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Background: Infection of burn wounds by multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of morbidity and mortality and remains one of the most challenging concerns for the burns unit. The aim of this study is purify and characterize the haemolysin produced by multidrug resistant P. aeruginosa PAO1 isolated from burn wounds. Methods: Isolation and identification of P. aeruginosa from burns was done by standard bacteriological methods. P. aeruginosa PAO1 was identified by PCR amplification and sequencing of the 16S rRNA gene. The haemolysin of P. aeruginosa PAO1 was purified by 70% ammonium sulphate precipitation followed by gel filtration on Sephadex G-100, and separation by SDS-Poly Acrylamide Gel Electrophoresis. In vivo toxicity of the purified haemolysin was determined by intraperitoneal injection of Swiss albino mice, and in vitro toxin-antitoxin neutralization test was performed as previously described. Results: The pure haemolysin had a molecular weight of 37 kDa, with maximum activity at 25°C for 30 minutes and stable within pH range of 4-9 (maximum activity at pH 7). The haemolysin was activated by Ca2+, Fe3+ and Cu2+. Intraperitoneal injection of mice with 0.5ml of haemolysin (128 HU/ml) caused 100% mortality while 0.5 and 0.1 ml of haemolytic titer (64 HU/ml) of the heated haemolysin (toxoid) caused 50% and 0% mortality respectively. In vitro toxin-antitoxin neutralization test revealed that anti-haemolysin antitoxin was present in the serum of the mice that were previously vaccinated with heated toxin. Conclusion: This study concluded that haemolysin can be a potential vaccine component for prevention of haemolysis caused by multidrug resistant P. aeruginosa in burn patients.Keywords: haemolysin, Pseudomonas aeruginosa, multidrug resistant organism

Most microorganisms form biofilm as protection mechanism that provides cells advantages as infectivity, antibiotic resistance and survivability of a variety of infections in humans. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen cause of infections in cystic fibrosis patients such as pneumonia and chronic lung infections. Although biofilm control is an important in medicine, clinically, there is not available effective inhibitors of biofilm formation but some environmental factors could influence on biofilm formation. This research investigated the effect of incubation periods, temperature and iron concentration on the formation of biofilm which is critical for virulence by multidrug resistant P. aeruginosa taken from the sputum of pulmonary infection patient. It was found that moderate biofilm was observed on day two and day one was low in formation of biofilm but on day three was the optimum period for P. aeruginosa to switch into a strong biofilm. We investigated the effect of temperature and found that biofilm showed the highest level at temp 37°C. After using different concentrations of iron, it was showed that biofilm production was higher at the addition of FeSO 4 at 25 mM incubated at 37°C for 3 days. It concluded that some factors can affect and control the biofilm formation as a significant part of virulence system plays a critical role in antibiotic resistance in P. aeruginosa infection.