concentration in altering Salmonella infectivity patterns.
Salmonella infectivity in the presence of milk components and differentially-sourced
lipids is component and serotype-specific.
There is no single fatty acid-based dietary supplement capable of minimizing risk of all
Salmonella infections and the commitment to one requires careful consideration of
multiple variables.
Control
1% Fish oil
1% Mega red oil
1% Borage oil
-40
-30
-20
-10
0
10
20
Adhesion
Invasion
Salmonella / Caco2
Lumen
Lamina propria
INFLAMMATION
Control
1% Fish oil
1% Mega red oil
1% Borage oil
-100
-75
-50
-25
0
25
50
75
100
CFU/Caco2 cell
Lumen
Lamina propria
Hypothesis: The addition of lipid-based dietary supplements to an in vitro model of
Salmonella infection reduces Salmonella adhesion capacity to the host.
Control
1% Fish oil
1% Mega red
1% Borage oil
-100
-75
-50
-25
0
25
50
75
100
Adhesion
Invasion
cfu/Caco2
Models of Salmonella infection
a. In the absence of dietary supplements
a. In the presence of dietary supplements