Introduction: There is an increasing recognition that bacteria biofilm play a significant role in the morbidity and mortality associated with device related and wound infections[1]. There is an unmet need for new antimicrobial technologies that are effective against biofilm and won't select for antibiotic resistance. The goal of this study was to evaluate and compare the efficacy of two novel antimicrobial peptides against Staphylococcus aureus (SA) in preformed biofilms. A secondary focus was to evaluate the effect of pH and the presence of serum proteins on the concentration of each peptide required to eradicate biofilm.
Materials and Methods: Two proprietary cationic, amphiphilic antimicrobial peptides, ASP-1 and ASP-2 were synthesized by Bachem. A fresh overnight culture of SA Rosenbach (ATCC 10390) was diluted to ~107 bacteria in cation adjusted Muller Hinton broth (CAMHB) and used to inoculate pegs on the lid of a 96-well MBEC device (Innovotech). Biofilms were allowed to develop for 24 hrs at 37°C and 110rpm. Biofilms were challenged with serially diluted peptide solutions for 24 hrs at three pHs: acetate buffer saline (AcBS) pH6.0, phosphate buffer saline (PBS) pH7.4 and borate buffer saline (BBS) pH 8.9. The impact of biologically relevant proteins on peptide activity was also studied using buffers containing 1) Bovine serum albumin (BSA) at 30mg/ml, or 2) 50% heat-inactivated human serum with 0.05% peptone (HISP). All challenging media contained 2% CAMHB.
Minimum biofilm eradication concentration (MBEC) was determined by sonicating pegs in Dey-Engley neutralizer broth followed by a 24 hr re-growth period and OD reading at 600nm. MBEC was assigned to the lowest concentration that gave OD<0.1. Bacteria recovery was quantified by serially diluting sonicates and plating on TSB/Agar.
Results and Discussion:
ASP-1 and ASP-2 displayed nearly identical MBEC values ranging from 1.2 to 19 µg/ml, which are similar to previously reported minimum inhibitory concentrations for planktonic bacteria (Table 1)[2]. An increase in pH from 6.0 to 8.9 led to a 2 fold decrease in MBEC. Higher peptide concentrations were required to eradicate biofilm in the presence of proteins with an 8-fold increase observed for 50% HISP at pH 6.0 and 7.4.
As shown in the Figure 1, both peptides possess strong biocidal properties over a wide pH range demonstrating more than 3 log reductions in colony forming units at low micromolar concentrations even in the presence of albumin.
Conclusion: The two engineered antimicrobial peptides demonstrate potent activity against biofilm in a broad range of media simulating clinical conditions. These properties combined with high intrinsic stability and low likelihood for developing antibiotic resistance offer significant practical advantages and high potential for wide range of applications requiring infection prevention and control.
References:
[1] Mihai MM, Holban AM, Giurcaneanu C, Popa LG, Oanea RM, Lazar V, Chifiriuc MC, Popa M, Popa MI. Curr Top Med Chem 2015;15(16):1552-76.
[2] Deslouches B, Steckbeck JD, Craigo JK, Doi Y, Mietzner TA, Montelaro RC. Antimicrob Agents Chemother 2013;57(6):2511-21.