Optimizing and purifying extracellular amylase from soil bacteria to inhibit clinical biofilm-forming bacteria

Abstract

Background. Bacterial biofilms have become a major threat to human health. The objective of this study was to isolate amylase-producing bacteria from soil to determine the overall inhibition of certain pathogenic bacterial biofilms. Methods. We used serial dilution and the streaking method to obtain a total of 75 positive amylase isolates. The starch-Agar plate method was used to screen the amylolytic activities of these isolates, and we used morphological and biochemical methods to characterize the isolates. Optimal conditions for amylase production and purification using Sephadex G-200 and SDS-PAGE were monitored. We screened these isolates' antagonistic activities and the purified amylase against pathogenic and multidrug-resistant human bacteria using the agar disk diffusion method. Some standard antibiotics were controlled according to their degree of sensitivity. Finally, we used spectrophotometric methods to screen the antibiofilm 24 and 48 h after application of filtering and purifying enzymes in order to determine its efficacy at human pathogenic bacteria. Results. The isolated Bacillus species were Bacillus megaterium (26.7%), Bacillus subtilis (16%), Bacillus cereus (13.3%), Bacillus thuringiesis (10.7%), Bacillus lentus (10.7%), Bacillus mycoides (5.3%), Bacillus alvei (5.3%), Bacillus polymyxa (4%), Bacillus cir-culans (4%), and Micrococcus roseus (4%). Interestingly, all isolates showed a high antagonism to target pathogens. B. alevi had the highest recorded activity (48 mm) and B. polymyxa had the lowest recorded activity (12 mm) against Staphylococcus aureus (MRSA) and Escherichia coli, respectively. On the other hand, we detected no antibacterial activity for purified amylase. The supernatant of the isolated amylaseproducing bacteria and its purified amylase showed significant inhibition for biofilm: 93.7% and 78.8%, respectively. This suggests that supernatant and purified amylase may be effective for clinical and environmental biofilm control. Discussion. Our results showed that soil bacterial isolates such as Bacillus sp. supernatant and its purified amylase are good antibiofilm tools that can inhibit multidrugresistant former strains. They could be beneficial for pharmaceutical use. While purified amylase was effective as an antibiofilm, the isolated supernatant showed better results.