The Effects of Bdellovibrio bacteriovorus on the Toxicity of E. coli in Chick Embryos

Phil DeGaetano

Department of Biological Sciences, York College of PennsylvaniaTrial 1

Introduction

•All experimental groups were positive for bacteria

•Control Groups were negative

Lit Cited

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1. Lee, YK, Puong, KY, Ouwehand, AC, Salminen, S. 1. Lee, YK, Puong, KY, Ouwehand, AC, Salminen, S. Displacement of bacterial pathogens from mucus and Caco-2 cell Displacement of bacterial pathogens from mucus and Caco-2 cell surface by lactobacilli. surface by lactobacilli. J Med MicrobialJ Med Microbial. 2003; 52: 925-930.. 2003; 52: 925-930.

1.To determine the toxicity of E. coli against chick embryos using chick mortality

2.To determine if site of inoculation (air sac or allantoic sac) affected toxicity

3.100ul of 107 cells of E. coli (Migula) Castellani and Chalmers was inoculated into fertile White Leghorn chicken eggs

Experimental Design:

Results:

Conclusion:1.Because of allantoic mortality, an air sac

inoculation was used for trial #2

2.107 cells of E. coli were to be used in trial #2 to slightly increase mortality in the air sac

Trial 2

Experimental Design:

To determine if Bdellovibrio affects chick embryo response to an E. coli challenge following air sac injections of E. coli.

1.Inoculations of 100ul of E. coli (107 cells) done via air sac

2.Bdellovibrio inoculations done immediately, or at 24, or at 48 hours after E. coli inoculation

3.E. coli and Bdellovibrio controls were used

4.N=7 for each experimental and control group

5.Chick embryo weights were taken at times of death to measure the effect of Bdellovibrio

Results:

Conclusion:

•The 24 hour Bdellovibrio treatment seemed to reverse any inhibitory effects of the other treatments (group 3 & 5), although none of the groups were significantly different in embryo weight.

•Allantoic inoculations earlier in development to be made in trial #3 to increase toxicity

Trial 3

Experimental Design:

To determine if Bdellovibrio affects chick embryo response to an E. coli challenge following allantoic injections of E. coli.

1.Inoculations of 100ul of E. coli (107 cells) done via allantoic sac

2.Bdellovibrio inoculated at 2 or 24 hours post E. coli inoculations.

3.Controls used as indicated

4.N=12 for each experimental and control group

5.Chick embryo weights were taken at times of death to measure the effect of Bdellovibrio

6.All samples were cultured

Results:

Conclusion:1.Bdellovibrio does alter the response of chick

embryo growth to an E. coli challenge

2.Allantoic site inoculations increased toxicity as well as the time of inoculations

3.Bdellovibrio seemed to increase the toxicity of E. coli

4.The Bdellovibrio treatment (2 hours post E. coli challenge) was more toxic than the Bdellovibrio treatment (24 hours post E. coli challenge)

Survival of E. coli inoculated Chick Embryos

Control 104 105 1060

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6 Air SacAllantoic Sac

# E. coli Cells

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ays

The potential for infectious bacteria to develop resistance against traditional antibiotics has become an important area of study in recent years, and has led to a hunt for alternative agents which might destroy these pathogens. As new and different antibiotics are discovered, the infectious bacteria simply mutate to become further resistant. It is obvious and apparent that an entirely new means of fighting pathogens needs to be discovered. One means would be to use harmless bacteria to fight against the infectious bacteria. This idea comes from the common application of using live probiotic bacteria to improve intestinal functions in humans.

Probiotics are simply live bacteria that beneficially affect a host animal by improving its intestinal microbial balance. These bacteria help extensively with digestion, and are an indication of good health. One such study shows how probiotic bacteria can competitively displace pathogenic bacteria on the mucus walls of the human intestine (Lee et al., 2003).

In my study I am proposing a novel means to use bacteria as a solution to antibiotic resistance. Unlike probiotic bacteria, the bacteria that I am proposing would be predacious bacteria. It would have to prey on other pathogenic bacteria but also be harmless to eukaryotic cells. These bacteria would also need to be fairly common and easily isolated. A bacterium that fits these criteria is Bdellovibrio bacteriovorus. A major benefit in using bacteria such as B. bacteriovorus is that they have the potential to mutate in response to a mutated pathogen (prey). The need to replace obsolete Bdellovibrio with other bacteria would be unnecessary.

Figure 3. Mean (+- S.D.) of embryo weights of inoculated eggs in trial 3. Un-

inoculated control group 7 is not shown. Non-parametric ANOVA, overall significantly different (p= 0.0002). (*) When compared to group 6. were significantly different (p< 0.01).

Figure 2. Mean (+- S.D.) of embryo weights of inoculated eggs

in trial 2. Non-parametric ANOVA, overall nearly significantly different (p= 0.0618, N= 7).

Figure 1. Number of embryos that survived after E. coli inoculations at 104, 105, and 106 E. coli cells/ml. Data was taken at 12th day of incubation. Inoculations were via the air sac and the allantoic sac.

Figure 1.

Figure 2. Figure 3.

Acknowledgements•Carolyn Mathur, Ph.D.

•Ronald Kaltreider, Ph.D.

•Sally Hoh, Laboratory Technician/Chemical Hygiene Officer

Embryo Weights of E. coli/Bdellovibrio Innoculated Eggs (trial 3)

Group 1 Group 2 Group 3 Group 4Group 5 Group 60.00

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Group 1: E. coli + Broth (2 hrs after)Group 2: Bdello + Broth (2 hrs after)Group 3: E. coli + Broth (24 hrs after)Group 4: Bdello + Broth (24 hrs after)Group 5: E. coli + Bdello (2 hrs after)Group 6: E. coli + Bdello (24 hrs after)

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Embryo Weights of E. coli/Bdellovibrio Innoculated Eggs (trial 2)

Group 1 Group 2Group 3 Group 4 Group 5 Group 60

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Group 1: BdelloGroup 2: E. coliGroup 3: E. coli + Bdello (immediately)Group 4: E. coli + Bdello (24hrs after)Group 5: E. coli + Bdello (48hrs after)Group 6: Control

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Hypothesis

I hypothesize that B. bacteriovorus significantly alters the response of chick embryos to an E. coli challenge.