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From: Food Quality & Safety magazine, April/May 2007

Super Surfaces

Pulverizing Polymers Out-Muscle Pesky Pathogens

by Richard Scott

Despite tremendous strides in the development of anti-infectives, most experts believe they are losing the war against microbes. Resistance to antimicrobial agents has been magnified to some degree in nearly every strain of bacteria pathogenic to humans and animals.

Methicillin-resistant Staphylococcus aureus (MRSA), already common in hospitals, is responsible for many untimely deaths. In 2002, this bacterium killed more than 800 Americans. That’s more lives than claimed by SARS and avian influenza combined. Almost weekly, one reads about outbreaks of “super-bugs” with resistance to vancomycin, which –until recently – was considered the antibiotic of last resort.

The increased use of antibiotics in food animals is a potential source of microbial resistance with immediate impact on public health. According to data from the Centers for Disease Control and Prevention (CDC), contaminated food causes 76 million illnesses, 325,000 hospitalizations and 5,000 deaths in the United States each year. Although more than 200 known diseases are caused by contaminated food, Toxoplasma, Salmonella and Listeria cause more than three-quarters of foodborne illnesses. The latter of the two are two of the 10 most common food pathogens. Although many of these organisms originate from the environment, the carry-over of resistant pathogens from slaughtered animals is always a concern.

Meat processors are not alone in the fear of foodborne. Recent recalls of spinach, high-end pet foods, peanut butter and baby food – in addition to hamburgers and other meat products –underscore the urgency of reducing sources of contamination to the greatest possible extent.

Novel antimicrobial materials, however, could revolutionize manufacturing of products that come into contact with foods during production, processing and storage. These products – amphiphilic antimicrobial compounds (AACs) –are chemical mimics of host defense peptides, molecules that occur naturally in higher organisms to ward off infection. AACs are available in small-molecule and polymeric format and suitable for use as both antimicrobial therapies and germicidal materials, respectively.

AACs have unique properties, which set them apart from traditional antimicrobial molecules and materials, including:

  • A novel mechanism of action that makes development of bacterial resistance unlikely;
  • Potent, ultra-broad spectrum activity against more than 150 Gram-positive and Gram- negative bacteria;
  • Unlike many antibiotic classes, AACs are bactericidal, not simply bacteristatic;
  • Faster acting than many antimicrobials, with killing times measured in seconds to minutes;
  • Straightforward manufacturing through known chemical and polymer synthesis;
  • Active against drug-resistant bacteria, including clinical isolates of multiple vancomycin- and methicillin-resistant strains;
  • Small-molecule AACs have shown excellent anti-microbial activity in animal studies.

Primitive life forms, such as molds, secrete defense compounds like penicillin to protect themselves from bacteria. Multi-cellular organisms, in particular mammals, possess a more complex, first-line immunity against bacterial infections. Host defense peptides are part of the non-humoral (that is, not involving antibodies) response that keep humans from rapidly succumbing to infections.

Biologists have discovered many different classes of natural host-defense peptides, most containing 20 to 40 amino acids. Although these molecules possess a diverse array of amino acid sequences, their physicochemical properties are similar. All are amphiphilic, meaning they exhibit affinity for both charged/polar and uncharged/non-polar environments. This property, rather than amino acid sequence, believed to be responsible for host defense peptides’ antimicrobial activity. Among the most common and well-studied antimicrobial peptides are the cationic, amphiphilic alpha helices, including the cecropins, magainins and many others.

AACs are designed to mimic the amphiphilic structure of the host defense proteins, but with completely synthetic, non-peptide backbones and, for therapeutic AACs, in small-molecule format. AACs directly cause bacterial cell membranes to rupture, a mechanism that is unique among known antimicrobial compounds. For this reason, antimicrobial agents based on specific compound designs will not engender bacterial resistance.

Antimicrobial Materials

Although sanitation in food processing is achieved principally through cleaning, the effect of materials used in food-processing surfaces, vessels and implements can be significant. Generally, bacteria have greater difficulty clinging to and persisting on smooth surfaces than on rough ones. Thus many food processing-surfaces particularly those made from metals, are polished to reduce surface roughness.

Materials also possess varying innate degrees of bacteria-friendliness. A recent study conducted by the Centre for Applied Microbiology & Research (Salisbury, U.K.) revealed that metals commonly used to manufacture food-contact surfaces harbor bacteria for much longer than an average work shift, some almost indefinitely. The most common food-processing surface, stainless steel, retains live bacteria for 34 days and copper, which is known to possess anti-microbial properties, retains viable pathogens for up to 14 hours. [See News & Notes, Food Quality, February/March, p. 18]

The activity of monomer or small-molecule AACs as therapeutics, while not directly applicable to food processing, illustrates the potential of the related polymers in the food marketplace. AACs suitable for human or veterinary drugs have demonstrated characteristics that positively distinguish them from host defense peptides or –for that matter – any other synthetic antibiotic or compound.

In studies using rodent models of bacterial infection, AACs have shown a level of safety and efficacy orders of magnitude more favorable than for host defense peptides, not to mention many marketed antibiotics. Some relevant characteristics of these molecules, which translate to the polymeric materials, include:

  • Systemic activity in animals against multiple bacterial diseases;
  • Selectivity for bacteria vs. human cells of 100 to greater than 10,000, compared with 10- to 20-fold for host defense proteins;
  • Well tolerated in animals at blood levels more than 300 times higher than bactericidal concentrations;
  • Excellent drug-like properties – pharmacokinetics, half-life, serum binding, and tolerability profiles are characteristic of safe, effective drugs;< /li>
  • Ease of manufacture – chemical synthetic schemes comparable to those for manufacturing drugs or polymeric materials.

AACs work by exploiting the unique chemical composition of bacterial cell membranes. Bacteria contain more negatively charged chemical groups on the outer surface of their membranes than do mammalian cells. Bacterial membranes also lack cholesterol, an essential component of all mammalian membranes. AACs home in on membranes that lack cholesterol and which contain large numbers of negatively-charged phospholipids – thus, they are specific and selective for bacterial cell membranes, but do not harm animal cells.

Much of the early work on AACs involved Compound 1, in Figure 1, which illustrates the amphiphilic character. This structure is amphiphilic by virtue of the hydrophobic t-butyl group projecting from the bottom of the repeat unit, and a charged amino group (at the top of the repeat unit).

Using this simple chemical scaffold, numerous series of analogs were synthesized as either defined oligomers and small molecules or heterogeneous polymers. Several of these oligomer and polymer series were discovered to have potent antimicrobial activity and high killing selectivity for bacteria versus mammalian cells.

Subsequently, through molecular modeling and chemical elaboration, we were able to improve the biological activities and selectivity of these compounds. Structurally defined oligomers and small molecules are under development for therapeutic applications, whereas the polymers and certain low-cost oligomers are under investigation as materials.

Powerful Antimicrobial Activity

In proof of principle experiments, acrylamide polymers similar in structure to compound 1 were painted onto glass and plastic surfaces at concentrations of 0.01 percent concentration. When exposed to bacteria under typical growth conditions, the coated materials completely inhibited bacterial growth. (See Figure 2.)

Antibacterial activity has also been demonstrated with polyurethane plastic films coated with antimicrobial materials. In one experiment, polyurethane was coated onto a glass slide, and an oligomeric AAC was adsorbed onto the surface at 0.1 percent concentration using dimethyl sulfoxide as the solvent. The resulting slides were then placed in a bacteria-rich nutrient broth for 72 hours to ascertain growth of bacterial colonies. In Figure 3, three photographs show a blank glass slide, a slide coated with polyurethane film alone (control, no polymer), and a polyurethane coated slide with 0.1 percent polymer.

In other experiments, depicted in Figure 4, active methacrylate-AAC copolymers have been dispersed, at 1 percent concentration, into polyurethane during the production process to yield solid plastic disks possessing inherent antimicrobial properties. These disks are then placed in a vessel, with bacterial-rich broth poured on top. After 24 hours in bacterial broth, the disks are removed, and stained for fluorescence imaging, with the following results. Bacterial colonies, indicated by the green fluorescence, are clearly visible over the entire surface of the control surface, but almost completely absent from the 1 percent AAC surface.

One application of AAC polymers with tremendous potential is their use to combat Stachybortus chartarum, the “black mold,” which causes approximately $90 billion in homes and commercial building damage annually. An AAC polymer can be used as additives to paints, drywall and other construction materials to prevent the growth of this troublesome and unhealthy fungus.

Two of the most promising compounds have shown outstanding activity against a broad range of antibiotic-resistant bacteria. One is a second-generation compound with a benzene ring backbone, and the other is a third-generation molecule with higher rigidity along the backbone.

Activity against most drug-resistant bacteria, as measured by minimum inhibitory concentration, is easily within therapeutic values for most organisms. The compounds are metabolically stable during in vitro studies and blood concentrations well above the therapeutic levels can be achieved in single dose toxicity experiments in rodents.

It is valuable to keep in mind that no antimicrobial surface will completely eliminate pathogenic bacteria from food that comes into contact with it. Rather, AACs can prevent bacteria from surviving and multiplying on contact surfaces, thus greatly reducing the opportunity for cross-contamination or persistent bacterial presence.

A Question of Resistance

Microbial resistance, while principally a phenomenon encountered during antibiotic treatment in humans and animals, is of increasing concern for antimicrobial agents used in consumer and industrial products. Provided working surfaces are maintained hygienically, AACs would probably have little impact on the emergence of resistant bacterial strains on these surfaces. Where these new materials will make a difference is in food that is already contaminated with resistant bacteria, which may have proliferated because of improper usage of veterinary antibiotics. AACs, with their powerful bactericidal activity against resistant and non-resistant bacteria, would lessen the opportunity for such organisms to multiply on food-contact surfaces, where food products could be cross-contaminated and people, ultimately, could suffer foodborne illnesses.

The “broth micro-dilution” method is the industry standard for measuring resistance to common antibiotics. A broth micro-dilution with four of the lead AACs was used against Staphylococcus aureus. In this experiment, the bacterium was exposed serially in the presence of sub-effective concentrations of four AACs. Two common fluoroquinolone antibiotic drugs, ciprofloxacin (Cipro) and norfloxacin, were employed as controls. Bacteria, including S. aureus, readily develop resistance to conventional antibiotics in this model.

Growing bacteria in the presence of increasing concentrations of the drug completes the experiment. The culture tube containing the highest concentration of drug where bacterial growth is seen after 24 hours is selected and the bacteria are re-passaged with a fresh dilution series of drug. This process is repeated every 24 hours for 16 passages and the minimum inhibitory concentration (MIC), the lowest dose required to kill the bacteria) is noted at every passage. Development of resistance is indicated by a progressive increase in the MIC over time (or number of passages).

Figure 5 illustrates results of this experiment. Conventional antibiotic drugs (purple and aqua lines) show significant bacterial resistance developing after three to five passages, and increasing resistance in subsequent passages. The three AACs, whose graphs do not budge from baseline, show no tendency whatsoever, within this experimental protocol, to foster bacterial resistance (interestingly, host defense proteins show no resistance either). These results have been verified by independent investigators and repeated, with nearly identical results.

Broad Applications

Polymeric AACs can be used as additives to materials to create self-sterilizing surfaces and bactericidal products, including paints, plastics, and textiles. Some of these may be developed rapidly at relatively low cost and risk for multi-billion dollar potential market opportunities.

The focus of polymer development is on two particular classes of compounds for materials applications, the polymethylacrylates and polynorborenenes. A small phenylalkyne oligomer, with broad-spectrum antimicrobial activity and which is easily synthesized, is also suitable for materials applications.

All AAC monomers and polymers compounds are produced from commercially available starting materials. Methylacrylate polymer synthesis is a one-pot, one-step co-polymerization of two monomers; one functionalized monomer bearing a positively charged side group and the other bearing a hydrophobic side group. The resulting random co-polymer is amphiphilic and antimicrobial activity is dependent on the percent composition of each of the methacrylate monomers. Polynorborenene polymers are made using a co-polymerization process with mixtures of two monomers that bear positively charged and hydrophobic side groups, or a polymerization process using one type of monomer that bears both positively charged and hydrophobic side groups.

AACs in development as drugs are synthesized as small molecules, while those used in industrial materials are prepared as polymers. The polymers are formulated into materials in one of several ways. Pure AAC polymers may be ground into fine powders and added, in low concentration, to polymer blends to create bulk polymer (e.g. polymethacrylate) with excellent antimicrobial properties. Another possibility is to co-polymerize AAC monomers with a suitable monomer to form a homogeneous material that is inherently bactericidal.

Many types applications are feasible for polymeric AACs within the following product classes:

  • Biomedical Applications: intravenous tubes, catheters, antiseptic lotions, bandages, implantable joints, medical devices, surgical gloves;
  • Industrial Applications: clothing, paper, paints, construction materials (walls, floors, benches), antifouling coatings, hospital surfaces;
  • Consumer Products: cosmetics, personal care products, toilet seats, toys, bedding, towels, carpeting.

AACs show tremendous potential as materials for manufacturing a broad range of food-contact products, including work surfaces, processing equipment, utensils and implements, handling and storage equipment, and packaging. Since these materials may be formulated as bulk polymers/copolymers, polymer additives, or films, AAC could be available for almost any plastic product that comes into contact with food, from the processing plant to the dinner table.


AACs are still very much development-stage products and work in progress. Applying new materials to critical applications like food handling and medicine requires review and/or registration with appropriate regulatory agencies, and a good deal of scientific data supporting the materials’ safety. AACs have not been rigorously tested for toxicity, leaching, de-polymerization, or other types of chemical effects that may occur in the presence of food, and thus prevent their broad use in the food industry. AAC applications in sanitary industrial paints and coatings, as well as for clothing, appear to be more near-term possibilities.

Richard Scott, Ph.D. is vice president of research for PolyMedix (Radnor, Pa.) He can be reached at 484-598-2336 or



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