Characterization of multi-drug
resistant Enterobacter spp. in
the Camp Creek, IA
David Wyker, Dr. Mahajan (Mentor)
Perspectives on antibiotic use
• CDC
– Center for Disease Control
• “a clear link between antibiotic use in animals and
antibiotic resistance in humans.”
• AMA
– American Medical Association
• “The AMA is opposed to the use of antimicrobials at
nontherapeutic levels in agriculture or as pesticides or
growth promoters”
• WHO
– World Health Organization
 “evidence shows that pathogens that have developed resistance
to drugs in animals can be transmitted to humans”
Antibiotic resistant bacteria in Iowa
Des Moines Register, Aug. 27, 2010
Egg recall: Salmonella found on Iowa farms, feed
Recall of over 500 million eggs.
Sickened estimated 2,000 people
Prevalence of Staphylococcus aureus and methicillin-resistant
Staphylococcus aureus (MRSA) on retail meat in Iowa
B.M. Hanson et al. (2011) J. Infect. Public Health, vol. 4, 169-174
 27 of 165 meat samples (~16%)
were contaminated with S. aureus.
 Two of these isolates were MRSA.
Des Moines Register, March 29, 2001
Antibiotic Distribution
Agriculture
 20% antibiotics
 7.25 million lbs
Humans
Excretion
 75-90% unaltered
Manure
Field
Application
Sewage
Sewage
Treatment
 Incomplete
removal
Leakage/Runoff
Soil
Groundwater
Drinking Water
Surface Water
Adapted from Kumar et al. (2005). Antibiotic use in
agriculture and its impact on the terrestrial
environment. Advances in Agronomy. 87, 1-54.
 80% antibiotics
 29.8 million lbs
Camp Creek
Watershed Profile
• EPA
impaired waterway (2010)
 Sediment, Nitrogen, Phosphorous
• MWA
 Leachate facility and Landfill (MPE)
• Livestock
 Pasture, feedlots, CAFOs
 Many horses, 2900 cattle, 7300 hogs
• 68%
• City
Row crops
of Mitchellville
 Sewage treatment facility
Methods
1) Field Sampling at
Camp Creek
• Upstream/Downstream
2) Bacteria enumerations
• +/- tet plates
3) Identification
• Selective media
• Biochemical tests
Methods
4) Antibiotic resistance profiles
• Tetracycline
• Ampicillin
• Penicillin
• Streptomycin
• Chloramphenicol
• Kanamycin
• Erythromycin
5) Plasmid isolation
• Amresco Prep kit
• Agarose gel
electrophoresis
www.gbiosciences.com
Antibiotic Resistance
Profiles
Kirby-Bauer antibiotic susceptibility test
Isolate
Antibiotic Resistance Pattern
1
Tet – Eryth – Pen
2
Tet – Eryth – Pen
Antibiotic
Concentration
3
Tet – Eryth – Pen – Amp
Ampicillin
10 µg
4
Tet – Eryth – Pen – Amp
Erythromycin
15 µg
5
Tet – Eryth – Pen – Amp
Chloramphenicol
30 µg
6
Tet – Eryth – Pen – Amp – Chlor
Kanamycin
30 µg
7
Tet – Eryth – Pen – Amp
Penicillin
10 µg
8
Tet – Eryth – Pen
Streptomycin
10 µg
Antibiotic Resistance Profiles
Isolate
Penicillin
Ampicillin
Erythromycin
1
50 µg
0 µg
100 µg
2
50 µg
0 µg
100 µg
3
100 µg
100 µg
100 µg
4
100 µg
10 µg
50 µg
5
100 µg
100 µg
100 µg
6
100 µg
100 µg
100 µg
7
50 µg
20 µg
100 µg
8
25 µg
0 µg
100 µg
Results
• Tetracycline
resistance was lost in samples stored in LB
without antibiotic for 12 weeks
• Plasmid
DNA
isolation tests produced no extrachromosomal
Results
• Ampicillin,
not lost
Penicillin, and Erythromycin resistances were
• Deductions:
1. Tetracycline resistance was located on a plasmid and lost
2. Fitness cost of plasmid too high in absence of antibiotic
3. Other ABRs are located on bacterial chromosome and fixed
Sign Epistasis and MABR
• Multi-resistant mutations are not independent
• Additional resistance plasmid or mutation often increases
fitness of a strain already resistant to antibiotics.
• Strains carrying additional resistance
mutation will outcompete strains without it
 Drives evolution of MABR
MacLean, C., et al. (2010). The population genetics of
antibiotic resistance: integrating molecular mechanisms and
treatment contexts. Nature Reviews Genetics; 11, 405-414.
Reflection
• Other
resistances may have been located on plasmid and
not identified
• Maintain
samples in presence of antibiotic or isolate
plasmids asap
• Sub-therapeutic
use of antibiotics banned in Europe
 Swedish Animal Health Service: effective if maximize hygiene
practices
Conclusion
• Limit
on antibiotic use in agriculture will decrease
selective pressure for tetracycline
• Plasmid-borne
resistances can be lost
• Resistance
encoded on chromosome = fixed
• Resistance
provided by more than one mechanism
• Future
Research:
 Characterize ARGs on bacterial chromosome
 Take additional samples and characterize plasmids
Acknowledgments
• Dr.
Keith Summerville
• Dr.
Chinh Dao
• Dr.
Pramod Mahajan
• Ellis
Pharmacogenomics Laboratory
• CPHS
• Research
was also support by a Grow Iowa
Values fund grant to PBM