Towards rapid detection of Staphylococcus aureus during blood culture World Congress and Expo on Applied Microbiology August 18-20, 2015, Frankfurt Vincent Templier, PhD Student CEA, INAC-SPrAM-CREAB, F Grenoble, France Contact :
The threat of bacteremia 2 Introduction Bacteremia or bloodstream infection (BSI) = presence of viable bacteria in blood 1,2. Affects mainly immunocompromized patients but not only 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 BSI 5. Bacteremia = life-threatening infection which needs rapid medical care. 1 Reimer, L.G. et al., Clin Microbiol Rev, 1997 ; 2 Wilson, M.L. et al., Clinical and Laboratory Standards Institute, 2007 ; 3 Bearman, G.L., Archive of Medical Research, 2005 ; 4 Dellinger, R.P. et al., Critical Care Medicine, 2013 ; 5 Timsit, J.F., et al., BMC Infect Dis, 2014.
Current procedure for microbial identification 3 Introduction Hemoculture 12-36h Empirical antibiotic treatment Patient Blood sample (5 to 10mL) Appropriate dilution Low contamination (1 CFU / 10mL) 1. Assessment of bacterial presence
Current procedure for microbial identification 4 Introduction Hemoculture 12-36h Gram coloration and Microscopic observation Growth and Isolation 12-24h Empirical antibiotic treatment Patient If positive Blood sample (5 to 10mL) 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 Introduction A delay or a misuse in antibiotic treatment (in case of antibiotic resistant bacteria) results in an augmentation of patient deaths 6,7. 6 Davey, P.G. et al., Clinical Microbiology and Infection, 2008 ; 7 Kumar, A. et al., Chest, Hemoculture 12-36h Gram coloration and Microscopic observation Growth and Isolation 12-24h Identification and Antimicrobial Susceptibility Testing 24h - 72h Empirical antibiotic treatment 72h – 96h Suitable antibiotic treatment Treatment adjusting Result Patient If positive Blood sample (5 to 10mL) 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 time Introduction Growth and Isolation 12-24h Identification confirmation and Antimicrobial Susceptibility Testing 24h - 72h Empirical antibiotic treatment 72h – 96h Suitable antibiotic treatment Treatment adjusting Result Patient If positive Blood sample (5 to 10mL) Appropriate dilution Possible treatment modifications based on identification results Low contamination (1 CFU / 10mL) 1. Hemoculture AND identification 2. Bacteria isolation on solid media 3. 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 Introduction 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 Introduction Grafting of bacteria specific antibodies by a simple electrochemical reaction. Antibody array Cuve Glass Prism Gold layer Antibody grafted to the surface Reactor Wet phase Prism Dry phase Assets of the SPR i : Direct and multiplex detection Label-free Real time monitoring Samples 1 &2
Culture – Capture – Measure approach 8,9 by SPR i 9 Introduction 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 8 Bouguelia, S. et al., Lab on a Chip, 2013 ; 9 Mondani, L et al., JAM, 2014.
Proof of concept in blood 10 Experimental results 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 Experimental results IgG control (non specific of S. aureus) looks positive. Simultaneous interaction on all antibodies
S.aureus detection in culture media 12 Experimental results 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 Experimental results
IgG cleavage for S. aureus detection 14 Experimental results Anti-S. aureus digested IgG are successfully binding to their target
IgG cleavage for S. aureus detection 15 Experimental results Workable but enzymatic digestion must be adapted to each antibody. Anti-S. aureus digested IgG are successfully binding to their target Non 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) 16 Conclusions & Perspectives
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 17 Conclusions & Perspectives
Thanks CREAB : 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. 18 Thank you for your attention Any questions?
Microorganisms responsible of BSI 19 Annexs From 5 Timsit, J.F., et al., BMC Infect Dis, 2014, 14,
Experimental device 20 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. Annexs
SPRi Principle 21 Annexs 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 Annexs Resonance angle shift with interaction Δ R (%) Incident Light Angle (°) Reflectivity (%) 1.Resonance angle Θ 2.Fixed working angle 12 Initial plasmon curve Plasmon curve after interaction