Framework for Commensal Evaluation

1
Framework for Commensal Evaluation

• Populations ABR chip Bioreporter
• Natural background of ABR
• Dispersal of prior selected ABR
  
• Selection (out growth)
  
• agriculture, aquaculture, sewage
• ABR transfer to pathogen
  
• density, available mechanisms, nutrition,
  selection

Acknowledgements Syed Hashsham, Thad
Stanton Carlos Rodriguez, Robert Stedtfeld, Brian
Campbell
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83 of total since 2000 67 of total since 2003
Of only 7 soil studies, 84 of unique
isolate/gene combinations in soil come from 2
studies (published in 2007 and 2008) Despite only
7 studies, isolate/gene combinations unique to
soil isolates represent 17 of the total
isolate/gene combinations from environmental and
animal sources much unexplored diversity!
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Molecular functional analysis in
soil microcosms
100 g of water-saturated soil
located next to manured soil, same soil type
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Quantification of tet (36) in soil microcosms
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Tetracycline Resistant Bacterial Isolates from an
Agricultural Field in Michigan
Screened for A, B, C, D, E, G, H, J, X, Y, Z, 30,
and 31

• Expanding the spectrum of genes found in
  isolates
• Identification of tet(Y) in Pseudomonas, tet(C)
  and tet(31) in Stenotrophomonas, tet(A) in
  Microbacterium, and tet(A) and tet(C) in
  Thermomonas
• Second identification of tet(X) in
  Sphingobacterium
• Moore et al., 2005 show that Tigecycline is
  modified by TetX
• Although modification was slight perhaps
  mutation could enhance activity?

Unpublished
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PCR amplification of class 1 integrons
variable region from manured soil DNA
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Amplification of class 1 integron variable region
(manured wheat field, 1 week after application)
temp gradient 65-54oC
control soil (no manure)
manured soil
Amplification of class 1 integron variable region
(manured wheat field, 4 weeks after application)
manured soil
control soil (no manure)
M 65o 64o 63o 62o 61o 60o 59o58o 57o 56o 55o
54o M
1636 bp
1636 bp
1018 bp
1018 bp
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Abundance of RPP genes in clone libraries after 1
month of application
Abundance of integron-associated ABR genes in
clone libraries after 1 month of application
after 1 month
after 1 week
GTP-binding (2)
cation transporters (4)
ribosomal protection (7)
metabolism (6)
hypothetical/putative (31)
14 of retrieved sequences corresponded to RPP
genotypes (N 50)
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Assessment of integron diversity by comparative
sequence analysis of conserved regions among
integron encoded integrases
Integrons are classified on the basis of the
divergence among their integrase genes
(Rowe-Magnus and Mazel, 2001).
hep35/hep36 Degenerate primers designed to
hybridize to a conserved region (approximately
500bp long) among integron encoded integrase
genes intl1, intl2 and intl3 (White et al,
2000).
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Diversity of environmental integrases
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Biotrove OpenArray Used for ABR Chip
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Success rate of primer design Validation of 110
VMG Assays
Stedtfeld et al., AEM 2008
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QPCR of AB resistance genes Experimental approach
Primer Design Approach

• Download aligned nucleotide sequences from FGPR
• Group sequences based on similarity and construct
  consensus sequences.
• Design primers from conserved regions of
  consensus sequence using Primer Express.
• Blast and select primers based on theoretical
  specificity of 3 end.

Experimental Approach

• Overall, 336 primer sets targeting 176 ABR genes,
  and 39 virulence genes.
• Used multiple displacement amplification to
  increase DNA for each sample.
• SYBR based QPCR was performed on all samples,
  which were tested simultaneously (in triplicate)
  using 3 BioTrove plates.

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QPCR results Genes amplified in 1 or more samples
Collaboration with Thad Stanton. Natl Animal
Disease Cntr
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Intersection of Soil Chemistry and Microbiology
Does tetracycline contamination in soil enhance
the development, maintenance, and transfer of
resistance genes?

• Tetracycline is commonly found in soils at
  concentrations ranging from 200 300 ppb. In a
  complex matrix such as soil, is tetracycline at
  environmentally relevant concentrations
  bioavailable to drive resistance gene expression?
  What factors control the bioavailability of
  tetracycline in environmental matrices?
• To begin to answer these questions
• Characterize bioavailability of tetracycline
  complexed with environmentally relevant cations
  in solution phase.
• Quantitatively measure promoter activity using a
  tetracycline inducible bioreporter and model
  equations developed by Leveau Lindow, 2001.
• Explain results in context of tetracycline
  solution chemistry
• Use this base knowledge and collaboration with
  soil chemists to expand to tetracycline cation
  mineral phase systems (e.g. smectite clays)

Courtesy of Dr. Lars Hansen (University of
Copenhagen)
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TC Bioavailability (LB)
1 mM Mg2
0.1 ug/mL TC
n/a
1 mM Mg2
0.1 ug/mL TC
n/a
7.5 mM Mg2
5 mM Mg2
2.5 mM Mg2
7.5 mM Mg2
5 mM Mg2
2.5 mM Mg2
10 mM Mg2
10 mM Mg2
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10 mM MgCl2 Added
10 mM EDTA Added