1. Jonathan Kang and Dr. Crosby W. Jones
Isolation of bacteriophages specific to Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in livestock fecal samples.
Jonathan Kang and
Dr. Crosby W. Jones

2. What are bacteriophages?
“Bacteria” + “Phage” (Greek: φαγ = Eaters)
Virus characteristics:
Replication
No metabolic activity
Uses of Bacteriophages:
Bacteriophage therapy2, 8, 13
Indicator organisms for bacteria presence4, 10
Phage typing3

3. INTRODUCTION Animal-to-Human Transmission: Escherichia coli
Shiga toxin-producing E. coli (STEC)11, 17, 18
O157:H7
Staphylococcus aureus
Methicillin-resistant S. aureus (MRSA)6, 14, 15
Mastitis = infection of mammary glands13, 20
Pseudomonas aeruginosa
Antimicrobial-resistant strains5
Mastitis19

4. INTRODUCTION Goals of study: Site: Non-human animal environment
Isolate bacteriophage
Isolate bacteria
Observe for correlations
Site: Non-human animal environment

5. MATERIALS AND METHODS
Study Site: Management, Instruction and Research (MIR) Center 6,000 acre Beef cattle, sheep and goats

6. MATERIALS AND METHODS Collection of environmental samples
Fecal Samples:
Thirteen X Beef cattle
Fifteen X Sheep
Twelve X Goats
Second Fecal Sample Subset:
Four X Beef cattle
Four X Sheep
Four X Goats
Field Aseptic Techniques

7. Bacteriophage Isolation
Processing samples22
Physiological saline solution (0.85% NaCl)
Ultrafiltration using 0.22 µm pore-sized membranes.
Phage enrichment8, 22
5.0 ml of filtrate + 40 ml of tryptic soy broth (TSB) + log phase culture
E. coli American Type Culture Collection (ATCC) strain 23848
S. aureus ATCC 13565
P. aeruginosa ATCC PA01
36-48 h incubation at 35°C
Phage isolation.
Lawn spotting2
Reconfirmed via plaque assay3

8. Bacteria Isolation: Selective media: Gram Staining3:
S. aureus E. coli P. aeruginosa
Selective media:
Gram Staining3:
Identification Tests:
Mannitol Salt Agar
MacConkey’s Agar
Cetrimide Agar G+
cocci-shaped G-
rod-shaped G-
rod-shaped
Latex agglutination3
Indole test16
Cytochrome oxidase3,7
UV fluorescence21

9. Bacteria Isolation: Bacteria Isolation Sampling Method:
Retentate vs. freshly-collected homogenate
Bacteria Enrichment:
S. aureus
10 µl freshly-collected homogenate + TSB with 7.5% NaCl

10. RESULTS: Phage Isolation

11. RESULTS: Bacteria Isolation

12. RESULTS: Comparisons
Positive correlation4,10 vs. negative correlation17,18
Negative correlations observed
Total Samples: 40
E. coli
S. aureus
P. aeruginosa
Bacteriophages only
10.7%
53.6%
3.6%
Bacteria only
25.0%
0.0%
14.3%
Both

13. RESULTS (Statistical Analysis)
Fisher’s exact test 1
Differences in phage isolation:
Between livestock types
Within each livestock types
Phage specific to
E. coli
S. aureus
P. aeruginosa
Goats
Sheep
Cattle
E. coli 1
0.696
0.4244
0.1107
0.4757
0.294

14. Nested Set Modeling Mixed Effects: Sample as random effect
Bacteriophage Isolation:
isolation ~ bacteria * livestock + (1 | sample)
AIC =
Chi-squared p-value =
Bacteria Isolation:
AIC =
Chi-squared p-value = 7.740e-09
isolation ~ bacteria * sampling + (1 | sample)
AIC =
Chi-squared p-value = < 2.2e-16

15. DISCUSSION Precedents: Negative correlations Possible Explanations:
First isolation of P. aeruginosa-specific phages from goats
Negative correlations
Possible Explanations:
Virulence of bacteriophages
Persistency of bacteria

16. DISCUSSION Negative correlations Virulence of bacteriophages:
Phage presence naturally reduces host bacteria populations12,17,18
Lytic phages vs. temperate phages2
Persistency of bacteria:
Aeration decreases bacteria survival
Reduces survival from 1 year to 48 days in E. coli 11

17. DISCUSSION Significance of livestock type:
Spatial Isolation of beef cattle from sheep and goats
S. aureus-specific phages in beef cattle vs. all three in sheep and goats
Opportunistic transmission and infection

18. Acknowledgements
We would like to give our appreciation to the following Thesis Committee Members: Dr. Crosby W. Jones Dr. Nicholas J. Negovetich Dr. Ned E. Strenth Dr. Bruce Bechtol Facilitating access to the MIR Center: Dr. Cody B. Scott for. Providing support to allow presentation at Texas Academy of Science: Dr. Russell Wilke, Head of the Department of Biology

19. Thank you

20. Major Reference Citations
2. Balogh, B., J. B. Jones, F. B. Iriarte, and M. T. Momol Phage therapy for plant disease control. Curr. Pharm. Biotechnol. 11:48-57.
3. Brown, A. E., and J. F. Connely (ed.) Benson’s microbiological applications: laboratory manual in general microbiology, 11th ed. McGraw-Hill, New York, NY.
6. Ferber, D From pigs to people: the emergence of a new superbug. Science 329:
8. Gill, J. J., and P. Hyman Phage choice, isolation, and preparation for phage therapy. Curr. Pharm. Biotechnol. 11:2-14.
11. Kudva, I. T., K. Blanch, and C. J. Hovde Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry. Applied and Environmental Microbiology 64:
12. Kudva, I. T., S. Jelacic, P. I. Tarr, P. Youderian, and C. J. Hovde Biocontrol of Escherichia coli O157 with O157-specific bacteriophages. Appl. Environ. Microbiol. 65:
17. Niu, Y. D., T. A. McAllister, Y. Xu, R. P. Johnson, T. P. Stephens, and K. Stanford Prevalence and impact of bacteriophages on the presence of Escherichia coli O157:H7 in feedlot cattle and their environment. Appl. Environ. Microbiol. 75:
18. Oot, R. A., R. R. Raya, T. R. Callaway, T. S. Edrington, E. M. Kutter, and A. D. Brabban Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces. Lett. Appl. Microbiol. 45:445–453.
22. Texas A&M University Protocol: Phage enrichments. Texas A&M University, College Station, TX.