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Antibiotic Resistance in Non-coliform Bacteria Isolated from a Cattle Farm
KIMBERLEIGH FOSTER, J. RENGEL, M. MILLER, J. ARNOLD, J. WOLOZYNEK, and J. CHAMPINE
Southeast Missouri State University, Cape Girardeau, MO 63701 ([email protected])
Abstract Q-184
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 zinc-hydrolases, or other yet unidentified resistance mechanisms.
Hypothesis 1 - Resistance due to gene transfer
•
Resistance highly specific to antibiotics used
•
Resistance genes may be carried on
plasmids
•
-Lactamase gene may resemble class A
TEM bla found in enterics
Hypothesis 2 - Resistance evolved in soil organisms
•
Broader resistance to 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
Nine Organisms Identified by 16S Sequence
Isolate
Source
Identification
GenBank #
ACP12
Cattle Pond 1
Chryseobacterium
AY464462
ACP14
Cattle Pond 1
Pseudomonas
AY464463
CPA30
Cattle Pond 3
Pseudomonas
Not yet prepared
CPC20
Cattle Pond 2
Pseudomonas
Not yet prepared
CPD30
Cattle Pond 3
Morganella
AY464464
CPD32
Cattle Pond 3
Escherichia
Not yet prepared
CPE30
Cattle Pond 3
Pseudomonas
AY484469
WC56
Wolf Creek
Aeromonas
AY484470
FDM13
Meat-packing plant
Pseudomonas
AY464123
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
MIC of Ampicillin for isolates was determined with Etest strips (upper left)
•
14 of the isolates had a MIC of greater than 256 µg/ml. CPB30 (96µg/ml) and CPC32 (128 µg/ml)
Kirby-Bauer Agar diffusion tests: -lactam (Ox, Cec, Cz, Ctx, Ipm, Cb) and non- -lactam (Lvx, Te, PB, E,
K, S, RA, NB).
•
All of the isolates were resistant to at least 1 non--lactam antibiotic, and 14 were resistant to >1.
•
None of the isolates showed resistance to imipenem, suggesting no metallo- -lactamase activity.
Organisms used
•
Cattle farm ampicillin resistant isolates (focus of this
study): 16 organisms including Chryseobacterium,
Pseudomonas, Aeromonas, Morganella, and Escherichia
•
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 (see Q-201): 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
Organism/Ab
Susceptible
Intermediate
Resistant
Total
Known/ β-lactam
6.7
0.0
5.4
12.2a
Known/ Non-β-lactam
8.1
9.7
14.0
31.8b
Cattle Farm/β-lactam
20.3
5.3
23.3
48.9
Cattle Farm/ Non-β-lactam
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
significantly more resistant than expected, α=0.005, df=2, Fcrit= 10.6
significantly less resistant than expected, α=0.005, df=2, Fcrit= 10.6
C significant variation among groups; α=0.005, df=10, Fcrit= 25.2
a
EcoRI-digested Chromosomal DNA probed with a 1064 bp
BstXI fragment of a putative metallo-β-lactamase 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.
*
*
*
E.coli
ACP12
CPD30
b
*
*
No plasmids detected
EcoRI Chromosomal DNA was probed with a 540 DdeI
internal fragment of the bla gene from pBR322. No
class A TEM type -lactamase detected.
Discussion
• Bacteria showed a wide range of antibiotic resistances, but specific (and extreme) resistance to -lactams
• Lack of imipenem resistance suggests no metallo--lactamase activity
• No plasmid DNA or TEM type bla detected
• Poor hybridization to metall- -lactamase probe suggests there may be a greater diversity of -lactamase
genes or defenses than currently understood (Class C and D genes not tested)