IN VITRO CHARACTERISATION OF GROWTH AND MESOTHELIAL CELL KILLING CAPACITY OF PSEUDOMONAS SPP. ISOLATED FROM PERITONEAL DIALYSIS-ASSOCIATED PERITONITIS EFFLUENT

C PEH 1, M KOPCZYK 1,2, K MULRONEY 1,2 A CHAKERA 1,2,3

1Translational Renal Research Group, Harry Perkins Institute Of Medical Research, Nedlands, Australia, 2Centre for Medical Research, The University of Western Australia, Nedlands, Australia, 3Renal Unit, Sir Charles Gairdner, Nedlands, Australia

Aim: To better understand in vivo growth characteristics of Pseudomonas aeruginosa and Pseudomonas stutzeri clinical isolates and investigate cytopathic effects of P. aeruginosa isolates on mesothelial cells.

Background: Peritonitis is the major complication of peritoneal dialysis (PD). While peritonitis caused by Gram-positive bacteria occurs more frequently, Gram-negative peritonitis often results in more severe infection and associates with higher rates of catheter loss and treatment failure. Of Gram-negative infections, those caused by Pseudomonas spp. are associated with especially poor patient outcomes.

Methods: Pseudomonas clinical isolates were grown in filter-sterilised peritoneal dialysis effluent (PDE) and a nutrient rich culture broth, tryptic soy broth (TSB) over 4 hours (typical dwell time for dialysis fluid in patients), to compare their growth characteristics. Next, for our in vitro infection assay, we exposed MeT-5A (ATCC® CRL-9444™) monolayers to doses of either 1.0 × 10⁴ or 1.0 × 10⁷ CFU / mL for 4 hours and measured cell death using Live/DEAD Fixable Near-IR viability stain and an Attune™ NxT flow cytometer..

Results: We observed significantly lower growth in PDE compared to TSB (p = 0.003) across all isolates tested, except for one P. stutzeri isolate which grew better in PDE.  Preliminary results from our infection assay, suggests inter-strain variability and dose-dependent killing of Met-5A cells by P. aeruginosa: at low dose, cells killed varied from 8% – 67%, and at high dose from 35% – 92%.

Conclusion: Inter-strain variability seen in both growth indices and cytotoxicity of Pseudomonas spp. isolates may associate with the current unexplained differences in outcomes for patients suffering from PD peritonitis. Future studies should broaden the range of organisms tested to understand the mechanisms associated with these observations.


Biography:

An undergraduate student researcher that was interning at the Translational Renal Research Group at Harry Perkins over the summer and continued over the last semester of my Bachelors in Biomedical Science (Microbiology and Immunology & Pathology and Laboratory Medicine). My project looks into studying peritonitis infection by observing the interactions in vitro of mesothelial cells and Pseudomonas spp.. I was involved in bacterial expression systems and the validation of novel diagnostic tools for detection/ monitoring of Acute Lymphoblastic Leukemia, during the course of completing my Diploma in Biomedical Research back in Singapore.

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