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Frequency of
Tetracycline Resistance Genes
in Bacterial Genomic DNA
of Swine Feces
Sharise Redmond & Jeannette Nguyen
Under the Direction of Candace Glendening
Antibiotics

ABX
• Agents made by bacteria/mold to inhibit
bacterial growth

Do NOT kill viruses
• Use



treat infections in humans and animals
growth promotion in animals
Bacteria
• Pathogenic - harmful/disease causing

e.g. Salmonella, Escheria coli O157:H7
• Beneficial – non harmful


Commensal - symbiotic relationship which benefits one
species while the other is unaffected
lots of commensal bacteria in gut (E. coli spp.)
History of Antibiotics

1800’s: “germ theory”
• Began search for ABX

1929: Fleming discovered Penicillin
• 1942 1st large scale use of Penicillin
• Used largely during WWII

1946: Penicillin widely available clinically
• Obtainable OTC by the public until the mid ‘50s


Dev’t of more ABX over next few decades
1970’s: Antibiotic Resistance (AR)
recognized as a real threat
• Meningitis & gonorrhea strains resistant to
penicillin
AB Resistance

Bacteria’s ability to produce a protein
that:
• disables an ABX or
• prevents transport of the ABX into the cell


Main hypothesis of AR:
Genetic + antibiotics in = antibiotic
mutations environment
resistance
N. gonorrhoeae (gonorrhea) resistance:
penicillin
tetracycline
flouroquinolones
SOON
cephalosporins
ABX Use in Animals



Does the use growth
Therapeutic
– treatment
promotional
levels of
of bacterial
infection ABX in food animals
lead to ARlevels
bacteria
in
Sub-therapeutic
– prevention
of
our food?
disease & growth
promotion
Antibiotics used for growth promotion 
pigs gain weight:


3.3-8.8% increased weight
2.5-7.0% feed efficiency
• Food Research Institute, Doyle, 1998

ABX use by farmers is not regulated

~25 million pounds annually used
History of Tetracycline

1948: introduction of tetracycline
• Made by Streptomyces bacterium in soil
• Chemical structure:


• “broad spectrum”
• low toxicity
1953: Shigella dysenteriae dev. resistance to tetracycline
Today 2nd to penicillin in the world in production and
use
• Treat: Respiratory tract infections, typhus, cholera,
brucellosis, anthrax, syphilis, Chlamydia, acne
• Also used widely for growth promotion in animals.
Tet Mechanisms of Action

Tetracycline inhibits
bacterial growth by
inhibiting
translation.
• It binds to the
ribosomal subunit
and prevents the
amino-acyl tRNA
from binding to the
A site of the
ribosome.
Inhibition of Protein
Synthesis by Tetracycline
Mechanisms of Tet Resistance
Tetracycline
Efflux Pump
plasmid
Ribosomal
Protection
Protein
Inactivation
Enzyme
Mechanism of resistance for
characterized tet and otr genes
Efflux
n = 23
Ribosomal
protection
n = 11
Enzymatic
Unknown
Inactivation n = 1
n=3
tet(A), tet(B), tet(C),
tet(D),tet(E) tet(G),
tet(H), tet(J), tet(V),
tet(Y), tet(Z), tet(30),
tet(31), tet(K), tet(L),
tetA(P), otr(B), tcr3
tet(33), tet(35),
tet(38), tet(39),
otr(C)
tet(M), tet(O), tet(X),
tet(S),
tet(34),
tet(W),tet(Q), tet(37)
tet(T), otr(A),
tetB(P)b, tet,
tet(32),
tet(36
tet(U)
Growth Promotion
 There has been a lack of serious
studies in the amounts of antibiotics
given to livestock and its link to the
increasing rates of resistance genes.
Previously Done Studies
Organism
of Origin
Organism
Studied
# Tet Res
Genes
#
Samples
Reference
1200
Bryan et al.
(2004)
Human &
various
animals
E. coli
tet: A, B, C, D,
E, G, K, L, M, O,
S, A(P), Q, X
Swine
Lactobacillus
tet: M
94
Gevers et al.
(2002)
Swine
E. coli
tet: A, B, C,
D,E, G, H, J, Y,
Z, 30
21
Aminov et al.
(2002)
Bovine
A. pyogenes
tet: W
20
Billington et
al. (2002)
Human
oral microflora
tet: M, W, O, Q,
S, L, A, K
Groundwater
outflow from
swine farm
Soil &
tet: O, Q, W, M,
gastrointestinal P, S, T, otrA
microbiota
105
Villedieu et al.
(2002)
22
Chee-Sanford
et al. (2001)
Central Question

Does the use of tetracycline as a
growth promotant affect tetracycline
resistance in swine fecal flora?
Central Hypothesis

The use of tetracycline as a growth
promotant will  frequency of
detecting Tet Resistance Genes in
swine fecal flora.
Effects of Growth Promotional use
of Chlorotetracycline (CTC)

Large-scale, multi-year study led by
Julie Funk @ Ohio State Univ. (OSU)
Epidemiological approach to studying
the use of CTC as a growth
promotant for swine

Looked for AR bacteria

CDC Year 1 Study Design
Temporally matched Barn Pair
Treatment
(50g CTC/ton of feed)
Control
(no antibiotics in the feed)
Treatment from 10 weeks (50 lbs) until 6 months old (250 lbs).
• Pigs sampled pre-slaughter
• 14 barn pairs total
• 96 pigs per barn
• 2688 total pigs sampled
Selected CDC Year 1 Results


Isolated 100
different Gram
Negative bacteria
(usually E. coli)
from each fecal
sample
Studied resistance
to 4 antibiotics
Found phenotypic
(tet res) difference
between these 2
groups
1.0
Proportion Resistant to CTC

Gram Negative
Fecal Flora Isolates
.90
.80
.70
.60
.50
.40
.30
.20
.10
0
No CTC
CTC
Treatment
n=268,800 isolates
Objectives

To study the distribution of tetracycline resistance
genes found in the fecal flora of pigs
• + CTC diet in their finishing phase.
• Ctrl: NO growth promotional use of ABX

Our Study Population
• 10 barn pairs
• 48 pigs per barn


480 total pigs sampled
Recall there are at least 38 tet resistance genes
• Are certain genes found more often under the selective
pressure of tetracycline?
Experimental
Design
200 mg poop
(frozen quickly)
Qiagen Stool
DNA Extraction Kit
(Bacterial Genomic DNA)
200 l genomic DNA
(from bacterial population)
1 l
1 l
1 l
1 l
Multiplex PCR
Group 1
Group 3
Group 2
Group 4
Methods: Multiplex PCR
2 (or more) sets of
primers in same tube
E-gel Marker

• Ex: Group 1:




tet(B) 659 bp
tet(C) 418 bp
tet(D) 787 bp
B
C
D
B/C/D
2000
800
Run each sample
through four separate400
Multiplex PCR
200
reactions.
480 samples
x 4 groups =
1920 rxns!
100
Ng et al., 2001
Genes Studied
Group
1
Efflux Pump
2
Efflux Pump
3
Ribosomal
Protection
Or
Efflux Pump
(+) Plasmid
Tetracycline
resistance gene
Amplicon size
(bp)
pRT11
tet(B)
659
pBR322
tet(C)
418
pSL106
tet(D)
787
pSL18
tet(A)
210
pSL1504
tet(E)
278
pJA8122
tet(G)
468
pAT102
tet(K)a
169
pVB.A15
tet(L)
267
pJ13
tet(M)
406
pUOA1
tet(O)
515
pAT451
tet(S)
667
tetA(P)
676
tet(Q)
904
tet(X)
468
4
pJIR39
Ribo. Protection
pNFD13-2
Or Enzyme
pBS5
Inactivation
Sample Gel (Group 3)
(+) Controls
K
L M
O S
_
Individual Pigs from Farm Bailey 3 _
(-)
667
515
406
267
169
Individual Pigs from Farm Bailey 3
_
Sample Gel (Group 4)
(+) Controls
Individual Pigs from Farm Bailey 3
__
? Q X (-)
904
468
___
_
Individual Pigs from Farm Bailey 3
_______
Results
The Effect of CTC in Swine Finisher Feed on
Detection of Tet Resistance Genes
200
Neg
Pos
# Fecal Samples
180
160
140
120
100
80
60
40
20
0
CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl CTC ctrl
B
C
D
K
L
Tet Resistance Gene
M
O
S
Results
The Effect of CTC in Swine Finisher Feed on
Detection of Tet Resistance Genes
Neg
Pos
160
# Fecal Samples
140
120
100
80
60
40
20
0
CTC
ctrl
A
CTC
ctrl
E
CTC
ctrl
G
CTC
ctrl
A(P)
Tet Resistance Gene
CTC
ctrl
Q
CTC
ctrl
X
Results
The Effect of CTC in Swine Finisher Feed on
Proportion of Pigs with Tet Resistance Genes
90%
CTC
no CTC
80%
% Positive
70%
60%
*
*
50%
40%
30%
20%
10%
0%
B
C
.3
D
K
L
.33
Tet Resistance Gene
M
.49
O
5 E-10
S
1 E-4
Results
The Effect of CTC in Swine Finisher Feed on
Proportion of Pigs with Tet Resistance Genes
90%
80%
CTC
no CTC
% Positive
70%
*
60%
50%
*
*
40%
30%
20%
10%
0%
A
E
G
A(P)
6 E-4
Tet Resistance Gene
Q
2 E-5
X
3 E-7
Results
Tet Resistance Genes Grouped by Mechanism
350
*
*
300
CTC
ctrl
# Detected
250
200
150
100
*
50
0
Ribosomal Protection
.008
Efflux
.03
Enzymatic Inactivation
Tet Resistance Gene Type
3 E-7
Central Hypothesis
The Effect of CTC in Swine Finisher Feed on # of
Pigs with a Detectible Tet Resistance Gene
180
Pos
Neg
The use of tetracycline as a growth
140
81%
promotant
will  frequency
81% of
120
detecting Tet Resistance Genes in
100
swine fecal flora.
80
160
# Pigs

60
40
20
0
CTC
.94
Treatment
ctrl
Discussion


At least one tet res gene in 81% of both
treatment groups
5/8 tet res genes showed no statistical diff. btw
treatment groups or were not high in frequency
• tet(C), (L), (M) similar high frequency in both swine
groups
• tet(B), (D), (K) min. to 0 frequency in both swine
groups
• tet(S) mostly found in ctrl samples
• tet(O) mostly found in CTC samples

Group 4 AR genes in both treatment groups
• 3/3 tet res genes statistically diff. btw treatment groups


Most tet genes found in CTC groups
Group 2 data in progress
Future Work

Complete sample processing
• 4 more barn pairs


Look at more tet resistance genes
Try to quantitate the amount of each
tet gene present in the sample
References
1.
2.
3.
4.
5.
6.
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15.
16.
17.
Aminov, R. I.; Chee-Sanford, J. C.; Garrigues, N.; Teferedegne, B.; Krapac, I. J.;. White, B. A.; Mackie, R. I.
(2002) Development, Validation, and Application of PCR Primers for Detection of Tetracycline Efflux Genes of
Gram-Negative Bacteria. Applied and Environmental Microbiology, 68(4), 1786-1793.
Aminov, R. I; Garrigues-Jean, N; Mackie, R. I. (2000) Molecular Ecology of Tetracycline Resistance: Development
and Validation of Primers for Detection of Tetracycline Resistance Genes Encoding Ribosomal Protection Proteins.
Applied and Environmental Microbiology 67 (1), 22-32.
Billington, S. J.; Songer, J. G.; Jost, B. H. (2002) Widespread Distribution of a Tet W Determinant among
Tetracycline-Resistant Isolates of the Animal Pathogen Acranobacterium pyogenes. Antimicrobial Agents and
Chemotherapy, 1281-1287.
Bryan, A.; Shapir, N.; Sadowsky, M.J. (2004) Frequency and Distribution of Tetracycline Resistance Genes in
Genetically Diverse, Nonselected, and Nonclinical Escherichia coli Strains Isolated from Diverse Human and Animal
Sources
Chee-Sanford, J. C.; Aminov, R. I.; Krapac, I. J.; Garrigues-JeanJean, N.; Mackie, R. I. (2001) Occurrence and
Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production
Facilities. Applied and Environmental Microbiology, 67(4), 1494-1502.
Chopra, I.; Roberts, M. (2001) Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and
Epidemiology of Bacterial Resistance. Microbiology and Molecular Biology Reviews, 65(2), 232-260.
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Gevers, D.; Danielsen, M.; Huys, G.; Swings, J. (2002) Molecular Characterization of tet(M) Genes in Lactobacillus
Isolates from Different Types of Fermented Dry Sausage. Applied and Environmental Microbiology, 69(2), 12701275.
http://dictionary.reference.com
http://en.wikipedia.org/wiki/Tetracycline
Lefers, Mark and Holmgren Lab (2004) http://www.biochem.northwestern.edu/holmgren/Glossary/Definitions/DefA/antibiotic_resistance.html
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Mathews, K. H. (2001) Antibiotic Drug Use and Veterinary Costs in U.S. Livestock Production. United States
Department of Agriculture Economic Research Service, Agriculture Information Bulletin 766.
Ng, L.-K.; Martin, I.; Alfa, M.; Mulvey, M. (2001) Multiplex PCR for the detection of tetracycline resistant genes.
Molecular and Cellular Probes, 15, 209-215.
Rubkin, Roberts, Institute of Medicine (1998) Antimicrobial Resistance: Issues and Options. Washington, DC:
Harrison, P. R. and Lederberg, J. National Academy Press.
Villedieu, A.; Diaz-Torres, M. L.; Hunt, N.; McNab, R.; Spratt, D. A.; Wilson, M.; Mullany, P. (2002) Prevalence of
Tetracycline Resistance Genes in Oral Bacteria. Antimicrobial Agents and Chemotherapy, 47(3), 878-882.
White, D.G.; Zhao, S.; Simjee, S.; Wagner, D. D.; McDermott, P. F. (2002) Antimicrobial resistance of foodborne
pathogens. Microbes and Infection 4, 405-412.
Acknowledgements:



Grant!!!
Julie Funk, MS, DVM, PhD, Asst. Prof. @ OSU
School of Veterinary Medicine
Fecal Extraction Team
•
•
•
•
•



Andy Bowman
Luc Hesselschwardt
Andy Mack
Jodi Houser
Jamie Berning
Candace Glendening
Each Other
University of Redlands