Antibiotic Resistance in Non-coliform Bacteria Isolated from a Cattle Farm

1
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_at_semo.edu)
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
phenolchloroform 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-ß-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 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

• 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

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
  gt1.
• None of the isolates showed resistance to
  imipenem, suggesting no metallo- ?-lactamase
  activity.

2

• 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,
a0.005, df2, Fcrit 10.6 b significantly less
resistant than expected, a0.005, df2, Fcrit
10.6 C significant variation among groups
a0.005, df10, Fcrit 25.2
EcoRI-digested Chromosomal DNA probed with a 1064
bp BstXI fragment of a putative
metallo-ß-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.

• 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)