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Towards rapid detection of Staphylococcus aureus during blood culture Vincent Templier, PhD Student CEA, INAC-SPrAM-CREAB, F-38000 Grenoble, France Contact : [email protected] World Congress and Expo on Applied Microbiology August 18-20, 2015, Frankfurt The threat of bacteremia • Bacteremia or bloodstream infection (BSI) = presence of viable bacteria in blood1,2. • Affects mainly immunocompromized patients but not only. Introduction • 200 000-250 000 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 BSI5. Bacteremia = life-threatening infection which needs rapid medical care. 1Reimer, L.G. et al., Clin Microbiol Rev, 1997 ; 2Wilson, M.L. et al., Clinical and Laboratory Standards Institute, 2007 ; 3Bearman, G.L., Archive of Medical Research, 2005 ; 4Dellinger, R.P. et al., Critical Care Medicine, 2013 ; 5Timsit, J.F., et al., BMC Infect Dis, 2014. 2 Current procedure for microbial identification 1. Assessment of bacterial presence (5 to 10mL) dilution Hemoculture 12-36h Low contamination (1 CFU / 10mL) Patient Introduction Blood sample Appropriate Empirical antibiotic treatment 3 Current procedure for microbial identification 1. Assessment of bacterial presence 2. Bacteria isolation on solid media (5 to 10mL) dilution Low contamination (1 CFU / 10mL) Hemoculture If positive Growth and Isolation 12-36h 12-24h Gram coloration and Microscopic observation Possible treatment modifications Patient Introduction Blood sample Appropriate Empirical antibiotic treatment 4 Current procedure for microbial identification 1. Assessment of bacterial presence 2. Bacteria isolation on solid media (5 to 10mL) dilution Low contamination (1 CFU / 10mL) Hemoculture If positive Identification and Growth and Isolation 12-36h 12-24h 24h - 72h Gram coloration and Microscopic observation Possible treatment modifications Patient Antimicrobial Susceptibility Testing Empirical antibiotic treatment Result Introduction Blood sample Appropriate 3. Full identification of the causative bacteria Treatment adjusting 72h – 96h Suitable antibiotic treatment A delay or a misuse in antibiotic treatment (in case of antibiotic resistant bacteria) results in an augmentation of patient deaths6,7. Imperative need to shorten diagnosis time 6Davey, P.G. et al., Clinical Microbiology and Infection, 2008 ; 7Kumar, A. et al., Chest, 2009. 5 Goal : to perform hemoculture and identification in the same time 1. Hemoculture AND identification 2. Bacteria isolation on solid media 3. Full identification of the causative bacteria (5 to 10mL) Blood sample dilution Hemoculture AND If positive Growth and Isolation 12-24h Identification Identification confirmation and Antimicrobial Susceptibility Testing 24h - 72h Low contamination Patient (1 CFU / 10mL) Possible treatment modifications based on identification results Empirical antibiotic treatment Result Introduction Appropriate Treatment adjusting 72h – 96h Suitable antibiotic treatment To obtain reliable identification results during hemoculture 6 Use of protein biochip and optical detection Samples 1 & 2 Gold layer Glass Prism Antibody array Introduction Cuve Grafting of bacteria specific antibodies by a simple electrochemical reaction. 7 Use of protein biochip and optical detection Samples 1 &2 Gold layer Glass Prism Antibody array Introduction Cuve Grafting of bacteria specific antibodies by a simple electrochemical reaction. Antibody grafted to the surface Reactor Wet phase Prism Dry phase Assets of the SPRi: • Direct and multiplex detection • Label-free • Real time monitoring 8 Culture – Capture – Measure approach8,9 by SPRi Introduction Blood dilution with suitable culture media and artificial contamination Detection in simple and complex media (food matrix) Non destructive method which enables further testing (plating, PCR…) after incubation time. 9 8Bouguelia, S. et al., Lab on a Chip, 2013 ; 9Mondani, L et al., JAM, 2014. Proof of concept in blood Experimental results Detection of 100 UFC.mL-1 of Salmonella enterica serotype Enteritidis in diluted human blood (mean of 3 spots) Specific signal Salmonella detection in blood is feasible in a few hours. What happens with S. aureus? 10 S .aureus detection in culture media Experimental results IgG control (non specific of S. aureus) looks positive. Simultaneous interaction on all antibodies 11 S .aureus detection in culture media Experimental results IgG control (non specific of S. aureus) looks positive. Simultaneous interaction on all antibodies Cause : Staphyloccocal protein A recognizing the Fc fragment of antibodies. Difficult to say if an antibody recognizes its target or if interactions are only "protein A" related. 12 Experimental results IgG cleavage for S. aureus detection 13 Experimental results IgG cleavage for S. aureus detection Anti-S. aureus digested IgG are successfully binding to their target 14 Experimental results IgG cleavage for S. aureus detection 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. 15 Conclusion Conclusions & Perspectives 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 Perspectives Conclusions & 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 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. Thank you for your attention Any questions? 18 Annexs Microorganisms responsible of BSI From 5Timsit, J.F., et al., BMC Infect Dis, 2014, 14, 19 Experimental device Annexs Prism and cuve with 2 chambers. Optical bench of the SPRi system. Experiment monitored on a dedicated software Device placed in an incubator with temperature fixed at 37°C 20 SPRi Principle Bacteria Antibody grafted to the surface Incident light LED Reflected light CCD camera Annexs Computer Plasmon surface wave Wave penetration at the interface Polarizer Resonance angle Θ 21 SPRi Principle 1. Resonance angle Θ 2. Fixed working angle Initial plasmon curve Plasmon curve after interaction ΔR (%) Reflectivity (%) Annexs 1 2 Resonance angle shift with interaction Incident Light Angle (°) 22