Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.
BACKGROUND
Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites.


METHODS
Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR.


RESULTS
Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility.


CONCLUSION
The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen.
['Galdino|Anna Clara M|ACM|', 'Viganor|Lívia|L|', 'Ziccardi|Mariangela|M|', 'Nunes|Ana Paula F|APF|', 'Dos Santos|Kátia R N|KRN|', 'Branquinha|Marta H|MH|', 'Santos|André L S|ALS|']
D001426:Bacterial Proteins D001826:Body Fluids D001938:Brazil D003550:Cystic Fibrosis D004492:Edetic Acid D004591:Electrophoresis, Polyacrylamide Gel D018093:Gelatinases D005798:Genes, Bacterial D006801:Humans D045726:Metalloproteases D009928:Organ Specificity D010196:Pancreatic Elastase D018410:Pneumonia, Bacterial D011480:Protease Inhibitors D011552:Pseudomonas Infections D011550:Pseudomonas aeruginosa D012007:Rectum D012137:Respiratory System D014774:Virulence D014946:Wound Infection
Proteases Brazilian clinical isolates Pseudomonas aeruginosa Elastase Gelatinases Aislamientos clínicos brasileños Elastasa Factores virulentos Gelatinasas Proteasas Virulence factors