external evaluation of animal experiments and tolerance to ...tcoenye/iuap/ciofuv2.pdfc max (µg/ml)...

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 : the rate of change

lung function.mov

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


Effect of exercise on symptom severity score and

bacterial load in the lung following intranasal inoculation with P. aeruginosa


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/

http://www.xenogen.com/

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.

http://www.ncbi.nlm.nih.gov/pubmed/22354300







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;



▲ 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


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


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

+5% OligoG

Groups

CF

U / L

un

g

1.E8

1.E7

1.E6

1.E5

1.E4

55


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

+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


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


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


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).


(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


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

external evaluation of animal experiments and tolerance to ...tcoenye/iuap/ciofuv2.pdfc max (µg/ml)...

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