Optical forward-scattering for identification of bacteria within microcolonies

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Health & Medicine

Published on January 15, 2014

Author: prmarcoux

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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.

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

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 |2 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 |3 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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) |4 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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. |5 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 |6 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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. |7 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 |8 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 |9 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 | 10 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 | 11 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 | 12 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 | 13 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 ∅ . | 14 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 | 15 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 | 16 P.R. Marcoux | Forward-scattering for bacterial identification | 13 May 2013 © CEA. All rights reserved

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 © CEA. All rights reserved

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