Experimental Murine Models of Infection
Animal models are usually divided into screening models, mono/polyparametric models, ex vivo models, and discriminatory models. Evaluating new therapeutic agents for the
treatments of infectious diseases requires animal models that are reproducible among different laboratories. Therefore, standardization of models is essential for achieving
reproducibility in results. Animal models at NAEJA have been standardized on the basis of certain criteria, such as - choice of animals, choice of organisms and strains, route of
infection, culture conditions, inoculums preparation, infection loads, immunosupression, tissues burden, route of administration of test material, period of treatment, and length
of study.
The animal models that have been successfully standardized and utilized in the drug discovery research at NAEJA Pharmaceutical Inc. are:
Septicemia models:
Staphylococcus aureus MRSA
Staphylococcus aureus MSSA
Candida albicans
Aspergillus fumigatus
Pneumonia model:
Streptococcus pnuemoniae
Aspergillus fumigatus
Thigh infection models:
Staphylococcus aureus MRSA
Staphylococcus aureus MSSA
Streptococcus pnuemoniae
Escherichia coli
Sepsis models:
Staphylococcus aureus MRSA
Staphylococcus aureus MSSA
Escherichia coli
Superficial Skin infection modes:
Staphylococcus aureus MSSA
Staphylococcus aureus deep skin infection model using suture technique
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SEPTICEMIA MODELS
Murine models of candidiasis
Candida albicans DAY185 was grown on SDA plates and then twice in YPD broth. Yeasts from log-phase liquid culture were washed and resuspended in sterile
saline containing 0.01% Tween 20. The cells in the suspension were counted by a hemocytometer and the suspension was diluted into 2.5x106 CFU/mL, which
was confirmed by plating serial ten-fold dilutions of the suspensions onto SDA plates in duplicate. 200L of this suspension was used to inject intravenously into
the mice through lateral tail veins at a concentration of 5x105 CFU per mouse. One hour post-infection, the mice were administered with test and reference
compounds and the vehicle control. For survival study, the mice were observed daily for a period of 7-10 days and the clinical symptoms were recorded and
any mouse became moribund was euthanized. For tissue burden study, the animals were euthanized at different time points after the administration of the
drugs, and tissues, such as kidney, lungs or spleen, were collected aseptically and processed for recovery of the yeast from them.
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
PNEUMONIA MODELS
Mouse model of pneumonia
Streptococcus pneumoniae (ATCC 10813) was grown on Blood Agar (BA) plates at 37°C and colonies from 36 hours culture were picked to prepare inoculums in
sterile saline containing 5% dextrose. The suspension was further diluted to a concentration predetermined by the OD580nm spectrophotometer reading and
viable counts. Mice were sedated with a cocktail of Ketamine and Atravet in saline, and inoculated with 40 µL of the suspension by intranasal instillation. Two
hours post-infection, the mice were administered with the test and reference compounds. For survival study, the animals were treated for 5 to 7 days and the
mice were observed for 7-10days. For tissue burden, a group of mice were euthanized after 20-24 hours post infection and lungs were collected aseptically and
processed for tissue burden. Efficacy was determined by either/both survival pattern and bacterial counts from the tissues.
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
NEUTROPENIC MOUSE THIGH INFECTION MODELS (Gram positive)
Murine pathogen free Balb/C male mice, 20-22 gm/mouse, were rendered neutropenic by injecting Cyclophosphamide at 150mg/kg and 100mg/kg by IP routes
on Day –4 and Day –1, respectively, prior to the infection. S. aureus Smith (ATCC13709) or MRSA was grown fresh from frozen stock onto MH agar plates at
37ºC. Single colony was picked to grow the organism in brain-heart infusion (BHI) broth in a shaking incubator. The culture was washed and then resuspended
in sterile saline containing 0.05% Tween 80. From a pre-determined calculation (at OD580), the suspension was further diluted to 1x10 6cfu/ml. On Day 0, i.e.,
on 5th day after the start of Cyclophosphamide injection, the mice were transiently anesthetized with Isoflurane and 100µL of the bacterial suspension was
injected into one thigh of each mouse, which was marked for later recognition. One hour post-infection, the treatment began and mice were injected with
test and reference compounds. The animals were scarified 24 hours after infection and the thighs were collected aseptically. The tissues were homogenized
for 1 minute using Brinkmann Polytron PT300 homogenizer at 24-28K rpm and the resulting homogenates were used to determine the tissue burden in thighs.
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
SEPSIS MODELS
E. coli sepsis model
Escherichia coli ATCC 25922 was inoculated onto MH plates and subcultured for one or two times. Colonies from 12 hours plates were harvested in sterile saline
containing Tween and washed few times before diluting in to an inoculums that is equivalent to 3.0 McFarland standards (i.e., 9x108cfu/ml), which was
confirmed viable counts on MH plates. The inoculums were further diluted in 7% sterile hog gastric mucin in saline and 0.5ml was used to infect the mice by
intraperitoneal injection, which would ≈ 2.25x108 cfu/mouse. One hour following the injection of E. coli, the animals were administered with the vehicle (as
negative control), reference compound, such as Gentamycin, (as positive control) and test compounds at desired concentrations and dosing intervals. The mice
were monitored daily for 7 days and clinical symptoms, such as - condition of the fur coat, the amount of facial grooming; the degree of physical activity and
the respiratory activity, of each animal were recorded. Animals that became moribund were euthanized by carbon dioxide inhalation.
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
MOUSE SUPERFICIAL SKIN & DEEP SKIN INFECTION MODELS
Mouse superficial skin infection with S. aureus & S. pyogenes
Inoculums preparation:
Staphylococcus aureus (ATCC 13709) was grown fresh from frozen stock onto Muller Hinton (MH) plates at 37°C. After checking the initial growth, single colony
was picked to grow overnight to a late log phase (no more than 12 hours) in Blood Heart Infusion (BHI) broth in a shaking incubator at 37°C. The culture was
washed 3x by centrifuging at 6000rpm and resuspending in saline. The final inoculum was diluted to a predetermined spectrophotometric absorbance (OD580nm)
so that 10µl of the suspension would have ~107 cfu of bacteria, which would be used to infect the skins of the mice.
Superficial skin infection:
Superficial skin infection was caused by both tape-stripping and scalpel blade injury methods, as described by Kugelberg et al (2005) and Scaramuzzino et al
(2000). Briefly, the mice were anesthetized by intraperitoneal injection of Ketamine cocktail (Ketamine at 100 mg/kg and Xylazine at 5 mg/kg in sterile saline)
before preparing the mice. An area of 2 cm 2 was on the back of the mice were shaved with a clipper and then remaining furs were removed by hair-removing
lotion (e.g., Veet). The area was tape-stripped 7-10 times with pieces of elastic surgical adhesive bandage (e.g., Elastoplast). After this procedure, the skin
was visibly damaged and was characterized by reddening and glistening of the skin but without regular bleeding. To enhance the superficial damage, the area
was further injured by scalpel blade by 10 horizontal and 10 vertical light cuts on the skin in a way that no visible bleeding occurred. Microscopically, these
procedures would result in the controlled removal or cuts of epidermal layer. After the superficial skin injuries were caused, 10µl of the inoculum containing
107 cells was applied on the injured skin by droplets and spread with a pipette tip.
Treatment:
The mice were treated topically with antibiotic in the form of ointments. The ointments were made in a Poly-mix base (60% Polyethylene glycol 400 and 40%
Polyethylene glycol 3350) and Fusidic acid (2%) and Erythromycin (0.5%, 1% and 2%) were used to treat the mice twice daily for 3 days. While placebo group
received vehicle only, the untreated group did not receive any treatment until the end of the experiment. The ointments were applied in a 0.1 volume to the
wound and were spread over the area uniformly. The first application was started 4 hours and the next one was 8 hours after the start of the infection. The
mice were treated twice daily (in the morning and the evening, with an 8 hours interval in between) for another 2 days (Day 2 and 3). The experiment was
terminated 18 hours after the last treatment, i.e., on Day 4, in order to avoid the carryover effect on the tissue.
Tissue processing:
On Day 4, the mice were euthanized by carbon dioxide inhalation and the infected skins were excised aseptically and collected in a round-bottomed tube
containing 3ml PBS. The tissues were homogenized by Brinkmann Polytron PT300 homogenizer at 24–28K rpm and the resulting homogenates were serially tenfold diluted (four to six times) in sterile saline. One hundred microlitres (100µL) of each dilution were plated onto MH in duplicate and the plates were
incubated for 24 hours at 35-37oC. The colonies were counted after 24 and also after 48 hours. The colony forming units (CFU/ml) were determined and Log 10
of the counts were calculated to analyze the data.
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
Mouse Staphylococcal deep skin infection model using the suture technique.
Staphylococcus aureus ATCC 13709 (SMITH) is a routine laboratory strain, maintained in cryogenic beads at -80C, cultured on Muller Hinton agar (MHA) and
Brain Heart Infusion (BHI) broth. Commercial cotton thread (Coates and Clarke’s, mercerized, size 8) monocontaminated with S. aureus was used as the
suture material to initiate and potentiate the experimental infection. The thread was cut into 5 cm segments and placed in boiling water for 3 minutes
for sterilization. The thread was removed from the water and blotted dry with sterile paper towels. The segments were then placed into a test tube
containing 8 ml of S. aureus suspension, mixed on a vortex for 10 seconds and allowed to soak for 30 minutes. The thread was finally removed from the
suspension and blotted dry with sterile paper towels. To determine the number of organisms adsorbed on the thread, a segment of thread was placed in a
test tube with 2 ml of sterile saline and placed on a shaker for 45 minutes. After the bacteria had been eluted from the thread, the resulting suspension
was diluted and plated on MHA. One day prior to infection, mice were anesthetized by intraperitoneal injection of a ketamine cocktail. The backs of the
mice were shaved with a fine-tooth electric clipper, and the remaining fur was removed with Nair hair removal lotion. On the day of infection, mice were
again anesthetized by intraperitoneal injection of a ketamine cocktail. Surgical wounds were produced on the backs of the mice by making a longitudinal
midline incision, 2.5 cm in length, and extending down to the panniculus carnosus. The skin on either side of the incision was retracted, and the wound
was infected by insertion of a contaminated segment of thread through the skin with a suturing needle in such a way that the thread lay diagonally across
the panniculus, with the ends extending slightly from the skin.
9
Mouse Staphylococcal deep skin infection model using the
suture technique
8
Log10 CFU/mouse
7
6
5
4
3
2
1
0
Untreated
Placebo
2.0% Fusidic acid
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
RAT THIGH INFECTION MODEL
Rat thigh infection model with Staphylococcus aureus
Experiment Design:
Animal:
Organisms:
Infection loads:
Reference compound:
Test compound:
Male Sprague-Dawley or Wister rats, weighing 150gm on average.
Staphylococcus aureus (Smith; ATCC 13709)
1x109cfu/mouse (by 200µl)
Vancomycin (100 and 10mg/kg)
Linezolid (80 and 20mg/kg)
Rat Neutropenia:
The rats were rendered neutropenic by two intraperitoneal (IP) injections of cyclophosphamide: the first dose of 150mg/kg at 96 hours and the second dose of
100mg/kg 48 hours prior to the infection, as described by Pantopoulou et al (1). This method induces absolute neutropenia (≤1000 WBC/mm2 of blood) in rat
for 3-4 days.
Inoculum Preparation:
S. aureus (Smith ATTCC 13709) was grown from frozen stock at -80ºC onto MH (Muller Hinton Agar) plates. The culture was checked for purity and a fresh
colony was picked to grow an overnight late log phase (not more than 12 hours) liquid culture in brain heart infusion (BHI) broth at 37ºC in a shaking incubator.
The culture was centrifuged at 6000rpm (6200g) by Omnifuge® at 4ºC and the pellet was washed by resuspending the organisms in sterile saline and
centrifuging again. The organisms were washed twice similarly. Finally, the cells were resuspended to 5x109cfu/ml by a predetermined absorbance at 580nm
Spectrophotometric reading. This suspension was confirmed by plating serial ten-fold dilutions onto MH plates in duplicate. The rats were anesthetized
transiently (with isoflurane) and one of the thighs was disinfected properly with 70% alcohol or similar disinfectant and a 200µl of the inoculum was injected
into the caudal end of the thigh.
Tissue Processing:
The rats were euthanized by halothane or carbon dioxide inhalation and the infected thighs were dissected aseptically to expose the site of infection. The
presence of an abscess (expected within 24 hours) at the site of the infection was looked for and the infected tissues were collected. The tissues were
collected in 3ml of sterile saline in round-bottomed tubes (Fisher Scientific, Canada). The tissues were homogenized for 1 minute using Brinkmann Polytron
PT300 homogenizer at 24–28K rpm and the resulting homogenates will be serially ten-fold diluted (five to six times) in sterile saline. One hundred microlitres
(100µL) of each dilution were plated on MH agar plates in duplicate and will be incubated at 37oC for 24 hours. The colonies were counted after 24 and 48
hours and the colony forming units (CFU) were determined. The Log10 of the counts were determined and presented in the result.
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
RAT PNEUMONIA INFECTION MODEL
Rat lung infection model with Streptococcus pneumoniae
Experiment Design:
Animal:
Organisms:
Infection loads:
Reference compound:
Male Sprague-Dawley rats of 225-250gm/each.
Streptococcus pneumoniae (ATCC 6303), Type 3 (serotype).
8x107cfu/mouse (by 300µl)
Linezolid (120mg/kg/day)
Rat pneumonia model was established by intratracheal instillation of S. pneumoniae (ATCC 6303) and the rats were treated with linezolid (suspended in 1%
CMC) by oral (PO) administrations for 5 days. Levofloxacin was administrered orally bid and lungs harvested after a single day dosing. The untreated group
received vehicle only for the same period, and the survival of the rats were observed for 10 days post infection in the survival model.
Inoculum Preparation:
Type 3 S. pneumoniae (ATCC 6303) was grown from frozen stock at -80ºC onto blood agar plate (BAP) at 37ºC in 5% CO2. The culture was checked for purity and
liquid culture was grown by adding few pure colonies from the plate into Todd-Hewitt broth containing supplemented with 2% yeast extract. The culture was
grown overnight (~12 hours) at 37ºC in 5% CO2 and on next day morning, the organisms were collected by centrifuging at 6000rpm at 4ºC. The pellet was
resuspended in 10ml endoxin-free PBS and was centrifuged again to collect the pellet. Then the suspension was resuspended in 3ml of PBS and
spectrophotometric reading at 580nm was taken, which will contain a known number of bacteria (from recent test). The inoculum was then diluted to 2.5x108
CFU/ml, so that a 0.3ml had around 8x107cfu of the bacteria. To confirm the count, the inoculum was checked by viable counts by serial dilutions of the
inoculum and plating them on BAP in duplicate.
Rat Pneumonia Model:
The rats were anesthetized and were maintained by controlled inhalation of isoflurane. A small mid-line incision was made on the skin of the neck overlying
the trachea and the trachea was exposed by blunt dissection. A needle was inserted between tracheal rings to make a hole on the trachea and a catheter
(inner diameter, 0.5 mm; external diameter, 0.8 mm) (e.g., P50) was passed through the hole until it is wedged in the bronchus. Through the catheter, a 0.3
ml of the pneumococcal suspension containing 8x107CFU (~50 times lethal load) was delivered by introducing a 23 gauge needle connected to a 1ml syringe.
After the instillation, the catheter was removed immediately and gentle pressure was applied for 5 sec to occlude the trachea. The skin was then sutured and
the animals were placed slanting 60º upward until they woke up. This facilitated the migration of the organism to the distal alveolar region by gravity. By this
technique, the untreated rats were expected to die within 10 days. All studies were conducted in the Provincial Microbiology Laboratory located at the
University of Alberta Hospital in accordance with the guidelines set out by the Animal Policy and Welfare Committee of University of Alberta and Canadian
Council on Animal Care (CCAC).
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
PPDM 07-144: Efficacy of Levofloxacin in Rat
pneumonia model with S. pneumoniae ATCC 6303
(Infection: 1.9x 107cfu/rat; Treatment: Levofloxacin at 100 mg/kg BID for 1 day)
9
8
CFU/lungs
7
6
5
4
3
2
1
0
Vehicle, 1%CMC
Levofloxacin, 100mg/kg
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Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
NEUTROPENIC MOUSE THIGH INFECTION MODELS (Gram negative)
Efficacy studies with Ceftazidime (CAZ) and FPI-1465 in murine neutropenic thigh infection
model with KPC-2 producing Klebselia pneumoniae (NPI-2679).
Experimental Design Overview:
Animals:
Organisms:
Infection loads:
Test compound:
Six- to 8-week-old female CD-1 mice (20-22gm)
K.pneumoniae (NPI-2679/KPC-2);
1x105cfu
Ceftazidime, NXL-104
The mice were rendered neutropenic by Cyclophosphamide injection. At 6h post-hour infection, the mice were euthanized; thighs were collected and processed.
The bacterial loads were calculated and the growth at 6h was determined.
Log cfu/thigh reduction of KPC-2 producing K.pneumoniae after treating with ceftazidime alone and in combination with NXL-104
Mean
SD
Log difference (form
vehicle)
Vehicle-BID
7.7304
0.1424
0.0000
NXL-104 (64mpk) BID
7.5622
0.1528
0.1682
CAZ: NXL(1024:256 mpk) BID
4.2207
0.2189
3.5096
CAZ: NXL(512:128 mpk) BID
5.6256
0.2007
2.1047
CAZ: NXL(256:64 mpk) BID
6.4527
0.5227
1.2777
CAZ: NXL(128:32 mpk) BID
6.8957
0.1893
0.8347
CAZ: NXL(64:16 mpk) BID
7.1558
0.1368
0.5745
CAZ (1024mpk) BID
7.0473
0.2348
0.6830
Treatment Groups
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
Evaluation of CAZ, NXL-104 alone and in combination at a ratio of 4:1 at three different dose level in a neutropenic murine thigh model with KPC-2 producing K. pneumonia.
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
Log cfu/thigh reduction of KPC-2 producing K.pneumoniae after treating with ceftazidime alone and in combination with FPI-1465
Mean
SD
Log difference from
vehicle
Vehicle-BID
7.4617
0.1928
0.0000
Ceftazidime (1024mg/kg) BID
CAZ: NXL(1024:256 mg/kg) BID
FPI-1465 (64mg/kg) BID
FPI-1465 (128mg/kg) BID
FPI-1465 (256mg/kg) BID
CAZ: FPI-1465 (256:64mg/kg) BID
CAZ: FPI-1465 (512:128mg/kg) BID
CAZ: FPI-1465 (1024:256mg/kg) BID
6.6344
4.4982
7.1637
7.4539
7.2685
5.3647
4.7189
4.1021
0.2838
0.4190
0.1965
0.2099
0.2857
0.6008
0.3062
0.2636
0.8273
2.9636
0.2980
0.0078
0.1932
2.0970
2.7429
3.3597
Treatment Groups
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Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com
Evaluation of CAZ, FPI-1465 alone and in combination at a ratio of 4:1 at three different dose level in a neutropenic murine thigh model with KPC-2 producing K. pneumonia.
17 | P a g e
Biological Sciences & Pharmaceutical Development. NAEJA Pharmaceutical Inc. 4290-91A Street, Edmonton, Canada, T6E 5V2
Tel: 780-462-4044; Fax: 780-461-0196; email: info@naeja.com