Tuesday, December 16, 2008

Who will win this war…….?

High in vitro antimicrobial activity of synthetic antimicrobial
peptidomimetics against staphylococcal biofilms.

Kristina Flemming, Claus Klingenberg, Jorun Pauline Cavanagh, Merethe Sletteng, Wenche Stensen, John Sigurd Svendsen and Trond Flægstad

Journal of Antimicrobial Chemotherapy (2009) 63, 136–145

It is more than 80 years when the first antibiotic was discovered by Sir Alexander Fleming (September 28, 1928). Since the mid 40’s antibiotics have been widely used to prevent bacterial outbreaks in humans, but they also play a role as growth promoting agents in agriculture. The years of positive selection pressure have caused the global spread of antibiotic resistance in the microbial population. Special threats are bacteria that form a biofilm. Biofilm is defined as microbial-derived sessile communities attached to a surface and embedded in a self-produced polymeric matrix. Bacteria in biofilms are usually less susceptible to antimicrobial agents than rapidly growing planktonic cells. There are several hypotheses to explain the strong antimicrobial tolerance of biofilm cells such as the limitation of agent penetration, the existence of dormant cells, phenotypic variations, a quorum sensing system, and multidrug efflux pumps. So there is always need to develop new effective antimicrobial agents that can kill bacteria.

Cationic antimicrobial peptides (CAPs) are widespread in nature and play an important role as part of innate immunity. In general, CAPs are fairly large molecules that carry a net positive charge and contain ~50% hydrophobic residues. Their mode of action involves binding to negatively charged structural molecules on the microbial membrane. Once bound, CAPs form pores that increase the cell membrane permeability and ultimately lead to cell lysis. There is also evidence for other antimicrobial mechanisms such as interaction with intracellular targets or activation of autolytic enzymes in the bacteria, or induction of the immune response in the host. CAPs have a broad spectrum of antimicrobial activity and development of resistance is rare. Unfortunately, CAPs are difficult and expensive to produce in large quantities and are usually sensitive to protease digestion. Modifications of CAPs have resulted in the development of extremely short synthetic antimicrobial peptidomimetics, also called SAMPs. SAMPs mimic the effect of CAPs, but have improved pharmacokinetic properties and are thus a promising new group of antimicrobial substances.

In this study the authors investigated the antimicrobial activity of clinically relevant antibiotics like linezolid, tetracycline, rifampicin and vancomycinand, and newly designed SAMPs against biofilms of three different staphylococcal species (six strains). They also evaluate a simple screening method to quantify the metabolic activity of biofilms before and after the biofilm had been subjected to treatment with antimicrobial agents.

Two methods were used for quantify the biofilm metabolic activity. The first method used Alamar Blue (AB) to measure metabolic activity. AB is a redox indicator that both fluoresces and changes color in response to chemical reduction that can be measured by monitoring absorbances at 570 and 600nm. The AB method showed excellent applicability and it is simple, fast, non-toxic and suitable for high-throughput quantification, at least for biofilms grown in microtitre plates. It shows also great reproducibility and good sensitivity which is very important in antibiotic effect studies. To confirm the killing properties of the antibiotics used, the second method based on LIVE/DEAD biofilm staining was used. This use two stains: Syto9 (green fluorescence) and propionium iodide (PI – red fluorescence). Syto9 stains DNA in living cells while PI reduces green fluorescence only in dead cells. Fluorescence is observed with a confocal laser scanning microscope (CLSM).

Using those two methods authors showed that all SAMPs were clearly more effective in reducing metabolic activity in staphylococcal biofilms at low concentrations compared with antibiotics, even though they generally had higher MICs under planktonic growth conditions. In general, antibiotics were rarely able to cause a complete suppression of metabolic activity. In contrast, SAMPs were frequently able to suppress metabolic activity completely, indicating effective killing. It seems that SAMPs caused damage of the bacterial cell membranes even in slow growing or dormant bacteria embedded in a biofilm. In contrast, the antimicrobial agents used in this study predominantly affected growing bacteria by inhibiting their cell wall development (vancomycin) or by inhibition of their protein synthesis (linezolid, rifampicin and tetracycline).

In conclusion the SAMPs are potential new therapeutic agents in biofilm-associated infections. They could be especially attractive for topical treatment of chronic wound infections. The possible clinical applicability of SAMPs to prevent medical device-associated staphylococcal infections warrants future in vivo studies.

So maybe we can win this war…….

dr Jaroslaw E. Krol

UoI, Moscow

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