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Identification down to the strain level:
classification rate = 75%
identification
Copyright © 2015 Pierre R. MARCOUX, pierre.marcoux@cea.fr
Jé ré my Mé teau, Valentin Genuer, Pierre R. Marcoux, Emmanuelle Schultz
CEA-LETI, Département des Technologies pour la Biologie et la Santé, 17 avenue des Martyrs, 38054 Grenoble cedex 9, France
Rapid label-free identification of pathogens with optical elastic scatteringRapid label-free identification of pathogens with optical elastic scattering
PRINCIPLE:
• As for Raman spectroscopy and intrinsic fluorescence, elastic scattering is a label-free method.
• A laser beam targets the microcolony to be identified, through closed lid. 1
• Noninvasive technique, requires little or no consumable , can be automated.
→ For any kind of culture of fungi or bacteria on transparent agar media
Label-free identification, directly on agar plate with a simple and low-cost instrumentation. Noninvasive (closed lid) and nondestructive
method, with a short acquisition time (1ms).
Measurement can be done on a single microcolony: forward scattering for growth on transparent media (TSA, SDA, etc.) and backward
scattering for opaque media, such as blood-supplemented agar media (COS, etc.).
Outline: 1
P.R. Marcoux et al.; Appl. Microbiol. Biotechnol.,
2014, 98, 2243-2254.
http://dx.doi.org/10.1007/s00253-013-5495-4
FORWARD SCATTERING BACKWARD SCATTERING
Acquisitions (1 ms) are
done with closed lid, agar
on top. The size of the
probed zone depends on
the Z position of the Petri
dish.
Prototype Microdiff: 15kg;
25k€; 55×43×59 cm
descriptor
( )nαα ...1
scattering
pattern
microcolony on Petri dish, after
6h at 37°C (8-200µm Ø)
• The packing of bacteria within microcolony induces
a periodic modulation of phase (refraction index) and
absorbance ⇒ it yields diffraction fringes.
laser
laser λ=532nm
features
extraction
supervised
learning
1ms
5-20 s
→ For cultures on transparent and opaque agar media
(e.g. blood-supplemented agar media, such as COS)
Laser
(532nm)
Petri
dish
camera
Closed lid, agar on top.
Closed lid,
agar below.
RESULTS FROM FORWARD SCATTERING
ATCC14053 ATCCC2091 ATCC10231 ATCC2001 ATCC14243 ATCC34449 ATCC13803 ATCC9763 ATCC25922 ATCC8739 ATCC35421 ATCC11775 ATCC13047 ATCC49741 ATCC12228
C. albicans C. albicans C. albicans C. glabrata C. krusei C. lusitaniae C. tropicalis S. cerevisiae E. coli E. coli E. coli E. coli E. cloacae S. epidermidis S. epidermidis <--- classified as
61,3 0,0 0,0 19,8 0,0 10,8 5,4 0,0 0,0 2,7 0,0 0,0 0,0 0,0 0,0 C. albicans ATCC14053
0,0 61,7 14,8 0,0 2,6 0,0 6,1 11,3 0,0 0,0 2,6 0,0 0,9 0,0 0,0 C. albicans ATCCC2091
0,8 9,8 54,5 0,0 3,0 0,0 22,0 9,8 0,0 0,0 0,0 0,0 0,0 0,0 0,0 C. albicans ATCC10231
7,1 0,0 0,9 83,9 0,9 5,4 0,0 0,0 0,0 0,0 0,0 0,9 0,0 0,9 0,0 C. glabrata ATCC2001
0,8 1,6 0,0 1,6 93,8 0,0 0,0 0,8 0,8 0,0 0,0 0,0 0,0 0,0 0,8 C. krusei ATCC14243
3,8 1,5 0,0 13,0 0,0 72,5 3,8 0,8 0,0 2,3 0,0 0,0 2,3 0,0 0,0 C. lusitaniae ATCC34449
3,4 5,0 22,7 0,8 4,2 0,0 59,7 4,2 0,0 0,0 0,0 0,0 0,0 0,0 0,0 C. tropicalis ATCC13803
0,0 6,0 21,1 0,0 3,0 0,8 3,8 65,4 0,0 0,0 0,0 0,0 0,0 0,0 0,0 S. cerevisiae ATCC9763
0,0 0,0 0,0 0,0 0,5 0,0 0,0 0,0 92,9 0,5 0,5 2,2 2,7 0,5 0,0 E. coli ATCC25922
0,8 0,0 0,0 3,0 0,0 1,5 0,0 0,0 2,3 89,5 0,0 1,5 1,5 0,0 0,0 E. coli ATCC8739
0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 8,9 0,0 83,9 0,0 7,1 0,0 0,0 E. coli ATCC35421
0,6 0,0 0,0 1,3 0,0 0,0 0,0 0,0 11,9 1,9 0,0 80,0 0,6 1,3 2,5 E. coli ATCC11775
0,0 1,0 0,0 0,0 0,0 3,1 0,0 0,0 13,5 7,3 9,4 5,2 60,4 0,0 0,0 E. cloacae ATCC13047
0,0 0,0 0,0 0,0 2,7 0,0 0,0 0,0 0,9 0,0 0,0 2,7 0,0 78,6 15,2 S. epidermidis ATCC49741
0,9 1,7 0,0 0,0 0,9 0,0 0,0 0,0 0,9 0,0 0,0 3,5 0,0 7,0 90,4 S. epidermidis ATCC12228
→ A database was collected at 532nm, after 6h of incubation (aerobic cond., 37°C), gathering Gram−, Gram+ and yeasts: 1900 scattering patterns, more than 120 patterns per strain.
Fungi Gram- Gram+ <---- classified as
96,8 2,8 0,4 Fungi
5,0 92,4 2,6 Gram-
3,9 3,4 92,7 Gram+
Classification rate = 94% for the discrimination
between Gram−, Gram+ and yeasts.
C. albicans ATCC14053 E. coli
ATCC25922
E. coli ATCC35421
94% for the discrimination between the 3 species
of Candida yeasts
86% for the discrimination between the 4 strains of
E. coli bacteria
87% for the discrimination between the 5 bacteria
species
Preliminary results (classification rates) with a
small database collected on 3 strains of bacteria
and 3 strains of yeasts:

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Rapid label-free identification of pathogens with optical elastic scattering

  • 1. Identification down to the strain level: classification rate = 75% identification Copyright © 2015 Pierre R. MARCOUX, pierre.marcoux@cea.fr Jé ré my Mé teau, Valentin Genuer, Pierre R. Marcoux, Emmanuelle Schultz CEA-LETI, Département des Technologies pour la Biologie et la Santé, 17 avenue des Martyrs, 38054 Grenoble cedex 9, France Rapid label-free identification of pathogens with optical elastic scatteringRapid label-free identification of pathogens with optical elastic scattering PRINCIPLE: • As for Raman spectroscopy and intrinsic fluorescence, elastic scattering is a label-free method. • A laser beam targets the microcolony to be identified, through closed lid. 1 • Noninvasive technique, requires little or no consumable , can be automated. → For any kind of culture of fungi or bacteria on transparent agar media Label-free identification, directly on agar plate with a simple and low-cost instrumentation. Noninvasive (closed lid) and nondestructive method, with a short acquisition time (1ms). Measurement can be done on a single microcolony: forward scattering for growth on transparent media (TSA, SDA, etc.) and backward scattering for opaque media, such as blood-supplemented agar media (COS, etc.). Outline: 1 P.R. Marcoux et al.; Appl. Microbiol. Biotechnol., 2014, 98, 2243-2254. http://dx.doi.org/10.1007/s00253-013-5495-4 FORWARD SCATTERING BACKWARD SCATTERING Acquisitions (1 ms) are done with closed lid, agar on top. The size of the probed zone depends on the Z position of the Petri dish. Prototype Microdiff: 15kg; 25k€; 55×43×59 cm descriptor ( )nαα ...1 scattering pattern microcolony on Petri dish, after 6h at 37°C (8-200µm Ø) • The packing of bacteria within microcolony induces a periodic modulation of phase (refraction index) and absorbance ⇒ it yields diffraction fringes. laser laser λ=532nm features extraction supervised learning 1ms 5-20 s → For cultures on transparent and opaque agar media (e.g. blood-supplemented agar media, such as COS) Laser (532nm) Petri dish camera Closed lid, agar on top. Closed lid, agar below. RESULTS FROM FORWARD SCATTERING ATCC14053 ATCCC2091 ATCC10231 ATCC2001 ATCC14243 ATCC34449 ATCC13803 ATCC9763 ATCC25922 ATCC8739 ATCC35421 ATCC11775 ATCC13047 ATCC49741 ATCC12228 C. albicans C. albicans C. albicans C. glabrata C. krusei C. lusitaniae C. tropicalis S. cerevisiae E. coli E. coli E. coli E. coli E. cloacae S. epidermidis S. epidermidis <--- classified as 61,3 0,0 0,0 19,8 0,0 10,8 5,4 0,0 0,0 2,7 0,0 0,0 0,0 0,0 0,0 C. albicans ATCC14053 0,0 61,7 14,8 0,0 2,6 0,0 6,1 11,3 0,0 0,0 2,6 0,0 0,9 0,0 0,0 C. albicans ATCCC2091 0,8 9,8 54,5 0,0 3,0 0,0 22,0 9,8 0,0 0,0 0,0 0,0 0,0 0,0 0,0 C. albicans ATCC10231 7,1 0,0 0,9 83,9 0,9 5,4 0,0 0,0 0,0 0,0 0,0 0,9 0,0 0,9 0,0 C. glabrata ATCC2001 0,8 1,6 0,0 1,6 93,8 0,0 0,0 0,8 0,8 0,0 0,0 0,0 0,0 0,0 0,8 C. krusei ATCC14243 3,8 1,5 0,0 13,0 0,0 72,5 3,8 0,8 0,0 2,3 0,0 0,0 2,3 0,0 0,0 C. lusitaniae ATCC34449 3,4 5,0 22,7 0,8 4,2 0,0 59,7 4,2 0,0 0,0 0,0 0,0 0,0 0,0 0,0 C. tropicalis ATCC13803 0,0 6,0 21,1 0,0 3,0 0,8 3,8 65,4 0,0 0,0 0,0 0,0 0,0 0,0 0,0 S. cerevisiae ATCC9763 0,0 0,0 0,0 0,0 0,5 0,0 0,0 0,0 92,9 0,5 0,5 2,2 2,7 0,5 0,0 E. coli ATCC25922 0,8 0,0 0,0 3,0 0,0 1,5 0,0 0,0 2,3 89,5 0,0 1,5 1,5 0,0 0,0 E. coli ATCC8739 0,0 0,0 0,0 0,0 0,0 0,0 0,0 0,0 8,9 0,0 83,9 0,0 7,1 0,0 0,0 E. coli ATCC35421 0,6 0,0 0,0 1,3 0,0 0,0 0,0 0,0 11,9 1,9 0,0 80,0 0,6 1,3 2,5 E. coli ATCC11775 0,0 1,0 0,0 0,0 0,0 3,1 0,0 0,0 13,5 7,3 9,4 5,2 60,4 0,0 0,0 E. cloacae ATCC13047 0,0 0,0 0,0 0,0 2,7 0,0 0,0 0,0 0,9 0,0 0,0 2,7 0,0 78,6 15,2 S. epidermidis ATCC49741 0,9 1,7 0,0 0,0 0,9 0,0 0,0 0,0 0,9 0,0 0,0 3,5 0,0 7,0 90,4 S. epidermidis ATCC12228 → A database was collected at 532nm, after 6h of incubation (aerobic cond., 37°C), gathering Gram−, Gram+ and yeasts: 1900 scattering patterns, more than 120 patterns per strain. Fungi Gram- Gram+ <---- classified as 96,8 2,8 0,4 Fungi 5,0 92,4 2,6 Gram- 3,9 3,4 92,7 Gram+ Classification rate = 94% for the discrimination between Gram−, Gram+ and yeasts. C. albicans ATCC14053 E. coli ATCC25922 E. coli ATCC35421 94% for the discrimination between the 3 species of Candida yeasts 86% for the discrimination between the 4 strains of E. coli bacteria 87% for the discrimination between the 5 bacteria species Preliminary results (classification rates) with a small database collected on 3 strains of bacteria and 3 strains of yeasts:

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