Underestimated potential of isothermal microcalorimetry for microbiology. Olivier Braissant (Uni. Basel)

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1 Underestimated potential of isothermal microcalorimetry for microbiology Olivier Braissant (Uni. Basel)

2 Isothermal microcalorimetry in a nutshell

3 The basics Nutrients DNA or RNA Biomass Metabolic byproducts (CO2, lactic acid... etc) Heat ~2pW per cell

4 The basics Cell type Heat production rate per cell detection limit (200 nw) Human erythrocyte 0.01 pw / cellxxxxxxxxxx cellsxxxxxx Human platelets 0.06 pw / cellxxxxxxxxxx cellsxxxxxx Human neutrophils 2.5 pw / cellxxxxxxxxxx cellsxxxxxx Human lymphocytes 5 pw / cellxxxxxxxxxx cellsxxxxxx Human T-lymphoma 8 pw / cellxxxxxxxxxx cellsxxxxxx Human adenocarcinoma (HeLa) 31 pw / cellxxxxxxxxxx cellsxxxxxx Human white adipocytes 49 pw / cellxxxxxvxxxx cellsxxxxxx Rat white adipocytes 40 pw / cellxxxxxxxxxx cellsxxxxxx Rat hepatocytes 329 pw / cellxxxxxxxxxx cellsxxxxxx Mouse fibroblasts 17 pw / cellxxxxxxxxxx cellsxxxxxx Mouse lymphocyte hybridoma 30 pw / cellxxxxxxxxxx cellsxxxxxx Mouse macrophage hybridoma 32 pw / cellxxxxxxxxxx cellsxxxxxx Saccharomyces cerevisiae 4.1 pw / cellxxxxxxxxxx cellsxxxxxx Fusarium roseum 40 pw / cellxxxxxxxxxx cellsxxxxxx Escherichia coli 2.1 pw / cellxxxxxxxxxx cellsxxxxxx Staphylococcus aureus 2 pw / cellxxxxxxxxxx cellsxxxxxx Klebsiella aerogenes 2 pw / cellxxxxxxxxxx cellsxxxxxx Bacillus megaterium 3 pw / cellxxxxxxxxxx cellsxxxxxx Data from: Kemp & Lampecht 2000; James 1987; Kimura & Takahashi 1985; Braissant et al

5 Validation of calorimetric measurments

6 Gas consumption and calorimetry

7 Recent clinical studies Detection of infections Drug susceptibility testing Research

8 Screening of Clostridium difficile - Clostridium difficile is the most frequent cause of healthcare associated diarrhea, and a significant cause of morbidity and mortality among hospitalized patients. - Characterizing C. difficile strains remains quite an issue and require several techniques (stool and culture immunoassays, and PCR) in addition of culture based techniques. - Still a rapid tool to evaluate virulence (agressiveness) and risk of complication could be useful in a clinical setting.

9 Heat production rate of C.difficile cultures

10 Overall

11 Possible cost of virulence - Overall there seems to be a energetic cost for more virulent strains of Clostridium difficile. - Still our study being underpowered it is difficult for now to link it with a specific feature (ribotype, or binary toxin production)

12 The case of Urosepsis - Urosepsis is defined as a sepsis (a systemic inflammatory response) originating from the urinary tract - Urosepsis is virtually always linked with a urinary tract infection with high concentration of pathogens in urine - In most of the cases (i.e. >95%), the pathogen isolated from blood (if any) is the same as the pathogen isolated from urine - Polymicrobial infection are rather rare (5-11%), thus ensuring that only the targeted pathogen is investigated

13 Rapid drug susceptibility testing

14 Same but faster - Good agreement between three techniques (OD, VITEK, Isothermal microcalorimetry) - IMC required only 7 hours to deliver an antibiogram - Only one very major error (i.e., determined susceptible instead of resistant). This is 1.7%. - Such error rate is similar to the one of the VITEK-2 system Consensus IMC : OD IMC : VITEK OD : VITEK n %

15 Solid media or samples Biofilms Implant surface

16 After Ojha and colleagues Mycobacteria the classical way

17 Mycobacterial biofilms our way BCG Allows to transfer the very same biofilm from vial to vial Surprisingly high activity of mycobacterial biofilms...

18 Solid tumor microtissues + =

19 Antimicrobial surfaces testing

20 Parasitic worms and drug testing

21 Thank you

22 Thank you

23 Growth rates in urine Name Medium µ max [h -1 ] µ [h -1 ] Tg [h -1 ] n M. smegmatis Sterilized urine 0.311± ± ±1.6 6 M. phlei Sterilized urine 0.178± ± ±2.7 6 M. kansasii Sterilized urine 0.019± ± ± M. kansasii Modified urine 0.038± ± ±3.1 6 M. tuberculosis Sterilized urine M. tuberculosis Modified urine 0.015± ± ± After Bonkat et al 2012, Urology

24 Urinary tuberculosis and biofilms M. kansasii (grey lines) M. tuberculosis (black lines) After Bonkat et al 2012, Urology

25 Enzymatic reaction but no growth

26 Biofilms in the calorimeter Pseudomonas aeruginosa

27 Chondrocyte growth

28 Validation with microscopy count

29 Describing calorimetric data after Braissant et al (2010), FEMS Microbiology Letters

30 Models for calorimetric data Calorimetry data can be used as proxy for microbiology data. The same models are applicable: Nt = N0 e µt which becomes Qt = Q0 e µt for heat over time data.