updates of current technique for identification of
TRANSCRIPT
MALDI-TOF m
ass
spect
rom
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Nut Nithimongkolchai
1st year PhD student
Department of microbiology,
Faculty of Medicine, Khon Kaen university
Updates of current technique for identification of Burkholderia pseudomallei using MALDI-TOF
IntroductionMALDI-TOF m
ass
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rom
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• Gram negative bacteria
• Natural resistant many drugs
• Found in environment
• Selective media: Ashdown's media
• Causative agent of Melioidosis
• Bioterrorism
Characteristic of Burkholderia pseudomallei(Bp)
https://microbe-canvas.com/uploads/image/bacterien/burkholderia-pseudomallei/b-pseudomallei_03_ f-350x220.jpg2
Burkholderia Pseudomallei epidemiology
Introduction
Northeastern Thailand
Southern Taiwan
Northern Australia
MALDI-TOF m
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Melioidosis
MALDI-TOF m
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Introduction
Infection by - inhalation- ingestion
Incubation time: ≈ 9 daysFatality rate of up to 50 %Symptoms
- Localized pain- Sepsis- Pneumonia- Liver abscess
https://www.cdc.gov/melioidosis/index.html4
Burkholderia pseudomallei detection
MALDI-TOF m
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Introduction
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Conventional method
Burkholderiapseudomallei
Specimen from pateint
Cuture on Ashdown’s media
Identification based on biochemical test
Drug susceptibility test(DST)
accuracy rate is 53-98%
occur false positive and negative
IntroductionBurkholderia pseudomallei detection
MALDI-TOF m
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Latex agglutination test
Immunoassay
Multilocus sequencing
Whole genome sequencing MALDI-TOF
16s rRNA detection
Molecular techniques
not proper for routine work
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IntroductionMatrix assisted laser
desorption/ionization time of flight mass spectrometry
(MALDI-TOF)
MALDI-TOF m
ass
spect
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Based on proteomic analysis by generate pattern of protein and compare with database
Biomolecular analysis such as
▪ DNA▪ Carbohydrates▪ Lipid ▪ Various organic molecules
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Pick a colony
Smear onto target plate
The MALDI TOF process is a two-phase procedure1. Ionization Phase2. Time of Flight Phase
IntroductionMatrix assisted laser
desorption/ionization time of flight mass spectrometry
(MALDI-TOF)
MALDI-TOF m
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spect
rom
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1. conventional method can take up to 3-4 days: use long turn around time
2. The Bp is not in the FDA-approved database and still in the basic research stage and has not been widely used in clinical practice
Problem
MALDI-TOF m
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Spectrum and SuperSpectrum
spectrum
spectrum
spectrum
SuperSpectrum for B. pseudomallei
spectrum
spectrum
spectrum
SuperSpectrum for B. cepacia
B. pseudomallei
B. cepacia
MALDI-TOF m
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https://upload.wikimedia.org/wikipedia/commons/e/ee/Bps_ close.JPG
https://www.microscopemaster.com/images/burkholderiaonsheepbloodagar.jpg
https://img.medicalexpo.com/images_me/photo-g/75820-16073733.jpg
https://www.frontiersin.org/files/Articles/144398/fmicb-06-00791-HTML/image_m/fmicb-06-00791-g001.jpg
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MALDI-TOF m
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Aim to establish a SuperSpectrum of B. pseudomallei in Hainan and evaluate its application value in rapid identification of clinical isolate
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MALDI-TOF m
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Materials and method
A total 99 B. pseudomallei
Identified by MLST typing, 16s rRNA, vitek2 compact
17 B. pseudomallei
Creation of SuperSpectraSelf-built
SuperSpectra
95 isolates▪ 82 B. pseumomallei▪ 8 B. thailandensis▪ 2 B. cepacia▪ 1 B. cenocepacia▪ 1 B. multivorans▪ 1 B. gladioli
Phylogenetic analysis
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13 Isolates• 8 B. thailandensis• 2 B. cepacia• 1 B. cenocepacia• 1 B. multivorans• 1 B. gladioli
82 B. pseudomallei
Verification of SuperSpectra
MALDI-TOF m
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A total 99 B. pseudomallei
Identified by MLST typing, 16s rRNA, vitek2 compact
17 B. pseudomallei
Creation of SuperSpectraSelf-built
SuperSpectra
95 validation isolates82 B. pseumomallei8 B. thailandensis2 B. cepacia1 B. cenocepacia1 B. multivorans1 B. gladioli
Phylogeny13 strains
8 Bth2 B cepacia1 B cenocepacia1 B multivorans1 B gladioli
82 B. pseudomallei
Verification of SuperSpectra
Results
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The correct identification rate of B. pseudomallei is 100%The confidence interval is 75.5 – 99.9%
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ResultB. pseudomallei
based on a comparison of the similarity of main spectra projection by calculated the pattern matching
The 95 Burkholderia strains were divided into three groups1. B. pseudomallei2. B. thailandensis3. other closely related species of Burkholderia.
MALDI-TOF m
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B. thailandensis
B. CepaciaB. CenocepaciaB multivoransB. gladioli
Phylogenetic analysis
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based on a comparison of the similarity of main spectra projection by calculated the pattern matching
The 95 Burkholderia strains were divided into three groups1. B. pseudomallei2. B. thailandensis3. other closely related species of Burkholderia.
MALDI-TOF m
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Result
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Could be well classified by MALDI-TOF MS at the protein level
Conclusion
1. MALDI-TOF is an efficient for the rapid identification of B. pseudomallei
2. the quality of the self-built laboratory database is
• Ideal
• Rapid
• Reliable
3. the identification time of bacteria from around 48 hr. to several minutes
MALDI-TOF m
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MALDI-TOF m
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Aim to create a SuperSpectrum for Burkholderia pseudomallei identification using VitekMS in RUO(research use only) mode
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Materials and methodMALDI-TOF m
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SMRU: Shoklo Malaria Research UnitCOMRU: Cambodia Oxford Medical Research UnitLMWRU: Lao-Oxford-Mahosot Hospital-WellcomeTrust Research Unit
B. pseudomallei 243 isolatesB. thailandensis 14 isolatesB. cepacia 8 isolatesnon-Burkholderia Gram negative 9 isolatesGram positive bacteria 6 isolates
SuperSpectracreation
AdditionalSuperSpectra
creation
Viability check
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Materials and methodMALDI-TOF m
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1 B. pseudomallei CHCA (matrix)
CHCA: α-Cyano-4-hydroxycinnamic acid
+Fully dry
Culture on blood agar
Viability check
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SMRU: Shoklo Malaria Research UnitCOMRU: Cambodia Oxford Medical Research UnitLMWRU: Lao-Oxford-Mahosot Hospital-WellcomeTrust Research Unit
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MALDI-TOF m
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provides some reassurance from a safety perspective
Viability check
Growth was not detected after incubation of the plates for 14 days
Result
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Materials and methodMALDI-TOF m
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217 reference isolates of B. pseudomallei
from13 Asia isolates and 4 Australia isolates
SMRU-SS-Bps
Test with 25 isolates from17 reference isolates of B. pseudomallei (MORU)5 clinical isolate of B. pseudomallei (SMRU)
3 reference isolates of B. thailandensis
SuperSpectra creation
Validation
SMRU-SS-BPs: SMRU-SuperSpectra B. sseudomallei
triplicate per isolates
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MALDI-TOF m
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ResultSuperSpectra creation
Study sites
Isolate origin
misidentified as B. thailandensis
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Materials and methodMALDI-TOF m
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Additional SuperSpectra creation
4 reference isolates of B. pseudomalleiFrom Townville (Australia)
34 reference and clinical isolates of B. pseudomallei
From Townville (Australia)
SMRU-SS-Bps2
SMRU-SS-Bps3
SMRU-SS-BPs: SMRU-SuperSpectra B. sseudomallei
Add 7 SuperSpectrafrom Townvilles study
Validation
triplicate per isolates
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MALDI-TOF m
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Result
100% identification rate
Additional SuperSpectra creation
Study sites
Isolate origin
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Containing 10 SuperSpectra
MALDI-TOF m
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Conclusions
1. The vitek MS can be used for rapid identification of BP with 3 spots per isolate recommended
2. the limitations of the SuperSpectrumcreation algorithm is the geographical variability of the organism
https://www.nrl.ae/en/news/view/microbial-identification-test-using-maldi-tof-ms-methodology.html
https://www.nature.com/articles/nmicrobiol20158?proof=t%2Btarget%3D 25
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Summary
The new trend for the Bp identification and it related species
- The effect of geographical variability
The MALDI-TOF technique
Rapid
Accurate
Reliable
- Cannot detect mixed infection
the limitations
Low cost of detection
- Poor reproducibility
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MALDI-TOF m
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Criticisms
Strong point Weak point
1st paper - The number of strains for established and verifying the database was more than before, has very good representativeness and stability.
- Did not show the comparison result between MALDI-TOF with 16s rRNA and MLST
2nd paper -There was a comparison of the BP between regions, at least three countries in two different geographical location
- They use the same strain that establish superspectra for validation- The sample is not cover the endemic area and there is still the possibility that different strains might fail to identify
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POW
ERPOIN
THUB.COM
Assoc. Prof. Kiatichai Faksri, PhD.
Acknowledgment
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Thank youfor your kind attention