Microorganism Identification
and Tracking for Biorepositories
Workshop on Biodiversity and Climate Change
Astana, Republic of Kazakhstan
April, 2015
Karen Frank, M.D, Ph.D., D(ABMM)
Chief of Microbiology Service
Department of Laboratory Medicine
Clinical Center
NIH
Accurate Tracking of Stored Organisms and
Relevant Isolate Information is Essential
http://best-pcdesktop.blogspot.com
Traditional Bacterial
Culture Methods
Klebsiella
http://www.microbelibrary.org
Phenotypic Identification with Biochemical Testing
Tube Based or Automated Cartridges
http://www.biomerieux-usa.com
Traditional Culture
and Microscopic Identification of Molds
http://www.microbelibrary.org
http://www.microbiologyonline.org.uk
PCR for 16S rDNA
or Fungal ITS (Internal Transcribed Spacer) Region
or Specific Genes
Can Be Useful
PCR Technology more
Accessible than Advanced
Sequencing
http://www.scientificdealers.com
Mass Spectrometry
Instruments for Microbial
Identification
MALDI BioTyper
(Bruker Daltonics)
VITEK MS
(BioMerieux)
http://www.biomerieux-usa.com
http://www.bdal.de/products/software/maldi-biotyper/overview.html
Seng et al. (2010) Future Microbiol. 5:1733-1754
MALDI-TOF MS
Matrix-Assisted Laser Desorption IonizationTime of Flight Mass Spectrometry
Organic Acid
(DHB)
Wieser et al. (2012) Appl. Microbiol. Biotechnol. 93:965-974.
Intens. [a.u.]
Unique Spectral Peaks Correlate with
Organism Identification
Aspergillus fumigatus
3000
2000
Intens. [a.u.]
1000
0
Bacillus subtilis
8000
6000
4000
2000
0
Candida albicans
1.0
0.8
0.6
0.4
0.2
0.0
Eschericia coli
2500
2000
1500
Courtesy of
Anna Lau
1000
500
0
3000
4000
5000
6000
7000
8000
9000
10000
Beads Improved Extraction for
Mycobacteria and Molds
M. avium
Beads
Score =
1.97
M. avium
No beads
Score = 1.63
Mold Extraction is Rapid (15 min)
Pure isolate.
Medium, temperature,
& age independent.
5mm piece of mold
placed into 250 uL
EtOH with beads
Add 50 uL
70% formic
acid
Spin 13,000 rpm 2
min, remove all
EtOH
Vortex 15 min
(1 min)
Vortex 5 min
Add 50 uL
ACN, vortex
(1 min)
5 min
Spin 13,000
rpm 2 min
(1 min)
1 uL of supernatant spotted onto plate
Recent Case Blood Culture Positive
Identification with MALDI-TOF
No additional
culture required
9 year old boy with
leukemia
Courtesy of Ann Lau
& Adrian Zelazny
Fusarium
http://haicontroversies.blogspot.com/2011_12_01_archive.html
Genome Sequencing Technology
Database completeness and Bioinformatics
Expertise is Very Important
Roche/454
Courtesy Julie Segre
PacBio
Illumina MiSeq Illumina HiSeq
PacBio Single Molecule Real-time Sequencing Provide
Detailed Plasmid Data
 Short 1 kb reads
 Long 10 kb read
plasmids
Courtesy Julie Segre
Bacterial
chromosome
https://www.qiagen.com
Genome Sequencing Successfully
Used to Understand Transmission
of Bacteria Between Patients
Epidemiological Data
Snikin et al., Sci Trans Med, 2012. 4(148):1-9
Genetic Data Added
Advancing Sequencing Distinguished Plasmids
Carrying Similar Resistance Genes
Klebsiella pneumoniae
Enterobacter cloacae
pKpQIL
no
match
KPC-2
KPC-3
Conlan et al., Sci Transl Med. 2014 Sep 17;6(254):254ra126
Reliable Freezer or Lyophilizer System with
Redundancy is Essential
http://www.immunochemistry.com/
http://www.copanusa.com
http://www.esquirebiotech.com
http://www.laboratory-equipment.com