up a NADH oxidase (NOX)-based assay to quantify the bactericidal activit

F. Van Bambeke

P. M. Tulkens

Y. Diaz Iglesias

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•
 
Bacteria :
 
S. pneumoniae
 
ATCC49619; suspension (0.5 McFarland) in caMHB+ 5% 
lysed
 
horse blood.
•
 
Models and antibiotic exposure  : 
•
 
Planktonic
 
model :
 
incubation of bacterial suspension with antibiotics (0.03 to 
8000 x MICbroth
 
) during 24h at 37°C, following the CLSI MIC microdilution
 
protocol.
•
 
Biofilm
 
model :
 
25µL of bacterial suspension added to 175 µL of medium 
(caMHB
 
+ 5% lysed
 
horse blood + 2% glucose7) in 96-well plates and incubated 
during 48h at 37°C in 5% CO2
 
atmosphere; biofilm
 
incubated with antibiotics 
(10-6
 
to 103
 
x MICbroth
 
) during 24h in the same conditions. 
•
 
CFU counting :
 
enumeration of CFUs
 
after plating on agar plates.
•
 
NADH oxidase-based assay :
 
measure of the reduction in NADH OD340nm
 
after 30 
min.of
 
incubation at 37°C (initial conc.: 0.17mM). Daptomycin
 
(2 mg/L) is used as 
positive control (100 % mortality)
•
 
Resazurin
 
assay : measure of the
 
increase in fluorescence signal (λex
 
560nm; λem
 
590nm) of resorufin
 
(pink, fluorescent) formed by resazurin
 
(0.001%; blue, non 
fluorescent) reduction by living bacteria after 30 min (planktonic
 
cultures) or 72 h 
(biofilms).
Setting up a NADH oxidase
 
(NOX)-based assay to quantify the bactericidal activity 
of an antibiotic in planktonic
 
cultures and biofilms
 
of S.
 
pneumoniae.
N.M. Vandevelde, Y. Diaz Iglesias, P.M. Tulkens
 
and F. Van Bambeke
Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université
 
catholique
 
de Louvain, Brussels, BELGIUM
INTRODUCTION & AIM RESULTS
Contact info: 
francoise.vanbambeke@uclouvain.be
This poster will be made available for download after the meeting at: http://www.facm.ucl.ac.be/posters.htm
•
 
Measuring the release of a soluble cytosolic
 
enzyme is widely used for 
cytotoxicity
 
assay in eukaryotic cells, but is rarely applied to bacteria.  Yet, it 
could help for the rapid screening of bactericidal antibiotics towards both
 
 
planktonic
 
cultures (PK) and biofilms
 
(BF). 
•
 
In  S.
 
pneumoniae, NADH oxidase
 
(NOX), a soluble flavoprotein
 
involved in   
NADH reoxidation, is essential for virulence.1
 
As NADH does not permeate 
through membranes,2
 
its oxidation when added to the medium will be 
proportional to the amount of NOX release from lysed
 
bacteria3.
•
 
The aims of the study were (i) to set-up a NOX-based assay using moxifloxacin
 
(MXF) and clarithromycin
 
(CLR) as representatives of bactericidal and
 
 
bacteriostatic
 
antibiotics, respectively,4,5
 
and (ii) to compare its performance vs. 
CFU counting (PK) and resazurin
 
reduction assay 6,7
 
(PK and BF).
24h planktonic
 
culture 48h biofilm
Resazurin
 
assay
CFU counting
Characterization of moxifloxacin
 
(MXF) and clarithromycin
 
(CLR) activities (concentration-effect curves)  
on
 
S. pneumoniae
 
ATCC49619 planktonic
 
cultures and biofilms, using 3 different assays
Antibiotic
Maximal changes in tested parameter
24h planktonic
 
culture 48h biofilm

 
log10
 
CFU 
(cell enumeration)
NOX assay
(membrane integrity)
Resazurin
 
assay
(metabolism)
NOX assay
(membrane integrity)
Resazurin
 
assay
(metabolism)
Moxifloxacin
(bactericidal)
-3.8 ±
 
0.5 (A) -100.4% ±
 
4.4 (A) -100.0% ±
 
1.6 (A) -98.3% ±
 
3.0 (a) -100.4% ±
 
12.1 (a)
Clarithromycin
(mainly bacteriostatic)
-0.7 ±
 
0.7
 
(B) -61.7% ±
 
6.6 (B) -100.0% ±
 
3.8 (A) -6.5% ±
 
6.4 (b) -87.4% ±
 
6.2 (a)
Statistical analysis : values with different letters are statistically different from each other (unpaired t-test, p<0.05). 
uppercase letters: planktonic
 
culture; lower case letters: biofilm
•
 
We set up a new sensitive method allowing for the rapid quantification of antibiotic bactericidal and bacteriostatic
 
activities in pneumococcal planktonic
 
cultures and biofilms.   
•
 
As this NADH oxidase-assay can be completed in 30 min and is amenable to performance in various media, it 
may offer advantages for the rapid screening  of antibiotic activities in different culture conditions. 
•
 
Application to other bacterial species warrants further investigation.
NOX
(BACTERICIDAL)     
NADH 0.17mM
NOX
NOX
NOX
NOX
Moxifloxacin Clarithromycin
(BACTERIOSTATIC)
NADH 0.17mM
NOX
NOX
NOX
NOX
(BACTERICIDAL)     
NADH 0.17mM
NOX
NOX
NOX
NOX
Moxifloxacin Clarithromycin
(BACTERIOSTATIC)
NADH 0.17mM
NOX
NOX
NOX
NADH
NADH oxidase
 
(NOX) assay
Resazurin
 
assay
NADH oxidase
 
(NOX) assay
EP288
MATERIALS & METHODS
1Auzat et al, Mol Microbiol. 1999;34(5):1018-28 ; 2Laliberté
 
et al, 1987; J Bioenerg. 
Biomembr. 19(1):69-81; 3Yu et al, Microbiology. 2001;147(Pt 2):431-8 ;  4Visalli et al, 
AAC,1997; 41(12):2786-9; 5Ednie et al, AAC,1996; 40(8):1950-2; 6Tote et al, Lett
 
Appl
 
Microbiol. 2008;46(2):249-54 ; 7Vandevelde et al, AAC 2014 Mar;58(3):1348-58
SUMMING UP & CONCLUSIONS
REFERENCES
NAD
+
ACKNOWLEGMENTS
N.M.V. is Boursière
 
of the Belgian Fonds
 
de la Recherche
 
dans
 
l'Industrie
 
et l'Agriculture
 
(FRIA) and FBV is Maître de recherches
 
of the Belgian Fonds
 
de la Recherche
 
Scientifique
 
(F.R.S-FNRS). 
NOX + H+
 
+ 2e-
R = ribose-P-P-ribose-adenine
resazurin resorufin
-3 -2 -1 0 1 2
-6
-5
-4
-3
-2
-1
0
1
2
CLR
MXF
log10 concentration (X MIC)

l
o
g
 
C
F
U
 
f
r
o
m
 
i
n
i
t
i
a
l
 
i
n
o
c
u
l
u
m
-3 -2 -1 0 1 2
0
20
40
60
80
100
120
CLR
MXF
log10 concentration (X MIC)
N
A
D
H
 
a
b
s
o
r
b
a
n
c
e
(
%
 
c
o
n
t
r
o
l
 
v
a
l
u
e
)
-3 -2 -1 0 1 2
0
20
40
60
80
100
120
CLR
MXF
log10 concentration (X MIC)
R
e
s
o
r
u
f
i
n
 
f
l
u
o
r
e
s
c
e
n
c
e
(
%
 
c
o
n
t
r
o
l
 
v
a
l
u
e
)
-7.5 -5.0 -2.5 0.0 2.5 5.0
0
20
40
60
80
100
120
CLR
MXF
log10 concentration (X MIC)
N
A
D
H
 
a
b
s
o
r
b
a
n
c
e
(
%
 
c
o
n
t
r
o
l
 
v
a
l
u
e
)
-7.5 -5.0 -2.5 0.0 2.5 5.0
0
20
40
60
80
100
120
CLR
MXF
log10 concentration (X MIC)
R
e
s
o
r
u
f
i
n
 
f
l
u
o
r
e
s
c
e
n
c
e
(
%
 
c
o
n
t
r
o
l
 
v
a
l
u
e
)

 
the resazurin-assay, which shows a very sharp slope, did not distinguish between bactericidal and bacteriostatic
 
antibiotics (both caused a concentration-
 
dependent reduction in signal that reached a minimum at low multiples of the MICs
 
for planktonic
 
cultures as well as in biofilms); 

 
conversely, in both planktonic
 
cells and biofilm, the NADH oxidase
 
assay allowed for a unambiguous differentiation between bactericidal (MXF) and 
bacteriostatic
 
(CLR) antibiotics.  

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