Kedar P Purnapatre, Aditi Agarwal; Jitendra A. Sattigeri, Nobuhisa Masuda, Dilip J. Upadhyay. Daiichi Sankyo India Pharma Private Limited, Village Sarhaul, ...
In vivo efficacy of a novel Leu-t-RNA synthetase inhibitor Compound A against MDR Pseudomonas aeruginosa 1594965 in a foreign body associated urinary tract infection model Manoj Kumar, Manisha Pandya, Madhvi Rao, Tarani Kanta Barman, Vattan Joshi, Sarath Kumar Sunkara, Paras Kumar Jha, Kedar P Purnapatre, Aditi Agarwal; Jitendra A. Sattigeri, Nobuhisa Masuda, Dilip J. Upadhyay Daiichi Sankyo India Pharma Private Limited, Village Sarhaul, Sector-18, Udyog Vihar Industrial Area, Gurgaon-122 015, Haryana (India)
No.41.126
Materials and methods
Introduction • •
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Catheter associated UTI is responsible for 40% of nosocomial infections P. aeruginosa is responsible for 12% of all nosocomial UTIs making it third most common organism isolated from UTI patients in a hospital setting Catheter associated bacteriuria leads to an increased length of hospital stay , causing 900,000 additional hospital days per year and complications associated with nosocomial UTI contribute to an estimated death of 7500 additional deaths per year Biofilms formed by MDR P. aeruginosa are a serious problem as they are usually untreatable by antibiotic therapy. When antibiotic treatment is over the biofilms again shed planktonic bacteria resulting in recurrent acute infection Compound A, a novel Leucyl-t-RNA synthetase inhibitor (LRSI) was screened against a mouse model of foreign body (catheter) associated MDR P. aeruginosa AUTI.
Materials and methods Bacteria: P. aeruginosa 1594965 was a clinical isolate obtained from Daiichi- Sankyo ( Tokyo) Culture collection. Standard antibiotic and Compound A: Meropenem was obtained from Astra zeneca ( Meronem ) and Compound A was synthesized by Department of Medicinal Chemistry, DSIN. Ampicillin (Sigma Chemical Co., St. Louis, Mo., U.S.A.) was used for treatment of animals after catheterization.
Results Figure 1 : Scanning electron Microgram of biofilm formed on catheters in 4h
Figure 2: Kidney bacterial counts after 7 days treatment with Compound A
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Log10 CFU/Kidney
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SC, mg/kg
0 Figure 3: Bladder bacterial counts after 7 days treatment with Compound A
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Pseudomonas aeruginosa 1594965
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Log10 CFU/Bladder
Introduction: Pseudomonas aeruginosa is one of the major pathogens causing morbidity and mortality in hospital acquired infections (HAI). MDR P. aeruginosa are resistant to 3 or more classes of antibiotics. In cUTI cases catheter associated infections are incurable by most of the antibiotics. Compound A a novel Leucyl-t-RNA synthetase inhibitor (LRSI) showed efficacy in mouse ascending urinary tract infection (AUTI) model against P. aeruginosa biofilms formed on urinary catheters. Methods and material: In this study we established a mouse model of foreign body associated AUTI. A spiral polyethylene tube (PT) was placed transurethrally into bladder without surgical manipulation, followed by transurethral inoculation with Pseudomonas aeruginosa 1594965 (MDR) strain. Scanning electron microscopy (SEM) was done after 4 hr of inoculation of bacteria and bacterial counts were taken in kidneys, bladders and catheter pieces for respective samples. In vivo efficacy of Compound A was tested using this established model against P. aeruginosa 1594965(Compound A MIC: 1 µg/ml, meropenem MIC: 32 µg/ml). Compound A was tested at 30 and 220 mg/kg SC q6h and meropenem was tested at human simulated dose of 60 mg/kg SC q6h. The treatment was initiated 4hr post infection and continued daily for 7 days. Bacterial counts of placebo 4 hr, 7 days and 7 days treated group’s samples of kidneys, bladders and catheters were calculated. Data was analysed using bacterial counts of each group and compared with those of 4 hr placebo control group. Results: SEM showed good establishment of biofilm in 4 hr samples. In foreign body associated mouse infection model. LRS inhibitor Compound A at 30, 220 mg/kg SC q6h showed log10 difference of -2.38, -2.18 log10 CFU/kidney respectively compared to 4h placebo and meropenem at 60 mg/kg SC q6h was inefficacious. In bladders this log difference was 2.30, -1.21, -0.78 respectively. On catheter only Compound A at 220mg/kg SC q6h showed log10 difference of -1.59 log10CFU/ml. No efficacy was observed in remaining groups. Conclusion: Efficacy of Compound A in foreign body associated mouse AUTI infection model against MDR Pseudomonas aeruginosa 1594965 proves its efficacy on biofilm. These results warrant its further investigation in hospital associated Pseudomonas aeruginosa infections.
Animals : All the procedures on female Swiss Webster mice were done for this study as per the protocols approved by Institute animal ethic committee. Establishment of Foreign body associated infection : Preparation of spiral polyethylene tube: • Approximately 1.0 cm long segment of polyethylene tube (PT; ID 0.40 mm, OD 0.80 mm) were cut • First, the PT was fitted onto a flexible metal stylet, which then was coiled two and a half times around an 18-gauge injection needle • To fix the shape of the spiral PT, it was placed in boiling water for 3 sec. • The PT and stylet were straightened and moved to another end of stylet, and another segment of PT, 3 mm in length, was fitted onto the stylet. • The PT segments and stylet were sterilized by dipping the PT segments and stylet into 80% ethanol for ~2 hr. Placement of spiral polyethylene tube in to mice bladder: • Mice were anesthetized by intramuscular injection with a mixture of ketamine hydrochloride (10 mg/ml final conc.) and xylazine hydrochloride (4 mg/ml final concentration) • The end of the stylet having the 1.0-cm-long PT was placed into the urethra until its leading end was in the bladder. • The 3-cm long PT then was pushed inside until the shorter segment was pushed off the stylet, thereby leaving the 1 cm long PT in the bladder. • The stylet with the remaining PT subsequently was removed. • The animals were given drinking water containing ampicillin (1 mg/ml) for 4 days after PT-insertion to prevent infection by contaminants. Bacterial inoculation: • P. aeruginosa 1594965( Meropenem MIC : 32µg/ml, Compound A MIC : 1 µg/ml) was grown overnight on a trypto-soy agar (TSA) plate at 37 + 2˚C. • The bacterial suspension was prepared using PBS and turbidity was adjusted to 0.19 at OD620. • The water of animals was removed one night before (~16 to 18 hr) the inoculation. • The animals were anesthetized by an intramuscular injection with a mixture of ketamine hydrochloride and xylazine hydrochloride as described above. • A 50 µl volume of inoculum was transurethrally introduced into the bladder by a syringe fitted with a 25-gauge needle with a blunt point. • The urethra was clamped for 4 hr to prevent urine flow. • Scanning electron microscopy (SEM) was done after 4 hr of inoculation of bacteria to visualize the biofilms formed and bacterial counts were taken in kidneys, bladders and catheter pieces for the samples. Water was given after 4 hr when treatment was initiated. • In established foreign body associated infection animals were treated with Compound A at 2 doses (30, 220 mg/kg) and Meropenem 60 mg/kg SC, q6h dose x 7 days • Bacterial counts of Kidney and bladders of the animals were taken on the 8th day and for dead animals during 7 days treatment.
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Figure 4: Catheter bacterial counts after 7 days treatment with Compound A
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Log10 CFU/Catheter
Abstract
Conclusion
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Compound A was found to be efficacious against foreign body associated mouse AUTI model against Pseudomonas aeruginosa 1594965 in the present study at the dose of 220mg/kg SC q6h x7 days
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This warrants its further investigation in hospital ssociated MDR P. aeruginosa infections
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Scanning electron microgram showed formation of biofilm by P. aeruginosa 1594965 on catheters Compound A was efficacious in foreign body associated AUTI model at 220mg/kg SC q6h x7 days dosing with 2.38, 2.3 and 1. 59 log10 reduction in bacterial count in kidneys, bladders and on catheters respectively compared to 4 h untreated control (4 h PC) Compound A at 30 mg/kg SC q6h x 7 days showed 2.15 and 1.21 log10 reduction in bacterial count in kidneys and bladders respectively; it did not show decrease counts on catheter at this dose Meropenem was inefficacious at 60mg/kg SC q6h x 7 days in the present study
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References
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• In Figures 2, 3, 4 : Open circles represent data of individual animal organs, solid circles represent data for dead animals during this study
Daiichi Sankyo India Pharma Private limited, Gurgaon, INDIA
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Rahul Mittal et al. Urinary tract infections caused by Pseudomonas aeruginosa : A minireview. Journal of Infection and Public Health ( 2009) 2; 2001-2011 Thomas Hooton et al. Diagnosis, prevention and treatment of catheter associated urinary tract infection in adults: 2009 International clinical practice guidelines from Infectious Diseases Society of India Yuichi Kurosaka et al. A nonsurgical rat model of foreign body associated urinary tract infection with Pseudomonas aeruginosa. Microbiol. Immunol, 45 (1), 915, 2001
