Combining 2 Powerful Technologies to Enable Further Discovery in Bacterial Studies

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Direct Absolute Count (NovoCyte)

Number of Bacteria (Titer)

Infection of Cells of Interest with Certain Amount of Bacteria

Monitoring of Cell Index Changes of Target Cells to Assess Bacteria Mediated Cytotoxic Effects (xCelligence)

Study Flow

Bacterial Count with NovoCyte

Significance of Bacterial Count

• Food manufacturers– Required by regulatory authorities e.g. FDA to monitor the number and type of

bacteria in their products

– Beer and wine companies monitor the growth of yeast in their distilling process

• Environmental concerns– Water treatment plants monitor the effectiveness of their sterilization process

• Biotechnology firms– Closely regulate bacterial growth to produce useful pharmaceutical products

• Clinical laboratories– Monitor the growth rate of bacteria from patients to determine their antimicrobial

sensitivity

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How to Count Bacteria with Precision

• By dilution and plating– Dead bacteria do not form colonies. Some bacteria occur as single cells while other

species hang together in chains or clumps of 2 or more baceteria

• Counting chambers– Consist of a special microscope slide with a coverglass

– Can't tell which bacteria are alive thus this method is useless in disinfection studies

• Membrane filters– Huge volumes of liquid (e.g. water) can be filtered to show a few bacteria per liter

– Filter can be rinsed with sterile water to remove anything that could potentially interfere with bacterial growth

• Photometers and spectrometers– Efficient, no need to wait overnight for the colonies on the agar plates

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Direct Absolute Counting using NovoCyte

• Syringe Pump Fluidicso Direct absolute cell/particle counts

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ACEA Novocyte (Volumetric)Competitor's Flow Cytometer with Reference

Beads

Total lymphocyte

CD3+ /ul

CD3+CD8+/ul

CD3+CD4+/ul

Total lymphocyte

CD3+ /ul

CD3+CD8+/ul

CD3+CD4+/ul

QC Blood

Sample 1 1937 1435 　 　 2057 1492 　 　

Sample 2 1570 1160 404 676 1563 1146 406 669

Sample 3 1605 1175 408 685 1558 1153 405 676

Fresh Blood

Sample 4 846 634 　 　 902 659 　 　

Sample 5 886 605 297 279 925 619 296 286

Sample 6 1710 888 288 578 1779 913 294 597

Direct Absolute Counting: NovoCyte vs Others

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Absolute Counting: NovoCyte vs Others (Cont’d)

Direct Absolute Counting of E. Coli with NovoCyte

Experiment Settings:FSC-H Threshold: 2000Volume: 30uLSample Flow Rate: 14uL/min

1:1000 Dilution 1:10000 Dilution

Sample Count in Gate P1 Abs. Count (/uL)

1:1000 135,727 4,5241:10000 15,529 518

Advantages of Direct Absolute Bacterial Counting with NovoCyte

o Low CV’s (±2%)

o High accuracy (±5%)

o Provides consistent results between sample runs

o Automatic cleaning (low carry over of <0.1%)

o “Plug-and-play” operation

o Efficient, up to 20,000 events/sec

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Bacteria Mediated Cytotoxicity (xCelligence)

Bacteria mediated toxicity

• Direct damage

– Results from the means of a bacteria utilizes to adhere to host, grow and evade host defences

– Usually the more minor form of bacteria mediated toxicity

• Hypersensitivity reactions

– An immune response that is excessive to a point where it leads to damage (as with endotoxins) or is potentially damaging to the individual host

• Toxin-induced damage

– About 220 bacterial toxins are known of which 40% disrupt plasma membranes

– Exotoxins, endotoxins

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Bacterial Assays

• Bacterial agglutination

– Commonly used to identify specific bacterial antigens, and in turn, the identity of such bacteria

– Important technique in diagnosis

• Bacterial count assays (please refer to the previous section)

– Absolute direct count using our NovoCyte

• Conventional asssys for bacterial-mediated cytotoxicity (e.g. manual cell count with Trypan blue, MTT, release of LDH, ATP assay, microscopic analysis)

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Bacteria species validated on xCelligence system

• Clostridium difficile

• Bacillus

• Vibrio cholera

• Vibrio vulnificus

• Neisseria meningitidis

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Study One

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Responses of four cell lines to Vibrio cholerae toxin

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Analytical sensitivity of CT diluted with pooled negative stool specimens

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Representative CT-RTCA results for isolates and clinical specimens

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Study Two

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RtxA1 causes acute necrotic cell death

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Summary – Key Benefits of xCelligence

• A simple alternative to traditional methods for measuring bacteria mediated cytotoxicity

• Sensitive readout on cell mophology and adhesion changes in response to bacterial infection

• Quantitative monitoring of onset and kinetics of bacterial mediated effects in real time for up to hundreds hours

• Indentify the optimal bacterial titer and assay time point for subsequent screening of inhibitory compounds, neutralizing antibodies or neutralizing serums.

• No labelling of cells or bacteria required

• No post-experiment cell handling, sample preparation, or data collection (infect and walk away!)

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Publication list

1. Real-time cellular analysis coupled with a specimen enrichment accurately detects and quantifies Clostridium difficile toxins in stool. Huang, B., Jin, D., Zhang, J., Sun, J. Y., Wang, X., Stiles, J., Xu, X., et al. (2014).Journal of clinical microbiology, 1(January). doi:10.1128/JCM.02601-13

2. In vitro assessment of marine bacillus for use as livestock probiotics. Prieto, M. L., O’Sullivan, L., Tan, S. P., McLoughlin, P., Hughes, H., Gutierrez, M., Lane, J. a, et al. (2014).Marine drugs, 12(5), 2422–45. doi:10.3390/md12052422

3. Quantitative Detection of Vibrio cholera Toxin by Real-Time and Dynamic Cell Cytotoxicity Monitoring. Jin, D., Luo, Y., Zheng, M., Li, H., Zhang, J., Stampfl, M., Xu, X., et al. (2013).Journal of clinical microbiology. doi:10.1128/JCM.01959-13

4. A bacterial RTX toxin causes programmed necrotic cell death through calcium-mediated mitochondrial dysfunction. Kim, Y. R., Lee, S. E., Kang, I.-C., Nam, K. Il, Choy, H. E., & Rhee, J. H. (2013).The Journal of infectious diseases, 207(9), 1406–15. doi:10.1093/infdis/jis746

5. Real-time impedance analysis of host cell response to meningococcal infection. Slanina, H., König, a, Claus, H., Frosch, M., & Schubert-Unkmeir, a. (2011).Journal of microbiological methods, 84(1), 101–8. doi:10.1016/j.mimet.2010.11.004

6. Assessment of Clostridium difficile infections by quantitative detection of tcdB toxin by use of a real-time cell analysis system. Ryder, A. B., Huang, Y., Li, H., Zheng, M., Wang, X., Stratton, C. W., Xu, X., et al. (2010).Journal of clinical microbiology, 48(11), 4129–34. doi:10.1128/JCM.01104-10

7. Neisseria meningitidis induces brain microvascular endothelial cell detachment from the matrix and cleavage of occludin: a role for MMP-8. Schubert-Unkmeir, A., Konrad, C., Slanina, H., Czapek, F., Hebling, S., & Frosch, M. (2010).PLoS pathogens, 6(4), e1000874. doi:10.1371/journal.ppat.1000874

8. An ultrasensitive rapid immunocytotoxicity assay for detecting Clostridium difficile toxins. He, X., Wang, J., Steele, J., Sun, X., Nie, W., Tzipori, S., & Feng, H. (2009).Journal of microbiological methods, 78(1), 97–100. doi:10.1016/j.mimet.2009.04.007

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Appendix: Facts About Bacteria

Bacteria - Basic Facts

• Prokaryotic microorganisms typically a few microns in length

• 40 million bacterial cells in a gram of soil and a million in a millilitre of fresh water

• Have a number of shapes, ranging from spheres (cocci) to rods (bacilli or vibrio for slightly curved rods or comma-shaped) and spirals (spirilla or spirocchaetes)

• Many exist as single cells, others associate in characteristic patterns

– Neisseria form diploids (pairs)

– Staphylococcus group together in “bunch of grapes” clusters

– Actinobacteria can be elongated to form filaments and are often surrounded by a sheath that contains many individual cells. E.g. Nocardia form complex branched filaments similar in appearance to some fungal mycelia

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Bacteria – Cellular Structures

• Extracellular

– Cell wall present on the outside of the cytoplasmic membrane

– Consists of peptidoglycan

– Essential to survival and the antibiotic penicillin kills the bacteria by inhibiting the synthesis of peptidoglycan

– Gram-positive (thick cell wall) vs Gram-negative (thin cell wall)

• Intracellular

– Usually no membrane-bound organelles (e.g lack of true nucleus, mitochondria, chloroplasts)

– A single circular chromosome located in the cytoplasm in an irregularly shaped body called the nucleoid

– Micro-compartments (e.g. carboxysomes, magnetosomes)

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Bacteria - Biofilms & Quorum Sensing

• Often attach to surfaces and form biofilms.

• Bacteria living in biofilms form secondary structures such as micocolonies to enable better diffusion of nutrients.

• Common in natural environments (e.g. soil, surfaces of plants) and during chronic bacterial infections or infections of implanted medical devices

• Bacteria protected within biofilms are much harder to kill than individual isolated baceteria

• In more harsh conditions (e.g. starved of amino acids), they would detect surrounding cells and migrate toward each other (quorum sensing), and aggregate to form fruiting bodies where they cooperate to perform separate tasks

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