Towards rapid detection of Staphylococcus aureus during blood culture

World Congress and Expo on Applied MicrobiologyAugust 18-20, 2015, Frankfurt

Vincent Templier, PhD StudentCEA, INAC-SPrAM-CREAB, F-38000 Grenoble,

France

Contact : vincent.templier@cea.fr

The threat of bacteremia

2

Intr

oduc

tion

• Bacteremia or bloodstream infection (BSI) = presence of viable bacteria in blood1,2.

• Affects mainly immunocompromized patients but not only.

• 200 000-250 000 cases / year in the USA

• Mortality can be as high as 20-50%3,4.

• S. aureus = major pathogen, accounting for almost 1/5 of bacteria involved in BSI5.

Bacteremia = life-threatening infection which needs rapid medical care.

1Reimer, L.G. et al., Clin Microbiol Rev, 1997 ; 2Wilson, M.L. et al., Clinical and Laboratory Standards Institute, 2007 ; 3Bearman, G.L., Archive of Medical Research, 2005 ; 4Dellinger, R.P. et al., Critical Care Medicine, 2013 ; 5Timsit, J.F., et al., BMC Infect Dis, 2014.

Current procedure for microbial identification

3

Intr

oduc

tion

Hemoculture

12-36h

Empirical antibiotic treatment

Patie

nt

Bloo

d sa

mpl

e

(5 to

10m

L)

Appropriate

dilution

Low contamination

(1 CFU / 10mL)

1. Assessment of bacterial presence

Current procedure for microbial identification

4

Intr

oduc

tion

Hemoculture

12-36h

Gram coloration and

Microscopic observation

Growth and Isolation

12-24h

Empirical antibiotic treatment

Patie

nt

If positive

Bloo

d sa

mpl

e

(5 to

10m

L)

Appropriate

dilution

Possible treatment modifications

Low contamination

(1 CFU / 10mL)

1. Assessment of bacterial presence

2. Bacteria isolation on solid media

Current procedure for microbial identification

5

Intr

oduc

tion

A delay or a misuse in antibiotic treatment (in case of antibiotic resistant bacteria) results in an augmentation of patient deaths6,7.

6Davey, P.G. et al., Clinical Microbiology and Infection, 2008 ; 7Kumar, A. et al., Chest, 2009.

Hemoculture

12-36h

Gram coloration and

Microscopic observation

Growth and Isolation

12-24h

Identification and

Antimicrobial Susceptibility Testing

24h - 72h

Empirical antibiotic treatment72h – 96h

Suitable antibiotic treatment

Treatment adjusting

Result

Patie

nt

If positive

Bloo

d sa

mpl

e

(5 to

10m

L)

Appropriate

dilution

Possible treatment modifications

Low contamination

(1 CFU / 10mL)

1. Assessment of bacterial presence

2. Bacteria isolation on solid media

3. Full identification of the causative bacteria

Imperative need to shorten diagnosis time

Goal : to perform hemoculture and identification in the same timeIn

trod

uctio

n

Growth and Isolation

12-24h

Identification confirmation and

Antimicrobial Susceptibility Testing

24h - 72h

Empirical antibiotic treatment72h – 96h

Suitable antibiotic treatment

Treatment adjusting

Result

Patie

nt

If positive

Bloo

d sa

mpl

e

(5 to

10m

L)

Appropriate

dilution

Possible treatment modifications based

on identification results

Low contamination

(1 CFU / 10mL)

1. Hemoculture

AND

identification2. Bacteria isolation on solid

media3. Full identification of the causative

bacteria

Hemoculture

AND

Identification

6

To obtain reliable identification results during hemoculture

Use of protein biochip and optical detection

7

Intr

oduc

tion

Grafting of bacteria specific antibodies by a simple electrochemical reaction.

Antibody array

Cuve

Glass Prism

Gold layer

Samples 1 & 2

Use of protein biochip and optical detection

8

Intr

oduc

tion

Grafting of bacteria specific antibodies by a simple electrochemical reaction.

Antibody array

Cuve

Glass Prism

Gold layer

Antibody grafted to the surface

ReactorWet

phasePrism

Dry phase

Assets of the SPRi:

•Direct and multiplex detection

•Label-free

•Real time monitoring

Samples 1 &2

Culture – Capture – Measure approach8,9 by SPRi

9

Intr

oduc

tion

Detection in simple and complex media (food matrix)

Non destructive method which enables further testing (plating, PCR…) after incubation time.

Blood dilution with suitable culture media and artificial contamination

8Bouguelia, S. et al., Lab on a Chip, 2013 ; 9Mondani, L et al., JAM, 2014.

Proof of concept in blood

10

Expe

rimen

tal r

esul

ts

Detection of 100 UFC.mL-1 of Salmonella enterica serotype Enteritidis in diluted human blood (mean of 3 spots)

Salmonella detection in blood is feasible in a few hours.

What happens with S. aureus?

Specific signal

S .aureus detection in culture media

11

Expe

rimen

tal r

esul

ts

IgG control (non specific of S. aureus) looks positive.

Simultaneous interaction on all antibodies

S .aureus detection in culture media

12

Expe

rimen

tal r

esul

ts

IgG control (non specific of S. aureus) looks positive.

Difficult to say if an antibody recognizes its target or if interactions are only "protein A" related.

Cause : Staphyloccocal protein A recognizing the Fc fragment of antibodies.

Simultaneous interaction on all antibodies

IgG cleavage for S. aureus detection

13

Expe

rimen

tal r

esul

ts

IgG cleavage for S. aureus detection

14

Expe

rimen

tal r

esul

ts

Anti-S. aureus digested IgG are successfully binding to their target

IgG cleavage for S. aureus detection

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Expe

rimen

tal r

esul

ts Workable but enzymatic digestion must be adapted to each antibody.

Anti-S. aureus digested IgG are successfully binding to their targetNon specific digested IgG are no longer recognized.

Conclusion

Specific antibodies are required for proper bacterial recognition

Influence sensitivity and specificity of the assay.

Bacteria detection on an antibody array by SPRi is working in a few hours.

Easy to operate and applicable in complex media (diluted blood sample)

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Conc

lusi

ons

& P

ersp

ectiv

es

Perspectives

Mammalian antibodies could be (partially) replaced by alternative probes such as:

•Chicken antibodies

•Aptamers

Work to be done : •Screening of specific probes

•Analytical comparison with existing devices

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ons

& P

ersp

ectiv

es

ThanksCREAB : My PhD supervisors, Yoann Roupioz (PhD) and Thierry Livache (PhD).D. Pulido for the work done together. CHU Grenoble : Pr M. Maurin and S. Boisset (PhD)

The CEA programme « Technologies pour la santé » for the funding of my PhD thesis.

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Thank you for your attentionAny questions?

Microorganisms responsible of BSI

19

Anne

xs

From 5Timsit, J.F., et al., BMC Infect Dis, 2014, 14,

Experimental device

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Device placed in an incubator with temperature fixed at 37°C

Experiment monitored on a dedicated software

Prism and cuve with 2 chambers.

Optical bench of the SPRi system.

Anne

xs

SPRi Principle

21

Anne

xs

Antibody grafted to the surface

LED CCD camera

Bacteria

Computer

Incident light

Reflected light

Resonance angle Θ

Wave penetration at the interface

Plasmon surface wave

Polarizer

SPRi Principle

22

Anne

xs

Resonance angle shift with interaction

ΔR

(%)

Incident Light Angle (°)

Refle

ctivi

ty (%

)1. Resonance angle Θ2. Fixed working angle

1 2

Initial plasmon curvePlasmon curve after interaction