Natural Anti-Biofilm Agents

(March, 2015)

Introduction

Biofilms are gelatinous masses of microorganisms capable of attaching to virtually any surface. According to the NIH, they factor into nearly 80% of all bacterial infections [Schachter, 2003] and are inherently resistant to antibiotics. Biofilms are what keep wounds from healing, and bladder infections recurring. They may also be why lyme disease lingers. Biofilms are at the heart (and lung) of bacterial pneumonia, and are the death of cystic fibrosis kids and burn patients. Biofilms cause tooth decay, gum disease, sinusitis, ear infections, and Legionnaires’ disease. Biofilms glom onto medical devices (e.g., heart valves, catheters, joint replacements) where they are deadly, or difficult to eradicate. Biofilms plague hospitals, and contribute greatly to our health care burden. [Hall-Stoodley et al., 2004]

Biofilms are also good for us. They line the digestive tract, especially the lower intestines, and the skin. Healthy biofilms contain many different species of bacteria working together to benefit humans. Many trillions of organisms protect us from pathogens and toxins, help boost our immune defenses, keep our plumbing working, steer us away from obesity, and may even make us think and feel better [Pollan, 2013; Rose, 2011]. An imbalance of bacteria in the gut – particularly from antibiotic usage, stress, or lack of fiber in the diet – leaves us susceptible to disease.

There are likely a number of stealthy biofilms that adversely affect the body in unknown ways. The list may include fetal development, autism, depression, chronic fatigue, Lyme disease, cognitive impairment, etc. [Janossy, 2015] Chronic Lyme disease may play a role in the development of dementia and Alzheimer’s. [McDonald, 2012] Many stealthy infectious agents have been identified (e.g., Borrellia, Mycoplasma, Bartonella, Babesia, Rickettsia), but some are still unknown or poorly understood. Chronic inflammation from biofilm infection can lead to cancer, cardiovascular disease, dementia, and other debilitating conditions. [Esser et al., 2015]

Recently, new anti-biofilm agents have been developed as adjuncts or alternatives to classical antibiotic treatment. Many of these novel agents show “resistance” to the emergence of antimicrobial resistance, and even enhance the activity of conventional antibiotics. Anti-biofilm substances may be synergistic with other antimicrobials to overcome persistent infectious threats. [Wu et al., 2004]  The following is a quick review of many of the natural anti-biofilm agents currently under study.

Biofilm-Disrupting Enzymes

Enzymes, like DNase I, α-amylase and DspB are biofilm-dispersing agents that degrade the biofilm matrix, permitting increased penetration of antibiotics. DNase I cleavage of extracellular DNA leads to alterations in biofilm architecture, which permits increased antibiotic penetration. [Tetz et al., 2009] A DNA-dissolving drug (Pulmozyme) has been used in cystic fibrosis patients to help disrupt the biofilm. α-amylase is a proven anti-biofilm agent against Staphylococcus aureus, Vibrio cholerae and Pseudomonas aeruginosa, not only inhibiting biofilm formation, but also degrading preformed mature biofilms [Kalpana et al., 2012]. DspB is a soluble β-N-acetylglucosaminidase with broad-spectrum activity to dissolve the biofilm matrix, and shows synergy with other antimicrobials [Darouiche et al., 2009].

Proteolytic enzymes like serrapeptase help the body break down protein involved in inflammation and mucous. It may also help disrupt the outer layers of biofilms and uncover hidden microbes.

Unfortunately, the high cost of industrial enzyme production makes their large-scale application as anti-biofilm agents unfeasible. [Sun et al., 2013]

Bacteriophages are viruses that produce a number of enzymes that negate the protection afforded by biofilms. Phages degrade the biofilm matrix and lyse bacteria, while leaving friendly bacteria unharmed. Phage modification of biofilm architecture also increases susceptibility to antibiotics. However, phage-resistant bacteria can evolve rapidly. [Sun et al., 2013]

Quorum-Sensing Inhibitors

Quorum-Sensing (QS) is a form of communication bacteria use to cooperatively build biofilm communities. Most bacteria produce QS signals, as well as QS inhibitors. Usnic acid, a lichen metabolite, possesses inhibitory activity against bacterial and fungal biofilms via QS interference. QS inhibitors can increase the susceptibility of biofilms to antibiotics. QS Inhibitors are generally regarded as safe in humans. [Sun et al., 2013]

Garlic inhibits the expression of several genes that control bacterial QS. The star in garlic’s arsenal is ajoene, the sulfur-containing compound produced when garlic is crushed. Ajoene inhibits production of rhamnolipid, which shields biofilms from white blood cells. Over 90% of biofilm bacteria were killed with a combination of ajoene and the antibiotic tobramycin. Garlic also has anti-viral, anti-fungal, and anti-protozoal properties, and benefits the cardiovascular and immune systems. [Jakobsen et al., 2012] These sulfur compounds from garlic quickly lose their activity upon exposure to oxygen. A willow bark extract, hamamelitannin, also inhibits QS. [Morgan, 2015]

Antioxidants

The anticancer, antioxidant, and anti-inflammatory effects of flavonoids are well established. Yet, their biofilm disrupting function is practically unknown. Flavonoids appear to suppress the formation of biofilms via a non-specific QS inhibition [Vikram et al., 2010]. The flavonoid phloretin inhibited biofilm formation in E. coli O157:H7, and ameliorated colon inflammation in rats without harming beneficial biofilms [Lee et.al., 2011]. Naturally occurring flavanols in cocoa may reverse memory decline significantly. [Brickman, et al., 2014] Their ability to inhibit QS might provide a clue for their action.

The anti-aging antioxidant resveratrol, associated with red wine, is produced by plants when under attack by pathogens. Resveratrol demonstrated significant antimicrobial properties on periodontal pathogens [O’Connor et al., 2011]

Cranberry has a reputation for keeping bacteria from sticking to surfaces. The red pigments in cranberries have been shown to inhibit biofilm formation. These proanthocyanidins [PACs] have been reported to possess antimicrobial, anti-adhesion, antioxidant, and anti-inflammatory properties. [Bodet et al., 2006] They prevent the attachment of pathogens to host tissues, and can inhibit the formation of biofilms in the mouth and urinary tract. [Labrecque et al., 2006] Cranberry PACs stopped the gum disease pathogen, Porphyromonas gingivitis, from adhering and forming biofilm, which markedly reduced its invasiveness. [La et al., 2010] These unique PACs also prevented adherence and biofilm formation by Candida albicans, the causative agent of thrush and yeast infections. [Feldman et al., 2012]  Cranberry juice extract, at low micromolar levels, inhibited tissue-destroying enzymes made by bacteria [La et al., 2010] and humans. [Bodet et al., 2007] Cranberry PACs also prevented dental plaque, by inhibiting biofilm-forming enzymes, [Steinberg et al., 2004] and keeping bacteria from aggregating. [Weiss et al., 1998; Yamanaka et al., 2004] Daily use of a cranberry-containing mouthwash for 6 weeks significantly reduced levels of mutans streptococci in human saliva. [Weiss et al., 2004] The anti-adhesive benefits of cranberry for urinary tract infections may be substantially increased by increasing the alkalinity of urine (https://thescienceofnutritiondotnet.wordpress.com/2015/07/17/beat-urinary-tract-infections-with-nutrition/).

Chlorogenic acids (CGA), largely from coffee, are cinnamic acid derivatives with important antioxidant and anti-inflammatory activities. [Farah et al., 2008] In vitro antibacterial and anti-biofilm activities of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia resistant to trimethoprim/sulfamethoxazole (TMP/SMX) was investigated. The MIC and MBC values ranged from 8 to 32 μg/mL. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC. [Karunanidhi et al., 2012]

Boswellic acids are pentacyclic triterpenes, produced in plants belonging to the genus Boswellia, with potent anti-biofilm properties. Acetyl-11-keto-β-boswellic acid, which exhibited the most potent antibacterial activity, was effective against all 112 pathogenic gram positive bacteria tested (MIC range, 2-8 μg/ml). It inhibited biofilms formed by S. aureus and S. epidermidis, and could also disrupt preexisting biofilms. Disruption of bacterial membranes is the likely mode of action. [Raja et al., 2011]

The leaf extract of Pongamia pinnata showed significant antibiofilm activity [Karlapudi et al., 2012]. The antimicrobial activity of the plant extract is attributed to the presence of phenolic compounds, such as alkaloids, flavonoids, terpenoids and polyacetylenes. [Shan et al., 2007]

Five Indonesian medical plant extracts were shown to inhibit Pseudomonas aeruginosa and Staphylococcus aureus biofilm formation at concentrations as low as 0.12 mg/mL. [Pratiwi et al., 2015]

Wheat bran extract exhibits anti-biofilm activity, inhibiting biofilm formation and destroying pre-formed S. aureus biofilm in dairy cows with mastitis. [González-Ortiz et al., 2014]

Farnesol and xylitol were shown to possess antibiofilm and antibacterial effects when used in root canal irrigants. [Alves et al., 2013] Xylitol is a low-carb sweetener found in toothpaste and diet sodas. When bacteria incorporate xylitol into the biofilm, it makes for a flimsy structure. [Morgan, 2015]

Aspirin and many other naturally-occurring salicylates have been shown to inhibit the macromolecules that make up the biofilm matrix [Domenico et al, 1990; Muller et al., 1998]. Salicylates are produced by many plants in response to infection.

Pro-oxidants can also be effective against biofilms. Oxidative agents are microbicidal, and offer possibilities for reducing the pathogenic activities of biofilms, especially those with an anaerobic component. In one study, 85% improvement was seen among 66 chronic Lyme disease patients with hyperbaric oxygen therapy, together with antibiotics. [Huang et al., 2014] Oxygen-supercharged Kaqun water, which has exhibited anticancer properties, may also prove useful. In contrast, nitric oxide, a signaling molecule involved in the immune system, promotes biofilm formation. [Plate & Marietta, 2012]

Fatty Acid Inhibitors

Several Salvia (Sage) species widely used as spices were evaluated for their antimicrobial activities, including their anti-adhesive and anti-biofilm effects. Salvia triloba extract demonstrated significant bacteriocidal activity against MRSA. Its volatile oil was active against all tested microorganisms except P. aeruginosa. S. triloba extract and volatile oil were active against biofilms, demonstrating anti-adhesion and anti-biofilm activities, respectively. The antimicrobial activities of other Salvia species were negligible. [Al-Bakri et al., 2010]

Short- and medium-chain fatty acids exhibit antimicrobial activity. Formic, capric, and lauric acids are broadly inhibitory for bacteria. Undecylenic acid is another medium chain fatty acid known for its anti-biofilm ability – including the disruption of troubling biofilms of Candida albicans. [McLain et al., 2000] These fatty acid inhibitors contribute to the formation and interaction of species within biofilms. [Huang et al., 2011]

Bacterial Anti-biofilm Inhibitors

Not surprisingly, bacteria compete with one another for turf. Certain substances on the surface of one bacteria work to inhibit biofilms from another. Extracellular polysaccharides (EPS)  are the essential building blocks for the biofilm matrix of most microorganisms. Thus, EPS is the stuff of biofilms, but can also inhibit their neighbors’ biofilms, from initial adhesion, dispersion, cell to cell communication, to matrix degradation. [Rendueles et al.,2013]

One example of this EPS anti-biofilm activity is in Actinobacillus pleuropneumoniae serotype 5. The EPS from these bacteria inhibits cell-to-cell and cell-to-surface interactions of other bacteria, preventing them from forming or maintaining biofilms. This is one of a growing number of natural bacterial polysaccharides that exhibit broad-spectrum, non-biocidal anti-biofilm activity. [Karwacki et al., 2013]

Numerous bacteria produce anti-biofilm agents. Extracts of a coral-associated bacteria induced a reduction in S. aureus and Serratia marcescens biofilm formation. A novel natural product, 4-phenylbutanoic acid, from the marine bacterium Bacillus pumilus, shows inhibitory activity against biofilms from a broad range of bacteria. [Nithya et al., 2011] Ethyl acetate extracts of the bacterium, Bacillus firmus—a coral-associated bacterium–show antibiofilm activity against biofilms formed by multidrug resistant S. aureus. [Gowrishankar et al., 2012]

Streptococcus salivarius, a non-biofilm, harmless inhabitant of the human mouth, uses two enzymes to inhibit the formation of dental biofilms, otherwise known as plaque. These enzymes were identified as fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA), which affected a decrease in EPS production. The large quantities of FruA that S. salivarius produces may play an important role in microbial interactions for sucrose-dependent biofilm formation in the mouth. [Ogawa et al., 2011]

Minerals that Affect Biofilm

Many enzymes in the body are metallo-enzymes that rely on iron, zinc, selenium, manganese, magnesium  and other minerals for activity. Toxic heavy metals (mercury, lead, cadmium) can displace the metal component of these metallo-enzymes and render them ineffective or non-functional. Some of these enzymes (e.g., SOD, catalase, glutathione reductase) play key roles in our antioxidant defenses. Toxic metal elimination and good mineral nutrition from organically-grown foods and dietary supplements can enhance our immune capabilities substantially to reduce the risk of infection.

Silver is an important antimicrobial agent used as a coating to reduce bacterial adhesion to biomaterials and prevent infections. Silver ions increase bacterial membrane permeability, induce de-energization of cells, promote leakage of cellular content, and disrupt DNA replication. [Marambio-Jones & Hoek, 2010] Many studies support an anti-biofilm component of silver. However, a recent study suggests that silver may indirectly promote bacterial adhesion [Carvalho et al., 2013].

Iron promotes EPS production and biofilm formation in many pathogenic, biofilm-producing bacteria. By tying up iron, lactoferrin could have anti-biofilm activity. Lactoferrin shows powerful anti-Candida and anti-bacterial properties. [DePas et al., 2012]

Bismuth is an element in the earth’s crust (atomic number 83) that we demonstrated had anti-biofilm activity. It’s the only non-toxic heavy metal on the Periodic Chart. That’s why you see it in such remedies as Pepto-Bismol, because it’s generally regarded as safe.  Bismuth seems to work by competitively inhibiting redox enzymes containing iron.[Domenico et al., 1996], which are involved in EPS production. Pepto-Bismol  is actually a combination of two anti-biofilm agents, bismuth and aspirin. Both interfere with energy production, which inhibits slime formation.[Domenico et al, 1991, 1992].  Pepto-Bismol works essentially by dampening biofilm overgrowth in the gut.

Bismuth anti-biofilm activity occurs at very small doses (<1 µg/mL).[Domenico et al., 1991, 1992]  However, Pepto-Bismol is taken in rather large doses (~15 mg/Kg). Bismuth potency can be enhanced up to 1000-fold with lipophilic thiols. [Domenico et al., 1997] Some thiols used to potentiate bismuth are naturally-occurring, and some are synthetic. Each shows a unique antibacterial spectrum that adds to the utility of these novel anti-biofilm compounds. Bismuth-thiols (BTs) have potent, broad spectrum activity, even against antibiotic resistant bacteria.  Additionally BTs prevent and eradicate microbial biofilms at low micromolar concentrations. [Domenico et al., 1999; Folsom et al., 2011] Topical BT administration to infected open fracture wounds potentiated the effect of systemically administered antibiotics, reduced infection rate and bacteria quantity associated with bone and orthopaedic implants. [Penn-Barwell 2015] Microbion Corporation’s lead compound, BisEDT, has been granted FDA Qualified Infectious Disease Product (QIDP) status, and is in Phase 2 clinical studies for treatment of orthopedic wound infections and chronic wounds.

The low toxicity of bismuth makes it quite different than most heavy metals, which weaken immunity and create an environment for unhealthy biofilms. Chelation therapy with EDTA removes many of these heavy metals and shows anti-biofilm effects. Chelating agents show biofilm dispersing qualities because the biofilm matrix is held together largely by minerals like calcium, magnesium, and iron. Phosphate is involved also, to solidify the biofilm structure. EDTA weakens the structure of biofilms to allow the immune system or antibiotics to gain access to the microbes hiding deep within biofilm community.  EDTA may supercharge antibiotics by 1000-fold. [Finnegan & Percival, 2014]

Another metal chelator, N-acetyl-L-cysteine (NAC), at low milligram levels, was found to decrease biofilm formation by a variety of bacteria and reduced the production of EPS matrix, while promoting biofilm disruption [Pézer- Giraldo et al., 1997]. Synergy of NAC with ciprofloxacin was shown against biofilm production and pre-formed mature biofilms from many pathogenic microbes on ureteral stent surfaces. NAC increased ciprofloxacin action by degrading the EPS matrix of biofilms. [El-feky et al., 2009]

A healthy gut maintains healthy biofilm communities that support the absorption of nutrients. Using detox agents like charcoal to mop up certain poisons and toxic metabolites, may conceivably protect the gut biofilm, and ward off pathogenic biofilms. Charcoal shows life-extending effects in laboratory rats.[Frolkis et al., 1989]

Other Modalities that Inhibit Biofilms

Antimicrobial Peptides (AMPs) are cationic, amphipathic substances that are part of the innate immunity in animals, plants, and some microbes. AMPs bind to and disrupt bacterial membranes, and efficiently kill biofilms.  AMPs from sea urchins, sea cucumbers and echinoderms have all been shown to disrupt biofilms. Their drawbacks are a sensitivity to salt, ionic strength, pH and proteolytic activity in body fluids. Synthetic AMPs have recently emerged as attractive anti-biofilm agents. Specifically targeted AMPs (STAMPs) are fusion peptides that target single pathogens, and are relatively stable under a range of physiological conditions. STAMPs can selectively eliminate the biofilm-forming, tooth-decay pathogen Streptococcus mutans from a mixed-species environment. [Sun, 2013]

Low-frequency ultrasound treatment in combination with antibiotics is promising for biofilm removal. [He et al., 2011] Ultrasound facilitates transport of antibiotics across biofilms, and increases sensitivity of biofilm-growing bacteria to antibiotics. [Carmen et al., 2005; Dong et al., 2013] It has been used as a treatment for chronic rhinosinusitis. [Bartley & Young, 2009] Ultrasound could conceivably be used in tandem with any one or more anti-biofilm agents.

Taurolidine is active against a wide range of microorganisms, including antibiotic-resistant bacteria, fungi, and mycobacteria.[Watson et al., 1995; Torres-Viera et al., 2000]  Taurolidine and its derivatives react with the bacterial cell wall, cell membrane, and endotoxins. Microbes are killed and the resulting toxins are inactivated, which reduces the inflammatory effect. Taurolidine is also used in the prevention and treatment of catheter related infections. [Zweich et al., 2013; Handrup et al., 2012; Chu et al., 2012; Diamanti et al., 2014] Taurolidine decreases bacterial adherence to host cells by destroying fimbriae (appendages used to stick to surfaces), which prevents biofilm formation. Bacterial resistance against taurolidine has yet to be observed. No systemic side effects have been identified. However, high concentrations (up to 5 mg/mL) are required for activity.

Derivatives of 2-aminoimidazoles have recently been developed as molecules that both inhibit biofilm formation and disperse bacterial biofilms [Richards & Melander, 2009]. Examples of natural products in this class include oroidin, ageliferin, and mauritiamine. [Mourabit & Potier, 2001; Huigens et al., 2007, 2008] These naturally occurring secondary metabolites are produced by marine plants and animals (e.g., sponges, coral) to keep them slime free [Kelly et al., 2003; Tsukamoto et al., 1996; Yamada, 1997]. 2-aminoimidazole/triazole conjugates (effective range, 50-150 µM) eliminate biofilm colonization, augment the action of conventional antibiotics, suppress multidrug resistance, and are not hemolytic at active concentrations. [Huigens et al., 2009] Unfortunately, the activity of these agents may be significantly impaired in vivo by calcium or manganese ions [Rogers et al., 2009; Rogers et al., 2010].

Baking Soda (sodium bicarbonate) is one of the most useful health tools around. It’s alkalizing effects notwithstanding, antibiofilm activity may be one of the important reasons for its wide ranging benefits.[Gawande, 2008] Bicarbonates also work well with bismuth thiols, which show optimum effects at alkaline pHs. [Domenico et al., 1997] Potassium bicarbonate may be the preferred oral form of baking soda, since potassium offsets the ill effects of a high-sodium diet, helps build bones, lowers blood pressure, etc. It also raises the pH of urine to significantly improve host defenses against biofilms in the urinary tract (see my blog on how to prevent urinary tract infections: https://thescienceofnutrition.me/2015/07/17/beat-urinary-tract-infections-with-nutrition/).

Mucus is also beneficial In the fight against bacteria. Polymers called mucins adhere to bacteria and prevent them from sticking together on a surface, making them harmless. [Caldara et al., 2012]  Mucin complexity makes the commercial production of synthetic mucin impractical. However, mucins from jellyfish may be a commercially viable source in the future. [Ohta et al., 2009]

All major religions promote fasting, and medical research is just beginning to appreciate the benefits of intermittent fasting, from normalizing hormone levels, improved mental clarity, to fighting infection. Fasting can starve microbes while improving immunity, and may help combat biofilms. [Janossy, 2015]

The overconsumption of sugar and refined, processed food has altered our gut biofilms in unhealthy ways, which predisposes us to disease. [http://www.nutraingredients.com/Research/Gut-microbiota-shifts-could-predict-diabetes-risk-suggests-study] Artificial sweeteners also alter the gut microflora in negative ways. [Suez et al., 2014] Sugar promotes the growth of pathogenic yeast and other fungal biofilms. [http://www.thecandidadiet.com/causes.htm]

On the other hand, sugar has been shown to boost the effectiveness of antibiotics against biofilms. Administering sugar with gentamicin cured mice with chronic urinary tract infections, and kept the bacteria from spreading to their kidneys. Perhaps by jump starting the germs’ metabolism with sugar, they can be coaxed out of the biofilm mode. [Allison et al., 2011]

Concluding Remarks

Antibiotic resistance is a crisis of historic proportions, and biofilms are a central part of that problem. Biofilm-related infections are inherently resistant to conventional antibiotic therapy, making them recurrent and chronic. Innovative therapeutic measures need to be developed to eradicate persistent infections. Effective treatments are still limited, and there’s much we don’t know about these new anti-biofilm agents. Many of the new agents are preferentially non-biocidal, so they won’t damage our friendly flora and work against us.

All told, the future appears bright for the anti-biofilm trade. Drug cocktails comprised of antibiotics and novel anti-biofilm agents will soon be developed to treat biofilm-associated infections. While this treatise is not an exhaustive analysis of all the possible anti-biofilm candidates under study, the anti-biofilm agents discussed may someday be part of the solution to some of medicine’s most urgent needs.

Follow Dr. Phil on Twitter: @drwillip

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