Combining 2 Powerful Technologies to Enable Further Discovery in Bacterial Studies

1
Combining 2 Powerful Technologies to Enable
Further Discovery in Bacterial Studies
2
Study Flow
3
Bacterial Count with NovoCyte
4
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

5
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

6
Direct Absolute Counting using NovoCyte

• Syringe Pump Fluidics
• Direct absolute cell/particle counts

7
Direct Absolute Counting NovoCyte vs Others
ACEA Novocyte (Volumetric) ACEA Novocyte (Volumetric) ACEA Novocyte (Volumetric) ACEA Novocyte (Volumetric) Competitor's Flow Cytometer with Reference Beads Competitor's Flow Cytometer with Reference Beads Competitor's Flow Cytometer with Reference Beads Competitor's Flow Cytometer with Reference Beads
Total lymphocyte CD3 /ul CD3CD8/ul CD3CD4/ul Total lymphocyte CD3 /ul CD3CD8/ul CD3CD4 /ul
QC Blood Sample 1 1937 1435 2057 1492
QC Blood Sample 2 1570 1160 404 676 1563 1146 406 669
QC Blood Sample 3 1605 1175 408 685 1558 1153 405 676
Fresh Blood Sample 4 846 634 902 659
Fresh Blood Sample 5 886 605 297 279 925 619 296 286
Fresh Blood Sample 6 1710 888 288 578 1779 913 294 597
8
Absolute Counting NovoCyte vs Others (Contd)
9
Direct Absolute Counting of E. Coli with NovoCyte
Experiment Settings FSC-H Threshold
2000 Volume 30uL Sample Flow Rate 14uL/min
Sample Count in Gate P1 Abs. Count (/uL)
11000 135,727 4,524
110000 15,529 518
10
Advantages of Direct Absolute Bacterial Counting
with NovoCyte

• Low CVs (2)
• High accuracy (5)
• Provides consistent results between sample runs
• Automatic cleaning (low carry over of lt0.1)
• Plug-and-play operation
• Efficient, up to 20,000 events/sec

11
Bacteria Mediated Cytotoxicity (xCelligence)
12
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

13
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)

14
Bacteria species validated on xCelligence system

• Clostridium difficile
• Bacillus
• Vibrio cholera
• Vibrio vulni?cus
• Neisseria meningitidis

15
Study One
16
Responses of four cell lines to Vibrio cholerae
toxin
17
Analytical sensitivity of CT diluted with pooled
negative stool specimens
18
Representative CT-RTCA results for isolates and
clinical specimens
19
Study Two
20
RtxA1 causes acute necrotic cell death
21
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!)

22
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).
  doi10.1128/JCM.02601-13
• 2. In vitro assessment of marine bacillus for use
  as livestock probiotics. Prieto, M. L.,
  OSullivan, L., Tan, S. P., McLoughlin, P.,
  Hughes, H., Gutierrez, M., Lane, J. a, et al.
  (2014).Marine drugs, 12(5), 242245.
  doi10.3390/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.
  doi10.1128/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), 140615. doi10.1093/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), 1018.
  doi10.1016/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), 412934. doi10.1128/JCM.011
  04-10
• 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.
  doi10.1371/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), 97100.
  doi10.1016/j.mimet.2009.04.007

23
Appendix Facts About Bacteria
24
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

25
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)

26
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