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Development of a Novel Antibody-Based Assay for Simultaneous Identification
of a Pathogen and Determination of its Antimicrobial Susceptibility
Jonathan Faro,1 Malika Mitchell,2 Yuh-Jue Chen,3 Sarah Kamal,2 Gerald Riddle,1 Sebastian Faro1
1) The Ob/Gyn Infectious Disease Research Center 2) UT Health Science Center at Houston, Medical College, Houston, TX
ABSTRACT
Figure 1 A-C. Determination of the limit of detection for the Time Zero Test
A
107 bacteria/ml 106 bacteria/ml 105 bacteria/ml 104 bacteria/ml
B
C
0.8
1.6
2
0.6
1.2
OD450
OD450
1.5
0.4
0.8
1
0.2
0.5
0.4
0
0
0
1.E+08
1.E+07
1.E+06
1.E+05
1.E+04
GBS/ml
1.E+03
1.E+02
1.E+01
1.E+08
1.E+07
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.00E+08
1.00E+07
1.00E+06
E. faecalis/ml
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
N. gonorrhoeae/ml
Figure 2A-C. Determination of the limit of detection following incubation at 37o Celsius
B 2.5
A 2.5
C
METHODS
Antibodies utilized:
All antibodies were purchased from either Virostat (Portland, ME) or
ThermoFisher (Waltham, MA), and were rabbit polyclonal antibodies (#1521
anti-GBS, #1524 HRP-anti-GBS, #3711 anti-Enterococcus, #3714 HRP-antiEnterococcus, #PA1-7233 anti-gonorrhoeae, and #PA1-73144 HRP-antigonorrhoeae).
Bacterial Detection and Competition Experiments:
Wells were coated with specified antibody overnight
Wells were blocked with StartingBlockTM
Bacterial dilutions were prepared, starting at 0.5 McFarland, and diluted out
serially from 108 bacteria/ml to 10-3 bacteria/ml
After 30 min, wells were washed with PBS-Tween
HRP-conjugated antibody was added for 20 min
Read OD450
3.75
2
Determination of Antimicrobial Susceptibility and limit of detection studies:
Bacterial suspensions were incubated in FastidiousBrothTM for specified timepoints, either 6 hours (GBS and Enterococcus antibiotic susceptibility), 9 hours
(inducible resistance of GBS to clindamycin), or overnight for determination of
the limit of detection for all three organisms or determination of N.
gonorrhoeae antibiotic susceptibility.
B
A
B
A
B
Clindamycin
0 mg/ml
Erythromycin
1.0 mg/ml
++++
++++
++++
++++
++++
++++
++++
+++
Clindamycin
0.5 mg /ml
Erythromycin
1.0 mg /ml
-
++++
-
++
-
+
-
+
Clindamycin
0.05 mg /ml
Erythromycin
1.0 mg /ml
-
++++
-
+++
-
+
-
+
Clindamycin
0.005 mg /ml
Erythromycin
1.0 mg /ml
-
++++
-
+++
-
+
-
+
CONCLUSIONS
3
1 hr
1:1
1:10
2 hr
1.5
1.5
1:1000
1
6 hr
1) Identification of a pathogen WITH SIMULTANEOUS determination of its
antimicrobial susceptibility
OD450
1:100
OD450
4 hr
1
2.25
2) Limit of detection RIVALS that of PCR
1.5
3) Unlike PCR, NO PRE-ENRICHMENT STEP NEEDED
24 hr
0.5
0.5
0.75
4) And unlike PCR, MAY DETERMINE LIVE VS. DEAD ORGANISMS
0
1.E+07 1.E+06 1.E+05 1.E+04 1.E+03 1.E+02 1.E+01 1.E+00
1.E-01
1.E-02
0
1.E-03
5) Results are MUCH FASTER than culture
0
1.00E-01
1.00E-02
GBS/ml
1.00E-03
1.00E+07
1.00E+06
1.00E+05
E. faecalis/ml
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
N. gonorrhoeae/ml
Figure 3 A-C. Bacterial identification and demonstration of susceptibility to selected antibiotics following a 6-hour culture
B
A 4.5
C
2
6) Theoretically, this test allows for detection of de novo antibiotic
resistance, which PCR CANNOT accomplish
7) Potentially ANY pathogen may be targeted, as long as an antibody
against it is available
2
0 ug/ml Penicillin
0 ug/ml PCN
0 ug/ml Vancomycin
IMPLICATIONS
3.75
0.06 ug/ml Penicillin
1.5
1.5
0.5 ug/ml PCN
4 ug/ml Vancomycin
0.12 ug/ml Penicillin
3
1 ug/ml PCN
2.25
OD 450
OD 450
OD 450
0.25 ug/ml Penicillin
Competition Studies: Dilutions of commonly isolated bacteria were added to
wells at a steady concentration of 108 bacteria/ml, and the amount of test
bacteria was diluted out serially.
A
This NOVEL assay allows for the following:
OD450
Bacterial strains utilized:
GBS clinical isolates 12386 (clindamycin susceptible) and 01.12.76 (clindamycin
resistant)
E. Faecalis ATCC 29212 (Vancomycin susceptible), ATCC 51299 (Vancomycin
resistant)
N. Gonorrhoeae Clinical isolate 1279 (penicillin susceptible), ATCC 31426
(penicillin resistant)
E. coli, S. aureus, C. albicans and Groups A, C, F, and G streptococcus were all
clinical isolates
B
Table 1. Detection of GBS following a 9-hour incubation in the presence of
erythromycin and dilutions of clindamycin. Isoate A (non-inducible resistnace to
clindamycin) compared with Isolate B (inducible resistance capable). ++++ OD > 3.0.
+++ OD 2.0-2.9. ++ OD 1.0-1.9. + OD 0.1-0.9. – OD 0-0.9.
0.5 hr
2
A
2
2.5
OD450
BACKGROUND: Elucidation of a pathogen’s antimicrobial susceptibility
traditionally requires subculture after the organism is first isolated. This process
takes several days, requiring patients to be treated with broad-spectrum
antibiotics until a pathogen is identified. This empirically based approach has
contributed to the development of bacterial resistance, and a call has been
placed for the development of new methods in targeting the treatment of
infectious diseases.
METHODS: By modifying a simple immunosorbent assay, we have developed a
tool allowing for the simultaneous identification of a pathogen and
determination of its antimicrobial susceptibility. Microtiter wells were coated
with a polyclonal antibody targeting the pathogen of interest. After a specified
incubation period, bacterial suspensions were added in the presence/absence
of selected antibiotics. After washing, captured bacteria were detected with a
horse-radish peroxidase-labeled secondary polyclonal antibody, and the optical
density was read.
RESULTS: Group B streptococcus (GBS), Enterococcus faecalis, and Neisseria
gonorrhoeae were each detected at 105 bacteria/ml following a 20-minute
incubation period. Susceptibility to select antibiotics was discernable following
a 6-hour incubation period (GBS and Enterococcus), and inducible resistance
against clindamycin was shown when GBS was cultured in the presence of both
erythromycin and clindamycin. Sensitivity was increased to 10-2 bacteria/ml for
GBS, 10-1 bacteria/ml for E. faecalis, and 101 bacteria/ml for N. gonorrhoeae
following an 18-24 hour culture.
CONCLUSIONS: This novel assay allows for the highly sensitive and specific
identification of a pathogen and simultaneous determination of its antimicrobial
susceptibility in a reduced time.
RESULTS
1
• By simultaneously identifying a clinical pathogen, the physician may
more rapidly utilize the appropriate antibiotic
• This may then decrease the use of broad-spectrum antibiotics which
would decrease the development of resistance and improve outcomes
1
1.5
0.5
ACKNOWLEDGEMENTS
0.5
0.75
0
0
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
E. faecalis/ml
GBS/ml
1.00E+03
1.00E+02
1.00E+01
•
Partial funding for this research was provided for by Nanologix, Inc, and has been
developed under the N-Assay trademark.
•
Jonathan Faro is named as a co-inventor of this technique, US20140315219 A1.
•
Manuscript has been accepted for publication in the journal of Infectious Diseases
in Obstetrics and Gynecology
0
1.00E+07
1.00E+06
1.00E+05
N.gonorrhoeae/ml
1.00E+04
1.00E+03