Updates of current technique for identification of

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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

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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

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Melioidosis

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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

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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

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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)

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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

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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

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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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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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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

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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.

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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.

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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

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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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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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Result

100% identification rate

Additional SuperSpectra creation

Study sites

Isolate origin

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Containing 10 SuperSpectra

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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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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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Assoc. Prof. Kiatichai Faksri, PhD.

Acknowledgment

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Thank youfor your kind attention