Abstract

EFFECT OF PROTEASES SECRETED BY STAPHYLOCOCCUS AUREUS ON BIOFILM INTEGRITY

Saja Ali Shareef* and Dr. Abdulwahid B. Al-Shaibani

Abstract

This study aimed to use protease enzymes produced by Staphylococcus aureus in medical applications through the investigation of their antimicrobial activity on biofilm integrity. For such purpose, a total of 100 samples were obtained from patients of both genders and various ages who were referred to two teaching hospitals in Baghdad suffering from general infections of different severity and location of injuries in their body. Only 98 (98%) of the samples were positive for giving bacterial growth. The samples were cultured on blood agar and mannitol salt agar as a primary step for cultivation of bacteria. The results showed that more than 40 bacterial isolates were obtained. After the identification of these isolates by cultural, microscopic and biochemical examination, the results showed that 34 isolates were identified as Staphylococcus aureus. Moreover, Vitek 2 system was used which ensured that all of the 34 isolates belonged to this species. When the ability of these isolates to produce proteases was examined, the results showed that all the 34 isolates of S. aureus were able to do so but with variable degrees of production. Among them, an isolate symbolled S. aureus S28 was the most superior in protease production with a value of specific activity reaching 41.93 U/mg proteins. When the 34 S. aureus isolates were subjected to the antibiotics susceptibility test toward eleven different antibiotics, the results declared that all the isolates were totally resistance to penicillin, vancomycin, cephalothin, gentamicin and tetracycline, but completely susceptible to amikacin, ciprofloxacin, chloramphenicol, imipenem, novobiocin and rifampicin. To improve its production of proteases, the selected isolate S28 of S. aureus, was subjected to investigate its optimization conditions. The results declared that after supplementation of pH 8.5 with 0.5% lactose (as a carbon source), 1.5% peptone (as a nitrogen source), and 0.1% NaH2PO4 (as a phosphate source) when incubated at 37ºC in a shaker incubator (150 rpm) for 24 h, the maximum protease production was reached. To order to investigate their ability to form biofilm, fifteen 15 of the S. aureus isolates that gave the highest specific activity of proteases were used. The results showed that 14 of the isolates were able to produce a moderate production of biofilms at a rate of 93.3%, while the remaining one was a weak biofilm producer with a rate of only 6.7%. Upon studying the activity of proteases secreted by the selected Staphylococcus isolate on the biofilm integrity, the results declared that proteases treatment with different concentrations decreased the biofilm formation as the concentration increases. The lowest percentage of residual biofilm (29.4%) was achieved by using 1000 μg/ml of protease.

Keywords: Staphylococcus aureus, Protease, Biofilm.