AbstractBackground: 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 INVF’ 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.

Antibiotic Resistance Transfer in Agriculture

Cow Manure

Enteric Bacteria

Fecal Coliform

Use of antibiotics for feed and treatment selects for AbR phenotype

Class A TEM bla genes

Non-enteric soil bacteria

Lateral gene transfer – Conjugation, transformation, transduction

Non-enteric soil bacteria

Resistance evolved over timeClass B metallo--lactamase genes

?

?

Investigation into Antibiotic Resistance in Bacteria in Agricultural Settings

Hypothesis 1 - Resistance due to lateral 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 (null) - Resistance evolved in soil microorganisms– 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

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

OrganismsIsolate 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/ β-lactam 8 4 38 50

Reference/ Non-β-lactam 32 11 7 50Cattle Farm/β-

lactam 6 1 73 80Cattle Farm/

Non-β-lactam 37 8 35 80Chat Pile/ β-

lactam 21 4 25 50Chat Pile/Non-

β-lactam 36 4 10 50Total 137 32 191 360

Chi-square Contingency TableOrganism/Ab Susceptible Intermediate Resistant Total

Reference / β-lactam 6.7 0.0 5.4 12.2a

Reference / 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

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

Plasmid DNA Isolation Studies

DNA was isolated from each bacterium, as well as FDM13 (an antibiotic resistant bacterium known to harbor plasmids).

– Techniques used: Wizard miniprep (shown here), Qiagen spin kit, Qiagen miniprep kit

– Smears likely due to glycosylated DNA, but no distinct bands

No plasmids detectedλH

ind

III

FD

M1

3

CP

A2

0

AC

P1

4

MR

55

CP

D3

2

WC

24

CP

C3

2

CP

E3

0

AC

P1

2

λH

ind

III

WC

24

CP

D3

0

CP

C3

0

CP

C2

0

WC

20

WC

56

CP

A3

0

CP

B3

0

23kbp

4kbp

2kbp

500bp

23kbp

4kbp

2kbp

500bp

16S rDNA Hybridization - Control 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.

λ H

ind

III

E.c

oli

WC

42

WC

56

WC

20

AC

P14

AC

P12

CP

B30

CP

C20

CP

A30

CP

C32

MR

55

WC

2 4

CP

A20

CP

E30

CP

D32

CP

C3 0

CP

D30

λ H

ind

III

23kbp

2kbp500bp

23kbp

2kbp

500bp

Metallo-ß-Lactamase Hybridization 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.

λH

ind

III

G.

meta

llir

ed

ucen

s

AC

P1

2

WC

56

AC

P1

4

CP

D3

0

CP

E3

0

E.c

oli

23kbp

4kbp

2kbp

500bp

*

*

* * *

Lack of TEM1 Hybridization

λH

ind

III

Hem

2b

MR

55

WC

20

AC

P1

4

CP

A2

0

CP

D3

0

CP

C3

0

CP

D3

2

CP

E3

0

23kbp

4kbp

2kbp

500bp

E.

coli

• 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-β-lactamase or some other unidentified mechanism.

Pseudomonas resistant to cefotaxime, this resistance may be due to chromosomal AmpC.

Acknowledgements 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