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 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. Abstract Q-184 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 IsolateSourceIdentificationGenBank # ACP12Cattle Pond 1ChryseobacteriumAY ACP14Cattle Pond 1PseudomonasAY CPA30Cattle Pond 3PseudomonasNot yet prepared CPC20Cattle Pond 2PseudomonasNot yet prepared CPD30Cattle Pond 3MorganellaAY CPD32Cattle Pond 3EscherichiaNot yet prepared CPE30Cattle Pond 3PseudomonasAY WC56Wolf CreekAeromonasAY FDM13Meat-packing plantPseudomonasAY Nine Organisms Identified by 16S Sequence 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 a significantly more resistant than expected, α=0.005, df=2, Fcrit= 10.6 less resistant b 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/AbSusceptibleIntermediateResistantTotal Known/ β-lactam a Known/ Non-β-lactam b Cattle Farm/β-lactam Cattle Farm/ Non-β-lactam Chat Pile/ β-lactam Chat Pile/Non-β-lactam Total c 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. λHindIII G. metallireducens ACP12 WC56 CPD30 CPE30 E.coli * * * * * ACP14 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)