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METHODS
Comparison of Tobramycin and Ciprofloxacin efficacy
in a rat model of chronic lung infection
using clinically relevant Pseudomonas aeruginosa RP73
S. Gandi1, I. Cranston1, K. Maclellan1, G. McLachlan2, S. Wright2, J. Baily1, S. Madden1, M. Whitmarsh3, A. Young3, M. McElroy1#
1Charles River Laboratories - Edinburgh, 2The Roslin Institute, University of Edinburgh, 3Charles River Laboratories – Harlow
1INTRODUCTION 4CONCLUSION
3
2RESULTS
Pseudomonas aeruginosa infection is amajor complication for patients with cystic fibrosis.
Currently, it is difficult to eradicate pseudomonas from lungs, leading on to persistent
infection that can ultimately cause respiratory failure and/or mortality. We have developed a
chronic model of P. aeruginosa-induced pneumonia using strain RP73 (kind gift by
Jens Klockgether,Germany).Here we compare the efficacy of ciprofloxacin and tobramycin
in a rat model of chronic pseudomonas pneumonia.
Direct lung administration of tobramycin (3 mg/kg, daily) was an effective treatment in the chronic model of
pseudomonas pneumonia while ciprofloxacin (500 mg/kg, oral, daily) was ineffective although both strains
showed sensitive MIC values. Also, tobramycin-treated animals showed improved clinical signs and reduction
in the extent of inflammatory response by Day 5. These data highlights the importance of employing an
in-vivo model in the development of antibacterial agent for clinical use. Also the data supports the use of
tobramycin as standard of care reference item in a model of chronic pseudomonas pneumonia.
Strain Antibiotic MIC value
(µg/mL)
PA RP73
in beads
Ciprofloxacin 0.5 µg/mL
Tobramycin 0.25 µg/mL
PA RP73 from
O/N culture
Ciprofloxacin 0.5 µg/mL
Tobramycin 0.25 µg/mL
There was no difference in the
MIC value between PA RP73 in
beads and from the O/N culture
(standard procedure).
12345 12345 12345 12345 12345
0
10
20
30
40
50
60
70
80
90
100
Days
Percentage, %
20%
80%
40%
Tobramycin reduced
Lung CFU by Day 5
2 5
10 2
10 3
10 4
10 5
10 6
10 7
10 8
Days
CFU/Lung
*
Tobramycin improved
respiratory signs
2 5
10 2
10 3
10 4
10 5
10 6
Days
CFU/Lung
*
Tobramycin reduced
BALF CFU by Day 5
Tobramycin improved
body weight gain (%)
Tobramycin reduced
the extent of inflammation in BALF
Tobramycin reduced
BALF lymphocyte counts
Tobramycin reduced
BALF neutrophil numbers
Tobramycin reduced
BALF total protein
2 5
0
4
8
12
16
20
24
Days
Total Cells, x106
2 5
0
4
8
12
16
20
24
Days
Total Cells, x106
2 5
0.0
0.1
0.2
0.3
0.4
0.5
Days
Total C ells, x106
*
Microscopically, subacute inflammatory changes at Day 7were locally
extensive, of moderate to marked severity and contained ahigh
proportion of neutrophils. By Day 14,chronic inflammatory foci were
rare, of minimal severity and more organised with an increase in
lymphocytes and areduction in neutrophils.Day 7inflammatory foci
were positive for alcian blue staining which is indicative of biofilm
formation.Administration of sterile beads was associated with minimal
inflammation at Day 7which had resolved by Day 14 (data not shown).
Day 7- Lungs of animals administered
P. aeruginosa laden beads. Foci of
inflammation are highlighted in green
in animal A. H&E stain.
A B C
Day 14- Lungs of animals administered
P. aeruginosa laden beads. Foci of
inflammation are highlighted in green in
animal D. H&E stain.
D E F
MIC of ciprofloxacin and tobramycin were determined using the BSAC macro dilution method.Sprague Dawley rats were dosed with 6.5 x 107CFU/mL PA RP73-embedded agar beads (n=5 per timepoint) and sterile beads for the control group.Tobramycin
was dosed at 3mg/kg, once daily, directly to the lungs and ciprofloxacin dosed at 500 mg/kg twice daily, orally, from Day Minus 1to 4. Colony forming units (CFU) were measured in the lungs and bronchoalveolar lavage fluid (BALF) 24hpost infection (Day 2)
and 4days post infection (Day 5). Total and differential cell counts were measured in the BALF using the Sysmex XT-2000isystem.Total protein content in BALF supernatant was also quantified by BCA Assay.Body weight and clinical signs were monitored
daily.Histopathology was assessed to quantify lung inflammation.Data presented as Mean ± S.D. Statistical comparisons were performed against PA RP73 in beads with Vehicle group using two-tailed unpaired parametric t-test. *p≤0.05 **p≤0.01
Tobramycin (solid green bars) significantly reduced lung and
BALF CFU compared to the untreated controls (solid red
bars) on Day 5 (4 days post infection).Tobramycin (solid
green bar/line) also reduced the extent of tachypnea and
body weight loss compared to untreated controls (solid red
bar/line).No pseudomonas CFU or incidence of tachypnea
were detected in the animals dosed with sterile beads.
Total white blood cells in BALF were increased in
pseudomonas-challenged animals (solid red bars) but the
extent of this increase was reduced in tobramycin-treated
animals (solid green bars) on both Day 2and 5. Tobramycin
treatment reduced the number of neutrophils, lymphocytes
and total protein content on Day 5.
-2 -1 12345
-3
0
3
6
9
12
Days
% D ifference
Sterile beads + OP Vehicle
PA RP73 in beads + Tobramycin
(3 mg/kg, QD, via OP)
PA RP73 in beads + OP Vehicle
PA RP73 in beads + Ciprofloxacin
(500 mg/kg, BID, via Oral)
PA RP73 in beads + Oral Vehicle
2 5
0
2000
4000
6000
8000
Days
Total protein content (µg)
**
29th ECCMID, Amsterdam
Poster number: P1107
Session: Infection in cystic fibrosis, PS065
14th April 2019, 1.30pm to 2.30pm
#Corresponding author: Mary.McElroy@crl.com
