Transcript Emerging Diagnostics - A Sneak Peak at How Tomorrow’s

Emerging Diagnostics - A
Sneak Peak at How
Tomorrow’s Technologies are
Being Used at the University of
Wisconsin Today
Sharon C. Long
Today’s Objective
To get you thinking about new tools
and approaches to diagnosing the
biology in your processes
 Activated sludge
 Biosolids
 Source water
 Distribution systems
 Etc.
“Omics”
 Metabolomics – aims to compare differences
between biological samples based on their
metabolic profiles
 Genomics – using sequences of DNA and RNA
to develop useful biological knowledge
 Proteomics –used to relate microbial activities
to the identity of organisms in multispecies
communities
Today’s Examples
 Using ATP profiles to evaluate overall
microbial community changes
 Recognition of short sequences of DNA to
evaluate presence of certain target
microorganism - FISH
 Measuring the presence of short sequences of
DNA to evaluate presence of certain target
microorganism - PCR
 Sequencing the DNA of environmental
samples to assess community structure
ATP
 Transports chemical energy within cells for
metabolism
 Present in all living cells
 Amount in a sample is roughly proportional to the
number of cells present
 Measure the amount of light produced using
firefly enzymes
Study of Microbial Occurrence
in Wells
370
ME/ml
45,300
ME/ml
12,600
ME/ml
1,912,000 801,000 241,000
ME/ml
ME/ml
ME/ml
860
ME/ml
8,620
ME/ml
460
ME/ml
5,200
ME/ml
From: Andrew Jacque, PhD, PE, Water Quality Investigations LLC and Town & Country Engineering
Well Study
370
ME/ml
45,300
ME/ml
12,600
ME/ml
860
ME/ml
From: Andrew Jacque, PhD, PE, Water Quality Investigations LLC and Town & Country Engineering
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Fluorescent in situ
Hybridization (FISH)
 Use short sequences of single stranded DNA
complimentary to a sequence unique or
general to the target organism(s)
 Attach a fluorescent dye
 Prepare a sample
 Permeation
 Hybridization
 Quantify using microscopy
Applications of FISH
 Diagnosis of foaming in biosolids anaerobic
digesters
Probe
Specificity
Sequence (5’-3’)
EUB338 I
Universal bacteria
GCTGCCTCCCGTAGGAGT
EUB338 II
Universal bacteria
GCAGCCACCCGTAGGTGT
EUB338 III
Universal bacteria
GCTGCCACCCGTAGGTGT
ARC915
Archea
GTGCTCCCCCGCCAATTCCT
CREN499
Crenarchaeota
CCAGRCTTGCCCCCCGCT
MPA60
Microthrix parvicella
GGATGGCCGCGTTCGACT
MPA223
Microthrix parvicella
GCCGCGAGACCCTCCTAG
MPA645
Microthrix parvicella
CCGGACTCTAGTCAGAGC
Klare Keadle, Zachary Carroll, Daniel R. Noguera, and Sharon C. Long
Foaming and FISH Results
 Provided qualitative confirmation of the
presence and relative density of target
filaments
 M. parvicella was confirmed to be numerous
 Other known filamentous Archeae also
present in digesters
Applications of FISH 2
 Characterize microorganisms in aerated
anoxic Enhanced Biological Phosphorus
Removal
Probe
Specificity
Sequence (5’-3’)
RHC439
Candidatus Accumulibacter Phosphatis
& other Rhodocyclus-related organisms
CNATTTCTTCCCCGCCGA
PAO462b
Candidatus Accumulibacter Phosphatis
CCGTCATCTRCWCAGGGTAT
TAAC
PAO651
Candidatus Accumulibacter Phosphatis
CCCTCTGCCAAACTCCAG
PAO846b
Candidatus Accumulibacter Phosphatis
GTTAGCTACGGYACTAAAAGG
Ramesh K. Goel, Patricia Sanhueza and Daniel R. Noguera
EBPR FISH Results
120
Run II ( 0.3 %
2) O
100
80
120
%
60
Run III ( 0.6 %
2) O
More than 90 % Population was
Rhodocyclus related
100
40
% P removal
80
20
Run IV ( 1.2 %
2) O
% Rhodocyclus related
10
20
%
0
0
120
30
100
60
40
50
60
80
40
Time (days)
% 60
P removal
% Rhodocyclus related
%
20
40
0
0
5
10
15
2020
25
% P removal
% Rhodocyclus related
30
Time (days)
0
0
10
20
Time (days)
30
40
Polymerase Chain Reaction
(PCR)
 Use of Taq polymerase
 Target short (~100 to 400
Exponential Replication
bp), unique sequences of
DNA or RNA for species
identification
 Target broadly occurring
sequences of DNA for
organism family
identification
 Products may also be
sequenced to yield further
information
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2
3
4
5
Measure Presence of STEC in
Wastewater
Gene Target
stx 1
stx 2
ORF Z3276
16S gene
Assay
Interpretation
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+
+
+
+
+
+
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PCR Result
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+
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-
A
A
A
B
C
D
+
+
+
+
+
+
+
E
E
E
(A) E. coli O157:H7
(B) Non-STEC E. coli or Shigella
(C) E. coli absent
(D) Toxigenic organism, not STEC, not Shigella
(E) Non-O157:H7 STEC or Shigella
India Mansour, Mark Walter, and Sharon C. Long
DNA Sequencing
Trevor Ghylin
Database of Wastewater Microbes
(Proprietary)
EBPR Genomic Study
Cytophaga
8%
Rhodocylcaceae
8%
Chitinophagaceae
6%
Cytophaga
Rhodocylcaceae
Chitinophagaceae
Arcobacter Arcobacter
3%
Bacteroidetes
Bacteroidetes
Myxococcales
2%
Myxococcales Moraxellaceae
2%
Flavobacterium
Other
67%
MoraxellaceaeProsthecobacter
1%
Planctomycetes
Flavobacterium
Other
1%
Prosthecobacter
Planctomycetes 1%
1%
Acknowledgements