3rd International Conference on Bio-Sensing Technology 2013 This work won the Award for Outstanding Oral Presentation at the 3rd International Conference on Bio-Sensing Technology 2013. Pierre R. Marcoux, Mathieu Dupoy Department of Technology for Biology and Healthcare, CEA-LETI MINATEC, 17 avenue des Martyrs, 38054 Grenoble, France. Antoine Cuer, Joe-Loïc Kodja, Arthur Lefebvre, Florian Licari, Robin Louvet, Anil Narassiguin These authors contributed equally to this work. Ecole Centrale de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France. Frédéric Mallard bioMérieux SA, Innovation & Systems / Technology Research / Sample Prep & Processing Lab, 5 rue des Berges, 38000 Grenoble, France. The development of methods for the rapid identification of pathogenic bacteria is a major step towards accelerated clinical diagnosis of infectious diseases and efficient food and water safety control. Methods for identification of bacterial colonies on gelified nutrient broth have the potential to bring an attractive solution, combining simple optical instrumentation, no need for sample preparation or labelling, in a non-destructive process. Here, we studied the possibility of discriminating different bacterial species at a very early stage of growth (6 hours of incubation at 37°C), on thin layers of agar media (1mm of Tryptic Soy Agar), using light forward-scattering and learning algorithms (Bayes Network, Continuous Naive Bayes, Sequential Minimal Optimisation). A first database of more than 1000 scatterograms acquired on seven Gram-negative strains yielded a recognition rate of nearly 80%, after only 6 hours of incubation. We investigated also the prospect of identifying different strains from a same species through forward scattering. We discriminated thus four strains of Escherichia coli with a recognition rate reaching 82%. Finally, we show the discrimination of two species of coagulase-negative Staphylococci (S. haemolyticus and S. cohnii), on a commercial selective pre-poured medium used in clinical diagnosis (ChromID MRSA, bioMérieux), without opening lids during the scatterogram acquisition. This shows the potential of this method – non-invasive, preventing cross-contaminations and requiring minimal dish handling – to provide early clinically-relevant information in the context of fully automated microbiology labs.

1. 3rd International Conference on Bio-Sensing Technology
Optical forward-scattering for identification
of bacteria within microcolonies.
Antoine CUER
Joe-Loïc KODJA
Arthur LEFEBVRE
Florian LICARI
Robin LOUVET
Anil NARASSIGUIN
Mathieu DUPOY
Pierre MARCOUX
Frédéric MALLARD

2. Introduction: How a bacterium species can be identified ?
Molecular methods
Microscopy /
staining
Genomic analysis
Morphological
characteristics
ID based upon the
composition of cellular
membrane
Enzymatic activities
[P69] Non-invasive detection of
bacteria via the sensing of volatile
metabolites released by enzymatic
Biochemical
API L.H.
activity test Guillemot, M. Vrignaud, P.R.
tests
Marcoux, T.-H. Tran-Thi.
Antigenic
characteristics
ID based upon the
global cellular
composition
Spectral
fingerprint
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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3. Introduction: Rapid methods in diagnostic
Reference method
Rapid method under investigation
24h
API tests (bioMérieux):
Pathogen is identified
according to the results (+ or
-) of a series a biochemical
tests
6h
24h
a few
seconds
Raman spectroscopy:
Pathogen is identified
according to its Raman
fingerprint
• identification tests must be performed on a much smaller amount
of cells (1-103 cells), so as to reduce the time dedicated to growth
• identification tests must be faster
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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4. Introduction: optical methods for identification
1. Raman: inelastic scattering / Forward-scattering: elastic scattering
Elastic scattering yields much more photons:
exposure time in Raman: 30 s (spectrum on a single cell)
exposure time in forward-scattering: ∼1 ms (single-cell; microcolony)
2. Raman: vibrational spectroscopy, a peak is linked with a particular
spectroscopy
vibration mode of a type of covalent bond: e.g. υ(C=O); υ(C−Η)…
Forward-scattering is not a spectroscopy: it does not yield information
about cell composition, but rather about morphological characteristics.
Raman (inelastic
scattering)
Diffraction (elastic
scattering)
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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5. Introduction: optical methods for identification
3. As for Raman spectroscopy and intrinsic fluorescence, forwardscattering is a label-free method. Non invasive technique, requires
method
little or no consumable, can be automated.
automated
4. In direct space: the packing of bacteria
cells within microcolony induces a
periodic modulation of phase (refraction
index) and absorbance
⇒ in reciprocal space:
it yields diffraction
fringes.
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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6. Introduction: experimental process and setup
Elastic
diffusion
(laser)
microcolony
(6h of incubation)
(
Forward-scattering
(543 nm)
camera 1
(images in
direct space)
15
5
Multivariate
statistics
strain
EC21
HA4
PCA pl ot f or EC2 1 and HA4 scat t er i ng pat t er ns
classification
(for ex. Principal
Component Analysis)
PC# 2
Microcolony on
agar
camera 2
(acquisition of
scattering
pattern)
)
H. alvei
10
Laser (534nm)
Projection
onto a
basis
α 1 ... α n
functions
descriptor
(n-component vector)
0
E. coli
-5
PC# 1
0
20
40
60
80
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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7. Introduction
Let’s investigate the possibility of using forward-scattering as an
identification method on microcolonies after 6 hours of incubation
(37°C), directly on agar medium:
1. Scattering patterns: How are they formed ?
patterns
What kind of information do they give ?
2. Image analysis: How can we compare scattering patterns
analysis
quantitatively ?
3. Results: A first database of Gram- species at 6h
on TSA (Tryptic Soy Agar)
4. First results on two CNS (Coagulase-Negative Staphylococci) at 6h
Staphylococci
on ChromID MRSA.
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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8. 1. Scattering patterns : phenotypic information
A scattering pattern contains complex phenotypic information, which is a
sum of various parameters, such as:
1. Refraction index: of nutrient agar medium, of bacteria, of extracellular
matrix.
2. Cellular shape.
shape
3. Geometry of bacteria stacking within microcolony (the scattering of
planktonic cells, i.e. growing in liquid medium, yields a much less complex
pattern).
4. Shape of the whole microcolony: it acts as a micro-lens.
microcolony
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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9. 1. Scattering patterns : two kinds of fringes
 Fringes at low angles: corresponds to low spatial frequencies, the
angles
whole bacterial colony scatters. More light, but less complex shape.
Available from the start. Spatial periods: a few tens of µm.
4h50
 Fringes at high angles: corresponds to high spatial frequencies,
angles
scattering due to the stacking of cells. Much less photons (appears after
several hours of incubation), but more complex shape. Seems to yield
more discrimination.
Spatial periods: ∼ 1 µm and less.
LA
LA
HA
HA
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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10. 2. Image analysis: four strains of the same species at t=6h
E. coli ATCC25922
(EC10)
4h50
E. coli ATCC8739
(EC11)
How can we quantitatively compare
all these scattering patterns ?
E. coli ATCC35421
(EC21)
E. coli ATCC11775
(EC28)
E. coli ATCC8739
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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11. 2. Image analysis: calculation of descriptor
image
(scatterogram)
vector (descriptor)
V=(V1,V2,…,Vn) made of Anm projections:
4h50
Projection Anm of scattering pattern f(r,θ) onto Zernike polynomial Znm
= similarity coefficient between the image f and the basis function Znm
project
ion
Znm(r,θ )
ction
proje
projection
f(r,θ )
project
ion
The more similar f and Znm look, the
higher is Anm (Zernike moment).
Projections Anm (Zernike
moments) are calculated
for the first 120 Zernike
polynomials Znm
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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12. 3. Results: a first database on Gram- species (t = 6h)
Classified as
Imaged
scatterogram
EC8
EC8
48,8
7,9
0,0
11,0
EC10
6,4
49,1
14,5
EC11
0,0
7,3
EC21
12,9
EC28
EC10 EC11 EC21 EC28
HA4
CF7
17,3
0,0
15,0
0,0
25,5
2,7
1,8
89,1
0,0
2,7
0,9
0,0
0,0
0,0
81,9
0,0
0,0
5,2
20,5
24,2
1,5
0,8
42,4
0,0
10,6
HA4
0,8
0,0
0,0
0,8
0,0
86,0
12,4
CF7
7,8
0,0
0,0
1,7
0,0
3,5
87,0
sum
100%
(127)
100%
(110)
100%
(110)
100%
(116)
100%
(132)
100%
(121)
100%
(115)
Average classification rate (Naive Bayes) over
the whole database: 69%.
Forward scattering on
microcolonies (6h of
incubation, 37°C) growing on
a thin layer (1mm) of TSA
(Trypcase Soy Agar). Laser
beam: 100µm∅ on bacteria.
bacteria
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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13. 3. Results: a first database on Gram- species (t = 6h)
Can we discriminate the different
strains of the E. coli species ?
Principal Component Analysis
Supervised learning
(Naive Bayes Continuous)
⇒ Classification rate = 82% on average
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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14. 4. Results: distinguishing two species of Staphylococci
 Second step: with commercial Petri
dishes (5mm thick), scattering patterns
are acquired without opening lids
 no risk of cross-contamination
between samples
 We chose ChromID MRSA (bioMérieux)
as a nutrient medium: screening of
Gram+ strains resistant to methicillin.
methicillin
 Can we discriminate, after 6h of incubation, two species of
Staphylococci that grow on ChromID MRSA ?
Study on Staphylococcus haemolyticus and Staphylococcus cohnii, two
methicillin-resistant species (Coagulase-Negative Staphylococci).
 As we obtain a significantly slower growth, we reduce the laser beam
on bacteria down to 25µm ∅ .
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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15. 4. Results: distinguishing two species of Staphylococci
Forward scattering through the whole Petri dish (including lid).
6h of incubation (37°C). 2 species of methicillin-resistant Staphylococci
growing on ChromID MRSA (bioMérieux). Laser beam: 25µm∅ on bacteria.
S. cohnii
S. cohnii
S. haemolyticus
Principal Component Analysis
S. haemolyticus
Supervised learning (Naive Bayes Continuous)
⇒ Classification rates: 92% on average
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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16. Conclusion
Identifying pathogenic species is not enough: a complete diagnosis
must include Antibiotic Susceptibility Testing (AST).
→ To guide the selection and modification of antimicrobial therapy
Currently under investigation…
Towards label-free, non invasive (without
opening lids), non destructive, automated
methods.
Less than 6h for identification + AST
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P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
© CEA. All rights reserved

17. Acknowledgments
Mathieu DUPOY
n
atio
en t
m
stru
in
ical
opt
Antoine CUER, Joe-Loïc KODJA
Arthur LEFEVBRE, Florian LICARI
Robin LOUVET, Anil NARASSIGUIN
Charles-Edmond BICHOT
Frédéric MALLARD
Frédéric PINSTON
y
o lo g
bi
icro
m
g
inin
m
ata
d
and
is
alys
n
ge a
i ma
http://eric.univ-lyon2.fr/~ricco/tanagra/fr/tanagra.html
| 17
http://www.cs.waikato.ac.nz/ml/weka/
P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013
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