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 (jchampine@semo.edu)
Background: In order to assess the possibility that antibiotic resistance genes are being transferred from animals toenvironmental bacteria, non-enteric Ampicillin resistant (AmpR) bacteria were isolated from a cattle farm, a meat packing plantsewage lagoon, and the Mississippi river. Methods: Organisms were isolated on APT media containing 50 mg/L Amp, screenedfor cefinase activity, and the inability to ferment lactose to acid and gas in broth. MIC for Amp was determined using Eteststrips, and a profile of resistance to 17 antibiotics was determined using the Kirby-Bauer agar diffusion test. Chromosomal DNAwas extracted by phenol:chloroform separation in the presence of CTAB detergent and by DNeasy. Plasmid extractions wereperformed with the Qiagen mini-prep kit and the Wizard mini-prep kit. These DNAs were used in Southern hybridizationexperiments with probes for class A (TEM1-type) and class B (metallo-) -lactamases. Six of the isolates were identified bysequencing 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, containedplasmid DNA, but was not capable of transforming E. coli strains INVF’ or XL10 Gold. No plasmid DNA was detected in the16 isolates from the cattle farm and the Mississippi river. None of the chromosomal DNAs, or FDM13 plasmid DNA hybridizedwith 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 strongesthybridization with the metallo-β-lactamase probe. Conclusion: The lack of R-plasmids and the failure of hybridization with theTEM1 probe suggest that lateral gene transmission from enteric bacteria associated with animals to environmental bacteria is nottaking place. On the other hand, environmental bacteria that show a high degree of resistance to Amp were widespread, andresistance in these bacteria may be due to zinc-hydrolases, or other yet unidentified resistance mechanisms.
Abstract Q-184
Hypothesis 1 - Resistance due to gene transfer
•Resistance highly specific to antibiotics used
•Resistance genes may be carried onplasmids
•-Lactamase gene may resemble class ATEM bla found in enterics
Hypothesis 2 - Resistance evolved in soil organisms
•Broader resistance to variety of antibioticsencountered in soil over time
•Resistance may be plasmid or chromosomallyencoded
•Class B Metallo- -Lactamase observed inCaulobacter may be present
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
Nine Organisms Identified by 16S Sequence
Ampicillin resistance
•Plated on APT agar w/ 50 g/mlampicillin
•Single colony taken from eachplate with growth, unlessadditional morphotypes present
•Screened for cefinase activity
Non-coliform status (accepted if one ofthe 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 thisstudy): 16 organisms including Chryseobacterium,Pseudomonas, Aeromonas, Morganella, and Escherichia
•Reference organisms associated with soil (Lab teachingstrains): 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): 10organisms including Rhodococcus, Pseudomonas,Streptomyces, Ochrobactrum, and Arthrobacter
Antibiotics used
•-lactam: Ampicillin,Carbenecillin, Cefazolin,Cephatoxime, Cefaclor
•Non- -lactam: Erythromycin,Kanamycin, Polymyxin B,Streptomycin, Tetracycline
a significantly more resistant than expected, α=0.005, df=2, Fcrit= 10.6
less resistantb 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
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
EcoRI-digested Chromosomal DNA probed with a 1064 bpBstXI fragment of a putative metallo-β-lactamase fromG. 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, andreference strains showed no hybridization to thisprobe.
λHindIII
G. metallireducens
ACP12
WC56
CPD30
CPE30
E.coli
*
*
*
*
*
ACP14
No plasmids detected
EcoRI Chromosomal DNA was probed with a 540 DdeIinternal fragment of the bla gene from pBR322. Noclass 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 -lactamasegenes or defenses than currently understood (Class C and D genes not tested)