Transcript Abstract

Abstract
Background: In order to assess the possibility that antibiotic resistance genes are being transferred from
animals to environmental bacteria, non-enteric Ampicillin resistant (AmpR) bacteria were isolated from a
cattle farm, a meat packing plant sewage lagoon, and the Mississippi river. Methods: Organisms were
isolated on APT media containing 50 mg/L Amp, screened for cefinase activity, and the inability to ferment
lactose to acid and gas in broth. MIC for Amp was determined using Etest strips, and a profile of
resistance to 17 antibiotics was determined using the Kirby-Bauer agar diffusion test. Chromosomal DNA
was extracted by phenol:chloroform separation in the presence of CTAB detergent and by DNeasy.
Plasmid extractions were performed with the Qiagen mini-prep kit and the Wizard mini-prep kit. These
DNAs were used in Southern hybridization experiments with probes for class A (TEM1-type) and class B
(metallo-) -lactamases. Six of the isolates were identified by sequencing of PCR amplified 16S rDNA
(GenBank accession numbers). Results: A total of 17 non-enteric strains were studied, and 14 had MIC
values greater than 256 mg/L. Pseudomonas sp. FDM13 (AY464123), from the sewage lagoon, contained
plasmid DNA, but was not capable of transforming E. coli strains INVF’ or XL10 Gold. No plasmid
DNA was detected in the 16 isolates from the cattle farm and the Mississippi river. None of the
chromosomal DNAs, or FDM13 plasmid DNA hybridized with the TEM1 probe. Pseudomonas sp. CPE30
(AY484469), Aeromonas sp. WC56 (AY484470), Morganella sp. CPD30 (AY464464), Pseudomonas sp.
ACP14 (AY464463), and Chryseobacterium ACP12 (AY464462) showed the strongest hybridization with
the metallo-β-lactamase probe. Conclusion: The lack of R-plasmids and the failure of hybridization with
the TEM1 probe suggest that lateral gene transmission from enteric bacteria associated with animals to
environmental bacteria is not taking place. On the other hand, environmental bacteria that show a high
degree of resistance to Amp were widespread, and resistance in these bacteria may be due to zinchydrolases, or other yet unidentified resistance mechanisms.
Antibiotic Resistance Transfer in Agriculture
Use of antibiotics for feed and
treatment selects for AbR
phenotype
Cow
Enteric
Bacteria
?
Resistance evolved over time
Class B metallo--lactamase
genes
Manure
Fecal
Coliform
Class A TEM
bla genes
Non-enteric
soil bacteria
?
Non-enteric
soil bacteria
Lateral gene transfer – Conjugation,
transformation, transduction
Investigation into Antibiotic Resistance
in Bacteria in Agricultural Settings
 Hypothesis 1 - Resistance
due to lateral gene
transfer
 Hypothesis 2 (null) Resistance evolved in soil
microorganisms
– Resistance highly specific
– Broader resistance to
to antibiotics used
– Resistance genes may be
carried on plasmids
– -Lactamase gene may
resemble class A TEM bla
found in enterics
variety of antibiotics
encountered in soil over
time
– Resistance may be plasmid
or chromosomally encoded
– Class B Metallo- Lactamase observed in
Caulobacter may be
present
Water Collection Sites
 Meat Cattle farm in
Swinton, MO. Antibiotic
use reported as penicillin
only.
 100 ml water samples
were taken from 3 ponds
and a creek adjacent to
the farm ( )
 UTM 16 coordinates
shown for map datum
NAD 27 in CONUS.
 ArcMap used to plot the
points onto the
topographic map.
Isolation Approach
 Ampicillin resistance
– Plated on APT agar w/ 50
g/ml ampicillin
– Single colony taken from each
plate with growth, unless
additional morphotypes
present
– Screened for cefinase activity
 Non-coliform status (accepted
if one of the following are true)
– Gram positive
– No lactose fermentation on
EMB
– No gas from lactose broth
Organisms
Isolate
Source
Tentative Identification
GenBank Accession Number
ACP12
Cattle Pond 1
Chryseobacterium
AY464462
ACP14
Cattle Pond 1
Pseudomonas
AY464463
CPA20
Cattle Pond 2
CPA30
Cattle Pond 3
Pseudomonas
Not yet prepared
CPB30
Cattle Pond 3
CPC20
Cattle Pond 2
Pseudomonas
Not yet prepared
CPC32
Cattle Pond 3
CPC30
Cattle Pond 3
CPD30
Cattle Pond 3
Morganella
AY464464
CPD32
Cattle Pond 3
Escherichia senegalensis
Not yet prepared
CPE30
Cattle Pond 3
Pseudomonas
AY484469
WC20
Wolf Creek
WC24
Wolf Creek
WC42
Wolf Creek
WC56
Wolf Creek
Aeromonas
AY484470
MR55
Mississippi River
FDM13
Meat-packing plant sewage
Pseudomonas syringiae
AY464123
Documentation of Resistance


MIC of Ampicillin for isolates was determined with Etest strips (upper left)
– Result: 14 of the isolates had a MIC of greater than 256 µg/ml. CPB30 (96µg/ml)
and CPC32 (128 µg/ml) were slightly lower.
Kirby-Bauer Agar diffusion tests (upper right) were used to test for resistance to lactam (Oxacillin, Cefaclor, Cefazolin, Cefotaxime, Imipenem, Carbenicillin )and non-lactam antibiotics (Levaquin, tetracycline, Polymyxin B, Erythromycin, Kanamycin,
Streptomycin, Rifampin, Novobiocin).
– Result 1: All of the isolates were resistant to at least 1 non--lactam antibiotic, and
14 were resistant to 2 or more.
– Result 2: None of the isolates showed resistance to imipenem, suggesting no
metallo- -lactamase activity.
Comparison of Frequency of
Resistance to Various
Antibiotics
 Organisms used
– Cattle farm isolates (organisms
under study)
– Reference organisms associated
with soil (Lab teaching strains): B.
cereus, B. megaterium, B. subtilis,
B. brevis, B. pumilis, P.
aeruginosa, P. putida, P.
fluorescens, P. paucimobilis, P.
stutzeri
– Chat Pile Lead-mine tailings
isolates (non-selected
environmental isolates): 10
organisms including Rhodococcus,
Pseudomonas, Streptomyces,
Ochrobactrum, and Arthrobacter
 Antibiotics used
– -lactam: Ampicillin,
Carbenecillin, Cefazolin,
Cephatoxime, Cefaclor
– Non- -lactam: Erythromycin,
Kanamycin, Polymyxin B,
Streptomycin, Tetracycline
Frequency of Resistance among Isolates by
Antibiotic Group
Organisms/Ab
Susceptible
Intermediate
Resistant
Total
Reference/ βlactam
8
32
6
37
21
36
137
4
11
1
8
4
4
32
38
7
73
35
25
10
191
50
50
80
80
50
50
360
Reference/
Non-β-lactam
Cattle Farm/βlactam
Cattle Farm/
Non-β-lactam
Chat Pile/ βlactam
Chat Pile/Nonβ-lactam
Total
Chi-square Contingency Table
Organism/Ab
Susceptible
Intermediate
Resistant
Total
Reference / β-lactam
6.7
0.0
5.4
12.2a
Reference / Non-βlactam
8.1
9.7
14.0
31.8b
Cattle Farm/β-lactam
20.3
5.3
23.3
48.9
Cattle Farm/ Non-βlactam
1.1
0.1
1.1
2.3
Chat Pile/ β-lactam
0.1
0.0
0.0
0.2
Chat Pile/Non-β-lactam
14.1
0.0
9.9
24.1
Total
50.4
15.2
53.8
119.4c
a
significantly higher than expected, α=0.005, df=2, Fcrit= 10.6
b
significantly lower than expected, α=0.005, df=2, Fcrit= 10.6
C
significant variation among groups; α=0.005, df=10, Fcrit= 25.2
Antibiotic Testing Summary
 Non-imepenem resistance implies no metallo-lactamase activity
 Cattle farm isolates resistant to 3 or more classes
of antibiotics suggesting exposure to more than
just penicillin
 Cattle farm isolates are more resistant to -lactam
than non- -lactam antibiotics, suggesting a
specific mechanism of resistance
Molecular Approaches
 Isolate plasmids
 Isolate chromosomal
DNA
 Southern Blot
performed on each
isolate using probes
for TEM and metallo-lactamases
WC24
Plasmid DNA Isolation Studies
 DNA was isolated from each
bacterium, as well as FDM13 23kbp
(an antibiotic resistant
4kbp
bacterium known to harbor
2kbp
plasmids).
glycosylated DNA, but no
distinct bands
 No plasmids detected
500bp
WC24
– Techniques used: Wizard
miniprep (shown here),
Qiagen spin kit, Qiagen
miniprep kit
– Smears likely due to
23kbp
4kbp
2kbp
500bp
CPB30
WC20
WC56
WC42
16S rDNA Hybridization - Control
23kbp
2kbp
23kbp
2kbp
500bp
CPD30
CPD32
500bp
 Chromosomal DNA
was obtained with
DNeasy kit
 16s rDNA
hybridization was
used to determine if
DNA was suitable
for hybridization.
 RFLP can also be
used to determine if
some of the bacteria
are similar or the
same species.
E.coli
ACP12
 EcoRI-digested Chromosomal
DNA probed with a 1064 bp BstXI
fragment of a putative metallo-βlactamase from G. metallireducens
(positive control)
 ACP12 - 9kbp, WC56 - 3kbp,
ACP14 - 9kbp and 8kbp, CPD30 8kbp and 6kbp, and CPE30 - 8kbp
and 6kbp
 E. coli (negative control), WC24,
CPA30, MR55, CPA20, and
CPD32 showed non specific
hybridization.
 CPB30, WC42, CPC20, CPC32,
WC20, and CPC30, and reference
strains showed no hybridization to
this probe.
CPD30
Metallo-ß-Lactamase Hybridization
23kbp
*
4kbp
2kbp
500bp
*
*
*
*
23kbp
4kbp
2kbp
500bp
E. coli
CPD30
Lack of TEM1 Hybridization
• EcoRI Chromosomal DNA
was probed with a 540 DdeI
internal fragment of the bla
gene from pBR322.
•Hem2B is plasmid DNA
containing the bla gene
(positive control). E. coli is
negative control.
•No hybridization with this
probe was seen with any
ampicillin resistant
laboratory strains.
Conclusion
 Chromosomal DNA did not hybridize with
TEM1 probe.
 Interspecies gene transfer from enteric to
environmental bacteria may not be
occurring.
 Bacteria in the environment are already
resistant to antibiotics and are more
competitive than the transient fecal
organisms.
Conclusion
 Resistance may be due to metallo-βlactamase or some other unidentified
mechanism.
 Pseudomonas resistant to cefotaxime, this
resistance may be due to chromosomal
AmpC.
Acknowledgements
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Principal funding for this project came from
the Southeast Missouri State University
Grants and Research Funding Committee.
Additional funding to support 16S rDNA
sequencing costs came from the Southeast
Missouri State University Undergraduate
Research Program. Julie Rengel would like to
thank Dr. Allan Bornstein and Dr. Jane
Stephens for their support of undergraduate
research.
Funding for student travel was made
available through the Southeast Missouri
State University Student Professional
Development program (Drs. Rick Burns and
Christina Frazier).
Co-authors not in attendance: Julie Rengel,
Melanie Miller, Jennifer Arnold, and Josh
Wolozynek
Kimberleigh Foster’s thesis Committee: Dr.
Bjorn Olesen and Dr. Allen Gathman
Dr. Walt Lilly, Dr. John Scheibe, and Maija
Bluma
Dave Bridges for help with ARC Map