Detection of an autolysin produced by a Pseudomonas strain under anaerobic conditions.

TRIBELLI PAULA; LÓPEZ NANCY

Rosario, Santa Fe.

Congreso; V Congreso Argentino de Microbiología General; 2008

Sociedad Argentina de Microbiología General

Autolysins like antimicrobial agents are substances that have biotechnological and clinical interest. Autolytic enzymes cleave specific components of the bacterial cell wall, peptidoglycan (PG), and perform many important physiological functions during cell growth and development such as cell division, sporulation and competence. However, deregulated activity of these enzymes can also lead to autolysis under stressful circumstances such as those encountered during stationary phase. Pseudomonas sp. 14-3 is an Antarctic non pathogenic strain that is able to accumulate polyhydroxybutyrate. During anaerobic growth was observed an autolysis phenomenon at early exponential phase. The supernatant of a lysed culture was used for several assays. Zymogram was performed with purified Pseudomonas sp. 14-3 PG as substrate in renaturing polyacrylamide gels and autolytic activity was visualized as a clear zone in a background of PG stained with a Methylene Blue/KOH solution with an apparent molecular mass corresponding to 28 kDa. Lytic activity of the supernatant was tested in vivo in Pseudomonas sp. 14-3 and Pseudomonas aeruginosa PAO1. P. aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis. Exponential and stationary cells of both strains were exposed to the supernatant of a lysed culture and an inhibition growth zone was observed in aerobic and anaerobic conditions. A turbidimetric assay was used to monitor the time course of PG solubilization in both strains. Decrease in turbidity of a suspension of PG after exposition to autolysin was observed. Biofilm formation and persistence in an anaerobic environment is now considered crucial in chronic infection with P. aeruginosa. Microplate assay staining with crystal violet was used to analyze the effect of crude lysate on biofilm formation. We found that P. aeruginosa and Pseudomonas sp. 14-3 biofilms were dispersed. Identification of the possible autolysin induced during anaerobic growth of Pseudomonas sp. 14-3 by MALDI-TOF and PCR techniques followed by sequenciation is now in progress. Further studies of autolytic enzymes in combination with antibiotics therapy could be interesting in the treatment of Pseudomonas infections.