Presentation based on a research article published in the journal scientific reports in 2017, entitled as "A Novel Pathogen Capturing Device for Removal and Detection"

1. By-
Govinda R. Navale
A Novel Pathogen Capturing Device
for Removal and Detection
Gwangseong Kim1,2 Horatiu Vinerean3 & Angelo Gaitas1,2
Sci. Rep., 2017; Impact factor- 4.25
1. Kytaro, Inc., Miami, FL, 33199, USA.
2. Department of Electrical and Computer Engineering, Florida International
University, Miami, 33199, Florida, USA.
3. Office of Research and Economic Development, The Office of Laboratory
Animal Research, Florida International University, Miami, 33199, Florida, USA.
Received : 27th February 2017
Published : 17th July 2017

2. Summarized Abstract
 A simple technique that employs an antibody coated polydimethylsiloxane tube is
used for effective capturing of bloodborne and foodborne pathogens.
An effective and economical solution to microbiology techniques that rely on
enrichment, thereby expediting diagnostics.
 Using this method 80.3 ± 5.6% of Staphylococcus aureus with a starting
concentration of ~107 CFU/mL and 95.4 ± 1.0% of Methicillin-resistant
Staphylococcus aureus with starting concentration of ~104 CFU/mL within few
hours were captured.
This concept was extended to live rats with an induced bloodstream S. aureus
infection. A reduction of two fold bacterial load of the rats within a few hours.
Also effective in capture a food pathogen, Salmonella typhimurium, with starting
concentrations as low as ~100 CFU, from 100 or 250 mL of culture broth and in
ground chicken and ground beef.

3. Introduction
 In recent years, efforts have been taken to developed a devices and methods
for capturing of pathogens in fluids (blood, food matrices and water)
 It includes extracorporeal blood circulation methods to capture target
pathogens (e.g. circulating tumour cells) using immuno-capturing techniques
for diagnostics and therapies.
 Immuno-magnetic technologies using engineered Ab- remove food pathogens
and toxins from blood
 A hemofiltration cartridge was developed using the same engineered antibody.
It is evident that there is a multitude of applications and the specific
parameters for each may vary (for instance some applications require high
volume pathogen removal, such as food pathogen testing and environmental
testing

4.  Tube surface modification
 Bacteria Capturing in Blood - Small scale
-High Throughput
- Colony Counting
 S. aureus infections in rats and in vivo capturing S. aureus
 Bacteria Capturing at low Conc. & Large vol.
 Detection of S. typhimurium in Food matrix (Beef/Chicken)
 Fluorescence imaging of tube surface and Detection of pathogens by qRT-
PCR
 Detachment of Pathogens from Tube
 Experimental

5.  Tube surface modification
o A Polydimethylsiloxane (PDMS) tube (1.02 mm, 120 cm)
o Acidic Hydrogen peroxide solution (H2O:HCl:H2O2 in
5:1:1 volume ratio) 5min/RT
o Water Wash (5x) & dried
(Hydrophilic surface with (-OH) available for
functionalization)
o Amino-Propyl-Trimethoxysilane (APTMS) 10 min.
(primary amine group on the surface, confirmed by FITC
florescent dye)
Add
o S. aureus polyclonal Ab - PA1-7246,
o penicillin binding protein 2 A (PBP2a) monoclonal
Ab (10-P08B)- for MRSA capturing,
o BacTrace® Anti-Salmonella CSA-1 Ab
 Experimental
With Ab
a)

6.  Tube surface modification
 Bacteria Capturing in Blood - Small scale
-High Throughput
- Colony Counting
Bacteria
• S. Aureus (ATCC 12598), MRSA (ATCC 43300) and two S. typhimurium (ATCC
29630/14028)
• S. aureus and MRSA OD600 - 0.02–0.04 (about 107 CFU/mL) for
• S. typhimurium OD600 - 0.01 (about 107 CFU/mL) (serially diluted to
104 CFU/mL OR 100 CFU/mL
 Experimental

7. • Bacterial capturing in blood
• Small Scale-5 mL= 0.5 mL culture + 4.5 mL of blood, (107 CFU/mL; ) for S. aureus
and about 104 CFU/mL for MRSA)
• Large scale- 20 mL= 2 mL culture + 18 mL blood
0.5 mL/min
Results
5 mL/min

8. • Bacterial capturing in blood
• Small Scale-5 mL= 0.5 mL culture + 4.5 mL of blood, (107 CFU/mL; ) for S. aureus
and about 104 CFU/mL for MRSA)
• Large scale- 20 mL= 2 mL culture + 18 mL blood
0.5 mL/min
Results
5 mL/min
After 5h

9. • Wistar rats Infected by intraperitoneal (IP) injection with 2 × 1010 CFU of S. aureus.
• After 3 h, extracorporeal circulation through a 240-cm Ab-coated tube was performed for 5 h
at 0.5 mL/min flow rate
Results
 Ab-tube-Bacteria reduction-from 0.9 × 104 CFU/mL to 0.8 × 102 CFU/mL (n = 4)
Control- 0.9 × 104 to 1.0 × 104 CFU/mL (n = 8)
 This device effectively remove pathogens suppressing pathogen concentration in the
bloodstream.
 No blood clotting and clogging the tubes observed
 No adverse effect in the haematological blood profile was not observed
 In-vivo capturing S. aureus

10. Bacteria Capturing at low Conc. & Large volume
S. typhimurium
Initial Conc. Sample Vol Tube Length Total Time Replicated
10 CFU 100 mL 120 cm (30 cmx4) 7 h 2 (AB)
10 CFU 100 mL 120 cm (30 cmx4) 6 h 1 (C)
100 CFU 100 mL 120 cm (30 cmx4) 7 h 1 (D)
1 CFU 250 mL 120 cm (30 cmx4) 7 h 1 (E)
1.5 ml/min
captured S. typhimurium

11. Bacteria Capturing at low Conc. & Large volume
S. typhimurium
Positive RT PCR detection
• D (DNA extraction by bead beating)
• E (DNA extraction by commercial heating kit)
1.5 ml/min

12. (a) Capturing results under the following conditions:
(F-G) 10 CFU in 100 mL, 6 h of continuous flow,
40 cm tube.
(b) Positive RT PCR detection results from Condition
F and G (DNA extraction by heating in DI water).
• Effects of shorter tube length on tube capturing of very low concentration in large
sample volume

13. • Ground chicken and ground beef
• 25 g of ground meats in Seward filtered sample bag.
• Then 225 mL of BPW (Buffered Peptone water) -for ground chicken
Selective media Primary enrichment media with phage- for ground beef
• Blended for 5 minutes.
• Inoculation of S. typhimurium ~ 25 CFU.
• 7 h incubation at 35 °C
• The tube was connected to the filter bag inside the incubator at the beginning of the
incubation.
 Détection in Food matrix

14.  Détection in Food matrix
(a) Experiment set-up (b) RT PCR detection of S. typhimurium in
ground chicken at initial concentration ~25 CFU of S. typhimurium in 250 mL sample
containing 25 g ground chicken in BPW and ground beef in Selective Romer Labs’
primary media with phage supplement at the same concentration, capturing
completed within 6 or 7 hours (DNA extracted by magnetic bead method).

15.  Detachment of pathogen from tube
Two detachment solutions:
1. Detachment buffer from Pluriselect (neutral pH)
2. An antigen-antibody dissociation (IgG elution buffer) from ThermoFisher (pH 2.8)
 Two antibody coated tubes +200 µL, 10 CFU/mL S. aureus in broth, at 37 °C/ 5 h
 1 mL of 10 CFU/mL S. aureus solution was incubated for control.
 Bacterial solution was carefully removed from the tube and collected.
 Empty tubes + Pluriselect detachment buffer or the IgG Elution buffer for 15 min
 The solutions centrifuged at 3000 RPM for 5 min
 Pellets were resuspended in 20 µL PBS. 1 µL of syto 64 staining dye for 15 min.,
and images taken on flurescent microscope.
 Bacterial solution was recovered spread on agar plates for colony counting.

16.  Detachment of pathogen from tube
Fig. Confirmation of bacterial capturing by fluorescence imaging on detached bacteria
(a) Fluorescence images of detached S. aureus by Pluriselect (neutral pH) detachment
buffer,
(b) Detached by IgG elution buffer from Thermo Fisher Scientific (pH 2.8), both
stained with syto 64 dye.

17.  Summary of experimental parameters (The optimized
experimental parameters)

18. Conclusion
 It has been demonstrated that this technique can effectively capture a
wide range of bacterial quantities in blood and culture media in-
vitro and in-vivo.
 Capturing was verified by colony counts on agar plates, by direct
DNA extraction and subsequent real time PCR, by detachment and
subsequent imaging and plating, and by directly imaging inside the
tube.
 In-vitro and in-vivo results in constant flow, in low and high volume
capturing, and faster flow rates were demonstrated

19. Thank You !!!