Rapid label-free identification of pathogens with optical elastic scattering
1
Identification down to the strain level: classification rate = 75% identifica tion Copyright © 2015 Pierre R. MARCOUX, [email protected] Jérémy Méteau, 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 Rapid label-free identification of pathogens with optical elastic scattering 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/s00253-0 13-5495-4 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 n ... 1 scatteri ng pattern microcolony on Petri dish, after 6h at 37C (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 =532nm features extracti on supervis ed learning 1ms 5-20 s For cultures on transparent and opaque agar media (e.g. blood-supplemented agar media, such as COS) Laser (532nm ) Pet ri dis h 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., 37C), 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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Transcript of Rapid label-free identification of pathogens with optical elastic scattering
Identification down to the strain level: classification rate = 75%
identification
Copyright © 2015 Pierre R. MARCOUX, [email protected]
Jérémy Méteau, Valentin Genuer, Pierre R. Marcoux, Emmanuelle SchultzCEA-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 Rapid label-free identification of pathogens with optical elastic scatteringscattering
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 n ...1
scattering pattern
microcolony on Petri dish, after6h at 37C (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 =532nm
featuresextraction
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 dishcamera
Closed lid, agar on top.
Closed lid, agar below.
RESULTS FROM FORWARD SCATTERINGATCC14053 ATCCC2091 ATCC10231 ATCC2001 ATCC14243 ATCC34449 ATCC13803 ATCC9763 ATCC25922 ATCC8739 ATCC35421 ATCC11775 ATCC13047 ATCC49741 ATCC12228C. 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 ATCC140530,0 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 ATCCC20910,8 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 ATCC102317,1 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 ATCC20010,8 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 ATCC142433,8 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 ATCC344493,4 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 ATCC138030,0 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 ATCC97630,0 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 ATCC259220,8 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 ATCC87390,0 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 ATCC354210,6 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 ATCC117750,0 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 ATCC130470,0 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 ATCC497410,9 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., 37C), gathering Gram, Gram+ and yeasts: 1900 scattering patterns, more than 120 patterns per strain.
Fungi Gram- Gram+ <---- classified as96,8 2,8 0,4 Fungi5,0 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:
