Emerging Diagnostics - A Sneak Peak at How Tomorrow’s Technologies are Being

Used at the University of Wisconsin Today

Sharon C. Long

Today’s ObjectiveTo 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

860 ME/ml

460 ME/ml

1,912,000ME/ml

801,000 ME/ml

241,000 ME/ml

8,620 ME/ml

5,200 ME/ml

From: Andrew Jacque, PhD, PE, Water Quality Investigations LLC and Town & Country Engineering

7

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

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

Probe Specificity Sequence (5’-3’)EUB338 I Universal bacteria GCTGCCTCCCGTAGGAGTEUB338 II Universal bacteria GCAGCCACCCGTAGGTGTEUB338 III Universal bacteria GCTGCCACCCGTAGGTGTARC915 Archea GTGCTCCCCCGCCAATTC

CTCREN499 Crenarchaeota CCAGRCTTGCCCCCCGCTMPA60 Microthrix parvicella GGATGGCCGCGTTCGACTMPA223 Microthrix parvicella GCCGCGAGACCCTCCTAGMPA645 Microthrix parvicella CCGGACTCTAGTCAGAGC

Applications of FISH Diagnosis of foaming in biosolids

anaerobic digesters

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

Ramesh K. Goel, Patricia Sanhueza and Daniel R. Noguera

Probe Specificity Sequence (5’-3’)RHC439 Candidatus Accumulibacter

Phosphatis & other Rhodocyclus-related organisms

CNATTTCTTCCCCGCCGA

PAO462b Candidatus Accumulibacter Phosphatis

CCGTCATCTRCWCAGGGTATTAAC

PAO651 Candidatus Accumulibacter Phosphatis

CCCTCTGCCAAACTCCAG

PAO846b Candidatus Accumulibacter Phosphatis

GTTAGCTACGGYACTAAAAGG

EBPR FISH ResultsRun II ( 0.3 % O2)

Time (days)0 10 20 30 40 50 60

%

0

20

40

60

80

100

120

% P removal% Rhodocyclus related

Run III ( 0.6 % O2)

Time (days)0 5 10 15 20 25 30

%

0

20

40

60

80

100

120

% P removal% Rhodocyclus related

Run IV ( 1.2 % O2)

Time (days)0 10 20 30 40

%

0

20

40

60

80

100

120

% P removal% Rhodocyclus related

More than 90 % Population was Rhodocyclus related

Polymerase Chain Reaction (PCR) Use of Taq polymerase

Target short (~100 to 400 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

Exponential Replication

1 2 3 4 5

Measure Presence of STEC in Wastewater

India Mansour, Mark Walter, and Sharon C. Long

Gene Target PCR Resultstx 1 + - - - - + + + -stx 2 + + - - - + + - +

ORF Z3276 + + + - - - - - -16S gene + + + + - - + + +

Assay Interpretation 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

DNA Sequencing

Trevor Ghylin

Database of Wastewater Microbes (Proprietary)

EBPR Genomic StudyCytophaga

8% Rhodocyl-

caceae8%

Chitinopha-gaceae

6%Ar-

cobac-ter3%Bac-

teroidetes2%

Myx-ococ-cales2%

Moraxel-

laceae1%Flavob

ac-terium

1%

Pros-the-

cobac-ter1%

Planc-to-

mycetes

1%

Other66%

CytophagaRhodocylcaceaeChitinophagaceaeArcobacterBacteroidetesMyxococcalesMoraxellaceaeFlavobacteriumProsthecobacterPlanctomycetesOther

Acknowledgements