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External evaluation of animal
experiments and tolerance to
antibiotics
Oana Ciofu
Institute for International Health, Immunology and Microbiology
Faculty of Health and Medical Sciences University of Copenhagen
External evaluation of infection in animal model
• Clinical scores • Lung function • Visualization of light-tagged
microorganisms
Clinical scores • Parameters
– piloerection
– posture
– locomotion
– breathing
– curiosity
– nasal secretion
– dehydration
• Dysfunctions in single parameters assessed by zero, one or two points.
Clinical scores
• The overall fitness of the mice determined by adding the points resulting in the score of the mouse body condition:
• unaffected (0-1);
• slightly affected (2-4);
• moderately affected (5-7);
• severely affected (8-10);
• moribund (≥ 11).
Munder et al. Respiratory Research 2011, 12:148
Symptom severity scoring system
Eyes 0 – no signs, normal
1 – sore red eyes
Lesions 0 – none
1 – lesion on head
Fur 0 – well groomed
1 – ruffled fur
Neurological/ neuromuscular 0 – normal movement
2 – hunched back
2 – hind limb paralysis
3 – unresponsiveness
van Leeuwen Pauline B et al., PLoS One, 2013
Whole body plethysmography
http://buxco.com/Pulmonary.aspx?Page=Pulmonary
Loading the animal
Advantages: Conscious animals Non-invasive Allow long-term and longitudinal studies
Oana Ciofu WS Eurobiofilms 2011
What we measure ?
• The ”box” flow :
Nasal flow: Chest flow:
conditioning factor resistance factor
(temp., humidity) (negative pressure)
WBP parameters
Parameter derivations
Pause =Te-RT RT Penh (Enhanced pause)= PEF x Pause PIF
insp
irat
ion
ex
pir
atio
n
Rt= relaxation time: time to expire 65% of the ”volume”
Before infection
PIF
PEF
After infection
PEF
PIF
Increased Penh after infection
Infection
Bronchial inflammation
Airway obstruction of small airways
Journal of Medical Virology 81:2096–2103 (2009)
Effect of long-term voluntary exercise wheel running on
susceptibility to bacterial pulmonary infections in a mouse model
Oana Ciofu WS Eurobiofilms 2011 van Leeuwen Pauline B et al., PLoS One, 2013
Effect of exercise wheel running on lung function
van Leeuwen Pauline B et al., PLoS One, 2013
Effect of exercise on symptom severity score and
bacterial load in the lung following intranasal inoculation with P. aeruginosa
van Leeuwen Pauline B et al., PLoS One, 2013
Conclusion: increased P. aeruginosa infection load and symptoms after regular voluntary excercise
20
Non-invasive evaluation methods with lighting bacteria
real-time-view of the infection course
21 http://www.xenogen.com http://www.caliperls.com/products/optical-imaging/
JID 2010:201 (1 April)
Intranasal inoculation PAK lumi 1 x 107/50microliter PBS
bacteriophagePAK-P1 Bacteriophage-to-bacterium ratio of 10:1
28
Monitorization of biofilm infection in a mouse model (IVIS imaging system)
(Kadurugamuwa, J. Infection and Immunity, vol 71, 882-90, 2003)
Precolonized catheters with P.aeruginosa Xen 5 were implanted at subcutaneous sites
29
Bioluminiscense Candida albicans, chronic sepsis model
(Doyle, Microbial Pathogenesis, 40, 82-90,2006)
Biofilms non-susceptibility to antibiotics Tolerance: non-mutational, physiological condition that allows survival in the presence of
antibiotic concentrations above planktonic MIC
Resistance: mutational
• PK/PD biofilms
• OligoG_enhancers of antibiotics effect on biofilms
• Oxidative stress model
The killing of bacterial cells (planktonic) by antibiotics
Antibiotic PK/PD Target
Colistin-methansulfonat
Cmax/MIC Cmax/MIC> 8, largest effect Cmax/MIC>64
Beta-lactams (penicillins, cephalosporins, carbapenems)
T>MIC T> MIC 50% (but Cmax/MIC >10)
Fluoroquinolones AUC/MIC AUC/MIC>100 (Gram negative bacteria)
HØIBY 2002
Mucoid biofilm of P. aeruginosa in an alveolar surrounded by severely inflammed tissue. (PMNs, pneumonia). HE stain x 400
Planktonic growth Biofilm growth
The killing of biofilm-growing cells by antibiotics
Eurobiofilms 2011 Oana Ciofu
(Haagensen, J. et al. 2007; J. Bact.,189, 28-37) (Pamp, S. et al., 2008, Mol. Microbiol., 68, 223-40)
Tolerance to colistin
Mechanisms biofilm-specific: upregulation of PmrA-PmrB two-component regulatory systems upregulation of the MexAB-OprM efflux system
alive
dead
Tolerance to beta-lactams
alive
dead
Mechanisms biofilm-specific: Subpopulation with low metabolic activity
PAO1 day 3 biofilm treated with ceftazidime 512 X MIC, 24 h
Alginate beads for in vivo biofilm model
Biofilm formed on peg-lid of microplate
In vitro assay: killing of biofilm-growing P. aeruginosa by antibiotics
(Ceri H et al, J Clin Microbiol 1999; Moskowitz SM et al, J Clin Microbiol 2004) Alginate beads (SYTO 9 staining)
Minimal biofilm eradication concentration
Wang, H. et al AAC 2011
0
200
400
600
800
1000
1200
PAO1
PAO579
PDO300
planktonicday 1
day 3day 7
μg/m
l
Str
ains
Groups
Pharmakokinetic studies
0
20
40
60
80
100
120
140
160
180
0 20 40 60 80 100 120
Time (min)
Con
cen
trati
on
s of
Imip
enem
(μg/m
l)
serum of normal mice
serum of infected mice
lung of normal mice
lung of infected mice
Cmax (µg/ml) T1/2 (min)
Serum of normal mice 143.48 27.54
Serum of infected mice 148.81 38.85
Lung of normal mice 14.43 21.69
Lung of infected mice 36.39 29.08
Uninfected mouse Lung volume: 0.15±0.05 ml
Lung infected mouse Lung volume: 0.25±0.05ml
Pharmakokinetic of imipenem and colistin in a mouse model
MBEC: minimal biofilm eradication concentration MBIC: minimal biofilm inhibitory concentration
imipenem colistin
Neutropenic mouse lung infection model for study of PK/PD
• Mice were rendered neutropenic by injecting three doses of cyclophosphamide intraperitoneally at days 1 and 2 (150mg/kg), and day 4 (100mg/kg).
• At day 5, the neutropenic mice were challenged through trachea into the lower left lung with 0.04ml of either planktonic or alginate beads of P. aeruginosa PAO1 in 5×105cfu/ml, which caused 50% mortality 24h after challenge.
In vivo bacterial killing curve of colistin, neutropenic mouse
model
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
-2 0h 2h 4h 8h
Time of sampling
CF
U/L
un
g 0MIC
4MIC
16MIC
64MIC
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
-2 0h 2h 4h 8h
Time of sampling
CF
U / L
un
g
0MIC
4MIC
16MIC
64MIC
Biofilm PAO1
infection Colistin treatment
infection Colistin treatment
Concentration–dependent killing
Planktonic PAO1
In vivo killing curve of colistin, neutropenic mouse model
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
1.E+09
64MIC
×3
64MIC
×2
64MIC
×1
16MIC
×3
16MIC
×2
16MIC
×1
4MIC
×3
4MIC
×2
4MIC
×1
Control
Groups of Colistin
CF
U/L
un
g
Planktonic
Biofilm
• Mice treated with 64, 16, 4MIC dose of Colistin
• Administrated ×3, ×2, and ×1 (4 hours interval)
Dose-dependent killing of colistin
Hengzhuang, W. et al. Antimicrob Agents Chemother. 2012 May;56(5):2683-90.
Time-dependent killing of imipenem
Hengzhuang, W. et al. Antimicrob Agents Chemother. 2012 May;56(5):2683-90.
Conclusions PK/PD
• The old biofilms are more difficult to treat than young
biofilms: importance of early treatment
• The PK/PD for colistin is AUC above MIC (dose-dependent)
• The PK/PD for imipenem is time above MIC for biofilm infections (time-dependent)
• but much higher concentrations in vivo are required compared to planktonic cells
OligoG
An oligomer enriched from sodium alginate polysaccharides
– Sodium alginate, mainly guluronate monomers
– Number of monomers in the 5-20 range (MW 1,000-4,000)
– Inherent ability to interact with or bind multivalent cations
– In compliance with US & European pharmacopeia standards
– Water soluble and isotonic
– Processed and purified in compliance with cGMP regulations on an industrial scale.
– Regulatory approval for use in human respiratory tract
– API for CF Phase 2A clinical trial produced by FMC Biopolymer/NovaMatrix
The active compound
OligoG CF-5/20 is a linear sodium alginate oligomer with an average degree
of polymerisation Dp 13 comprising predominantly α-L-guluronate
α-L-guluronate (G) and β-D-manuronate (M)
Alginate oligomer structure
48
Ca2+
Ca2+
Ca2+
Ca2+
Biofilm dispersal
OligoG
Ca2+ Ca2+
The guluronic blocks of OligoG contain several electron
negative groups that interact (fully or partially charged)
with cations for example when bound to Na + or Ca 2+
One of many possible
mechanisms of action for
OligoG
Treatment
time (h)
Colistin (μg/ml)
+ 0%OligoG + 0.2%OligoG + 2%OligoG + 5%OligoG
1 hours >512 >512 512 512
2 hours >512 512 256 256
4 hours >512 128 32 16
8 hours 512 64 4 4
12 hours 256 32 < 4 < 4
24 hours 128 32 < 4 < 4
The effect of Colistin combined with 0.2%, 2%, 5%OligoG on
bioflm of alginate beads of P. aeruginosa NH57388A in vitro
MBEC (minimal biofilm eradication concentration) in saline
MIC of colistin: 0.094μg/ml (planktonic)
Biofilm lung infection model
Alginate beads for in
vivo biofilm model
Biofilm formed on peg-lid of microplate
Materials and Methods
Biofilm formed on microwell
Tracheotomy
Bacteriology and pathology
Anesthesia
Biofilm lung infection model
Anesthesia
Biofilm cells injection
Antimicrobial administration (IV, IP, IT, Oral)
Killing effect of intraperitoneal colistin combined with locally 5% OligoG
on alginate beads of P. aeruginosa NH57388A in vivo
▲ Groups (8 mice/group, total 9 groups)
Control (0h) , control (saline, at 24h time point), control (5%OligoG, at 24h time point)
0.4mg/kg colistin + saline; 0.4mg/kg colistin + 5% OligoG;
1.6mg/kg colistin + saline; 1.6mg/kg colistin + 5% OligoG;
6.4mg/kg colistin + saline; 6.4mg/kg colistin + 5% OligoG;
▲ The beads embedded NH57388A were administered by an intra-tracheal route to the
lower left lung of the mouse, with or without 5% OligoG (50mg/ml) at 0h time point
▲ One dose of colistin was administrated intraperitoneally 2hr after bacterial challenge
▲ Lungs were removed at sacrifice 24hr after bacterial challenge to determine bacterial
counts and macropathology Hoffmann et al. Infect and Immunity, 2005, p2504-2514
52
5%OligoG, 24h
Saline, 24h
Alginate beads of P. aeruginosa NH57388A treated with
local 5% OligoG in vivo
The biofilm of alginate beads of NH57388A were administered by the intra-tracheal
route to the lower left lung, with or without 5% OligoG (50mg/ml) at 0h time point .
Lungs were removed at 24hr after bacterial challenge
53
Groups
4MIC Colistin
+5%OligoG
4MIC Colistin
+Saline
Saline
CFU/Lung
1,E8
1,E7
1,E6
1,E5
1,E4
3
4MIC (0.4mg/kg)+ 5%OligoG, 24h
4MIC (0.4mg/kg)+ Saline, 24h
P <0.01
Alginate beads of P. aeruginosa NH57388A treated with
4MIC (0.4mg/kg) colistin and local 5% OligoG in vivo
▲ One dose of 0.4mg/kg colistin was administrated intraperitoneally 2hr
after bacterial challenge. 5% OligoG was administrated locally at 0h time
point. Lungs were removed at 24hr after bacterial challenge.
CF
U / L
un
g
1.E8
1.E7
1.E6
1.E5
1.E4
Saline 4MIC colistin
+Saline
4MIC colistin
+5% OligoG
Groups
54
Groups
16MIC Colistin
+5%OligoG
16MIC Colistin
+Saline
Saline
CFU/Lung
1,E8
1,E7
1,E6
1,E5
1,E4
3
16MIC(1.6mg/kg) + 5%OligoG, 24h
16MIC (1.6mg/kg) + Saline, 24h
P <0.01
Alginate beads of P. aeruginosa NH57388A treated with
16MIC(1.6mg/kg) colistin and local 5% OligoG in vivo
▲ One dose of 1.6mg/kg colistin was administrated intraperitoneally 2hr
after bacterial challenge. 5% OligoG was administrated locally at 0h time
point. Lungs were removed at 24hr after bacterial challenge.
Saline 16MIC colistin
+Saline
16MIC colistin
+5% OligoG
Groups
CF
U / L
un
g
1.E8
1.E7
1.E6
1.E5
1.E4
55
Alginate beads of P. aeruginosa NH57388A treated with
64MIC(6.4mg/kg) colistin and local 5% OligoG in vivo
64MIC(6.4mg/kg) + 5%OligoG, 24h
64MIC (6.4mg/kg) + Saline, 24h
Groups
64MIC Colistin
+5%OligoG
64MIC Colistin
+Saline
Saline
CFU/Lung
1,E8
1,E7
1,E6
1,E5
1,E4
1,E3
1,E2
19
3
P <0.01
▲ One dose of 6.4mg/kg colistin was administrated intraperitoneally 2hr after
bacterial challenge. 5% OligoG was administrated locally at 0h time point.
Lungs were removed at 24hr after bacterial challenge.
Saline 64MIC colistin
+Saline
64MIC colistin
+5% OligoG
Groups
CF
U / L
un
g
1.E2
1.E8
1.E7
1.E6
1.E5
1.E4
1.E3
56
Killing effect of antibiotics combined with 5% OligoG by local treatment
on alginate beads of P. aeruginosa NH57388A in vivo
▲ Groups (8 mice/group, total 9 groups)
Control (0h) , control (saline, at 24h time point), control (5%OligoG, at 24h time point)
32mg/L aztreonam + saline; 32mg/L aztreonam + 5% OligoG;
1.6mg/L colistin + saline; 1.6mg/Lcolistin + 5% OligoG;
48mg/L tobramycin + saline; 48mg/L tobramycin + 5% OligoG;
▲ The beads embedded NH57388A were administered by an intra-tracheal route
to the lower left lung of the mouse, with or without 5% OligoG (50mg/ml) at 0h
time point
▲ One dose of antibiotics was administrated by an intra-tracheal route to the
lower left lung 2hr after bacterial challenge
▲ Lungs were removed at sacrifice 24hr after bacterial challenge to determine
bacterial counts and macropathology
OligoG CF-5/20 combined with 16MIC (32mg/L ) local aztreonam
on biofilm of P. aeruginosa NH57388A in vivo
P <0.01
One dose of 0.04ml aztreonam (16MIC, 32mg/L) was administrated by an intra-tracheal route to
the lower left lung 2hr after bacterial challenge. 5% OligoG was administrated locally at 0h time
point. Lungs were removed at sacrifice 24hr after bacterial challenge to determine bacterial counts 57
0h Saline
24h
5% OligoG
24h
Saline
16MIC aztreonam
24h
5% OligoG
16MIC aztreonam
24h Groups
1.E9
1.E8
1.E7
1.E6
1.E5
1.E4
CFU
/Lung
58
OligoG CF-5/20 combined with 16MIC (1.6mg/L ) local colistin
on biofilm of P. aeruginosa NH57388A in vivo
One dose of 0.04ml colistin (16MIC, 1.6mg/L) was administrated by an intra-tracheal route to the
lower left lung 2hr after bacterial challenge. 5% OligoG was administrated locally at 0h time
point. Lungs were removed at sacrifice 24hr after bacterial challenge to determine bacterial counts
P <0.01
0h Saline
24h 5% OligoG
24h
Saline
16MIC colistin
24h
5% OligoG
16MIC colistin
24h
Groups
1.E9
1.E8
1.E7
1.E6
1.E5
1.E4
CFU
/Lung
59
OligoG CF-5/20 combined with 16MIC (48mg/L) local tobramycin
on biofilm of P. aeruginosa NH57388A in vivo
P <0.01
One dose of 0.04ml tobramycin (16MIC, 48mg/L) was administrated by an intra-tracheal route to
the lower left lung 2hr after bacterial challenge. 5% OligoG was administrated locally at 0h time
point. Lungs were removed at sacrifice 24hr after bacterial challenge to determine bacterial counts
0h Saline
24h
5% OligoG
24h
Saline
16MIC tobramycin
5% OligoG
16MIC tobramycin
Groups
1.E9
1.E8
1.E7
1.E5
1.E4
1.E6
CFU
/Lung
Conclusions
▲ Colistin-OligoG combinations were significantly better than
colistin alone in vitro and with two to three log reduction in
lung biofilm bacterial burden in vivo
60
▲ Aztreonam or tobramycin and OligoG combinations were
significantly better than aztreonam or tobramycin alone of local
treatment with almost two log reduction in lung biofilm
bacterial burden in vivo
▲ A two log reduction in lung bacterial burden was also
observed in the biofilm lung infection model when 5% OligoG
was administered alone
ROS Anti-oxidants
Oxidative stress
• PMN inflammation • CFTR related GSH deficiency • Incomplete correction of pancreatic insufficiency and malabsorption of fat-soluble anti-oxidants (vitE)
Anti-oxidants in CF: glutathione (GSH)
NH3 NH
HN
O-
O-O
O
O
SH
GSH
NH3 NH
HN
O-
O-O
O
O
S
GSSG
2
GSH + H2O2 H2O + O2 + GSSG
Oxidative stress model • Guinea pigs can not synthetize
the anti-oxidant vitamin C (like humans)
• An oxidative stress model by 6-8 weeks diet with low C vitamin was established
• Lung infection model by P. aeruginosa embedded in alginate (12 infected and 6 controls in each group)
Susceptibility to infection
• A significantly higher mortality of 6 out of 12 animals (p=0.03, Chi-square test) was observed after lung infection with alginate embedded P. aeruginosa in guinea pigs on ASC deficient diet compared to the animals on ASC sufficient diet
Jensen, P.Ø. et al. Basic & Clinical Pharmacology & Toxicology, 2012, 110, 353–358
Lung bacteriology: no difference between the two groups
1E4
1E5
1E6
1E7
CF
U/L
B 0
9.0
5
gruppeA (med vitC) gruppeB (uden vitC)
1E4
1E5
1E6
1E7
CF
U/L
B 0
9.0
5
gruppeA (med vitC) gruppeB (uden vitC)
CFU/ml lung homogenate forsøg II (aflivet 12.09.05)CFU/ml lung homogenate
Group A Group B (with vitC) (minus vitC)
Group A Group B (with vitC) (minus vitC)
Lung histology
Normal lung_control
Mononuclear cells (MN) dominated inflammation
Polymorphonuclear cells (PMN) dominated inflammation
Jensen, P.Ø. et al. Basic & Clinical Pharmacology & Toxicology, 2012, 110, 353–358
Increased ROS in PMNs from animals on ASC deficient diet
The spontaneous respiratory burst in peripheral PMNs from guinea pigs receiving an ASC sufficient (N=10) diet and ASC deficient diet (N=10).
Jensen, P.Ø. et al. Basic & Clinical Pharmacology & Toxicology, 2012, 110, 353–358
(Washko et al., J. Biolog. Chemistry, 268, 1993)
Excess ROS in CF
Bacterial infection
PMN activation
ROS
Lipid peroxidation Protein oxidation DNA damage
Activation of NF-kB Increase TNFα, IL1, IL-6
Hull, 1993 McGrath, 1999 Lagrange-Puget, 2004
Jensen, P.Ø. et al. Basic & Clinical Pharmacology & Toxicology, 2012, 110, 353–358
Plasma anti-oxidant capacity
Conclusions
• The animals receiving ASC deficient diet showed significantly higher mortality during infection and increased respiratory burst of peripheral PMNs compared to the animals receiving ASC sufficient diet.
• Higher PMN/MN ratios were present in animals on ASC deficient diet compared to animals on ASC sufficient diet
• the infection by itself decreased the antioxidant capacity of the plasma more than the ASC deficient diet, suggesting a high consumption of the antioxidants during infection.
• poor antioxidant status exacerbates the outcome of biofilm-related infections
Acknowledgements
Prof. Niels Høiby PK/PD studies • Wang Hengzuang • Hong Wu OligoG studies • Wang Hengzuang
Lung function measurements • Helle Krogh Johansen • Lieke de Vrankrijer • Pauline van Leeuvan
Oxidative stress model
• Prof. Jens Lykkesfeldt_ KULife
• Peter Østrup Jensen_RH
• Thomas Bjarnsholt_RH and IHIM
Tina Wassermann
Annie Bjergby Kristensen
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