Quorum Sensing and Biofilm Formation and Natural Compounds that Disrupt these Processes

by John & Barbara Connor, M.Ac., L.Ac.

Introduction
What is Quorum Sensing?
What are Biofilms?
Rationale for Using Anti-Quorum Sensing and Biofilm Disrupting Natural Compounds
Anti Quorum Sensing and Biofilm Disrupting Herbs, Spices, Foods and Essential Oils


Introduction
While researching the problem of antibiotic resistance John and I gained insight into the significance of the phenomena known as quorum sensing and biofilm formation. We would like to share with you some of the studies we came across on these two processes and their relationship to bacterial infections. We would also like to share with you some of the studies we have found that have been done on herbs, spices, foods and essential oils that can inhibit quorum sensing and disrupt biofilm formation. 

Alarming trends in the spread of antibiotic resistance among top pathogens, including Staphylococcus aureus, have placed mankind at the brink of what has been coined as the ‘post-antibiotic era’. Since the widespread introduction of antibiotics in the 1940s, the same storyline has repeated itself over and over again: a new antibiotic is introduced and then resistant variants emerge and quickly spread, effectively limiting the utility and lifespan of the drug. (Quave et al 2015)

As is well known, the major cause of mortality and morbidity in human beings is bacterial infection. However, bacteria have developed resistance to most of the antibiotics primarily due to large-scale and indiscriminate usage. (Kailia et al 2014)

What is Quorum Sensing?

Quorum sensing (QS) refers to the capacity of bacteria to monitor their population density and regulate gene expression accordingly: the QS-regulated processes deal with multicellular behaviors (e.g. growth and development of biofilm), horizontal gene transfer and host-microbe (symbiosis and pathogenesis) and microbe-microbe interactions. (Grandclement et al 2015)

The emergence of antibiotic-resistant bacterial pathogens, especially Gram-negative bacteria, has driven investigations into suppressing bacterial virulence via quorum sensing inhibition strategies instead of bactericidal and bacteriostatic approaches. (Gemiarto et al 2015)

The QS mechanism enables bacteria to detect their population density through the production, release, and perception of small diffusible molecules called autoinducers and to coordinate gene expression accordingly. A wide array of functions in bacteria ranging from bacterial cell motility to complex behaviors such as biofilm formation and production of virulence factors are regulated by QS in pathogenic bacteria.  (Husain et al 2015)

Typical QS involves the generation and release of small diffusible signal molecules—autoinducers; they accumulate in the environment to a certain threshold concentration, followed by recognition by receptor proteins that regulate the expression of a particular set of genes and control manifold activities. Since this mechanism is responsible for bacterial virulence induction, QS targeting could be a promising strategy to control pathogenic bacteria, and some medicinal plants are capable of inhibiting QS-related processes. (Deryabin & Tolmacheva 2015)

Quorum sensing cell communication is widely used by bacterial pathogens to coordinate the expression of several collective traits, including the production of multiple virulence factors, biofilm formation, and swarming motility once a population threshold is reached. (Castillo-Juarez  et al 2015)

Quorum sensing along with subversion of the immune system are the main factors that determine the bacterial infectious doses. Hence those bacterial pathogens that need small doses to infect, generally lack QS systems but are very effective at inactivating the immune response by killing professional phagocytes. In contrast, those bacterial pathogens that need high infectious doses rely on QS for the coordination of the expression of virulence. (Castillo-Juarez  et al 2015)

Several bacterial pathogens, like Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Vibrio cholerae, utilize quorum sensing cell communication to coordinate the expression of multiple virulence factors and associated behaviors such as swarming and biofilm formation, once a population size threshold is reached. (Castillo-Juarez  et al 2015)

What are Biofilms?

Biofilms can be defined as structured aggregation of surface-attached microorganisms encased in an extracellular matrix. In all these [four] phases of biofilm formation, quorum-sensing system is involved in the regulation of population density and metabolic activity. (Chung & Toh 2014)

Biofilms are responsible for most chronic and recurrent infections. Biofilm-related infections reoccur in approximately 65-80% of cases. Bacteria associated with the biofilm are highly resistant to antibiotics. (Venkatesan et al 2015)

Biofilms are colonies of bacteria encased within extracellular polymeric matrix. Sessile (immobile) biofilm bacteria are phenotypically different than planktonic (drifting or floating) bacteria, conferring increased resistance to desiccation, antibiotics, and the immune response. Antibiotics are able to kill the planktonic cells released by the biofilm after its maturation stages, but bacteria within the biofilm can persist, causing chronic infections. In biofilm formation, bacteria attach reversibly to a surface, and then begin to produce extracellular polysaccharides. As the bacterial number grows, quorum sensing allows a phenotypic change in the bacteria. The biofilm matures and grows. Eventually, proteins break down parts of the matrix so that bacteria within the biofilm can disperse. (Ulrey et al 2014)

About 80% of the infections caused by microorganisms are biofilm based. Biofilm architecture consists of structured and aggregated communities of bacteria encased in a self-secreted exopolymeric substance. Several studies have revealed that bacteria have developed resistance because of the prolonged treatment with conventional antibiotics possessing a broad-range efficacy via toxic or growth-inhibitory effects on target organisms rendering the traditional antibiotic treatment virtually ineffective. It has been found that bacteria living in the biofilm mode of growth are resistant to antibiotics up to 1000 times more than their planktonic counterparts. (Husain et al 2015)

The presence of biofilms has been mostly seen in medical implants and urinary catheters. Various signalling events including two-component signalling, extra cytoplasmic function and quorum sensing are involved in the formation of biofilms. The presence of an extracellular polymeric matrix in biofilms makes it difficult for the antimicrobials to act on them and make the bacteria tolerant to antibiotics and other drugs. (Gupta et al 2015)

Rationale for Using Anti-Quorum Sensing and Biofilm Disrupting Natural Compounds

Acinetobacter frequently causes infections associated with medical devices, e.g. vascular catheters, cerebrospinal fluid shunts or Foleys catheter. Biofilm formation is a well-known pathogenic mechanism in such infections. The potential of Acinetobacter to form biofilm may explain its exceptional antibiotic resistance and survival in the hospital environment. This study concludes that there a positive correlation between biofilm formation and multiple drug resistance in A. baumannii. (Badave & Kulkarni 2015)

Usage of antibiotics has caused pathogenic bacteria to become resistant and poses a global threat to public health. QS provides an alternative solution because by targeting bacterial communication the expression of the virulence phenotype is inhibited. (Tan et al 2013)

Blocking quorum sensing pathways are viewed as viable anti-virulent therapy in association with traditional antimicrobial therapy. Anti-quorum sensing dietary phytochemicals with may prove to be a safe and viable choice as anti-virulent drug candidates. (Kumar et al 2015)

Compounds that interfere with the QS system to attenuate bacterial pathogenicity are termed as anti-QS compounds. Such compounds neither kill the bacteria nor stop their growth and are less expected to develop resistance toward antibiotics. (Husain et al 2015)

The likelihood of bacteria developing resistance to quorum sensing inhibitors is less probable than that observed with conventional antibiotics. (Kalia et al 2014)

Natural products play a pivotal role for treating and preventing infectious diseases. Plant compounds usually target the bacterial QS system via three different ways, by either stopping the signaling molecules from being synthesized by the luxI encoded AHL synthase, by degrading the signaling molecules and/or by targeting the luxR signal receptor. (Koh et al 2013)

Plant-derived anti-QS compounds such as oroidin, ursolic acid, naringenin, cinnamaldehyde, salicylic acid, methyl eugenol, and extracts from garlic and edible fruits, have shown antibiofilm properties against several pathogens. (Olivero-Verbel et al 2014)

The following are summaries of 29 studies on anti quorum sensing and biofilm disrupting herbs, spices, foods and essential oils:

Anti Quorum Sensing and Biofilm Disrupting Herbs, Spices, Foods and Essential Oils

6-gingerol (a pungent oil of fresh ginger)
Agaricus blazei Murill (edible mushroom)
Ajoene (from Allium sativum)
Areca catechu
Armoracia rusticana (horseradish)
Centella asiatica (gotu kola)
Chamomile (Chamaemelum nobile)
Cinnamon oil (Ceylon type)
Citrus essential oil
Clove essential oil
Cranberry proanthocyanidins
Colloidal silver
Curcumin (from curcuma longa)
Eucalyptus essential oil
European Chestnut leaf (Castanea sativa)
Garlic
Geranium essential oil
Gnetum gnemon (belinjo leaves)
Imperata cylindrica
Lavender essential oil
Lemon essential oil
Marjoram essential oil
Menthol
Nelumbo nucifera
Nymphaea tetragona (water lily)
Oxyresveratrol
Panax notoginseng (root and flower)
Phyllanthus amarus (chanca piedra)
Piceatannol
Piper betle (betle leaves)
Piper nigrum (peppercorn), and Proanthocyanidin (from dried cranberry juice)
Propolis
Punica granatum
Prunella vulgaris
Prunus armeniaca
Quercetin
Resveratrol
Rose essential oil
Rosemary essential oil
Utica dioica (Nettles)
Wheat-bran 

6-gingerol (a pungent oil of fresh ginger) reduced biofilm formation experimentally, several virulence factors (e.g., exoprotease, rhamnolipid, and pyocyanin), and mice mortality. Further transcriptome analyses demonstrated that 6-gingerol successfully repressed QS-induced genes, specifically those related to the production of virulence factors. These results strongly support our hypothesis and offer insight into the molecular mechanism that caused QS gene repression. (Kim et al 2015)

Agaricus blazei Murill (an edible mushroom) is known to induce protective immunomodulatory action against a variety of infectious diseases. In the present study we report potential anti-quorum sensing properties of A. blazei hot water extract. (Sokovic et al 2014)

Armoracia rusticana (horseradish) stood out from a group of active crude extracts as highly active with respect to QSI activity against P. aeruginosa. Bioassay-guided fractionation and purification led to identification of 1-isothiocyanato-3-(methylsulfinyl) propane, commonly known as iberin, as an active QS inhibiting compound in horseradish. Real-time PCR (RT-PCR) and DNA microarray analysis of global gene expression revealed that iberin specifically and extremely effectively targets two of the major QS networks in P. aeruginosa, the LasIR and the RhlIR systems, and was found to downregulate QS-controlled rhamnolipid production in P. aeruginosa wild-type batch cultures. (Jakobsen et al 2012)

Centella asiatica (gotu kola) – The anti-quorum sensing (QS) nature of Centella asiatica herb can be further exploited for the formulation of drugs targeting bacterial infections where pathogenicity is mediated through QS. (Vasavi et al 2014)

Chamomile (Chamaemelum nobile) – The anti-QS property of C. nobile may play an important role in its antibacterial activity, thus offering an additional strategy in the fight against bacterial infections. However, molecular investigation is required to explore the exact mechanisms of the antibacterial action and functions of this phytocompound. (Kazemian et al 2015)

Cinnamon oil (Ceylon type) – This work is the first to demonstrate that cinnamon oil can influence various quorum sensing (QS)-based phenomena in Pseudomonas aeruginosa PAO1, including biofilm formation. (Kalia et al 2015)

Clove oil – The results of this study confirmed that the QS systems play an important role in the pathogenicity of P. aeruginosa infections. Consequently, compounds that attenuate QS may offer significant promise as potential therapeutic agents. These compounds provide alternative medicine for treating emerging bacterial infections without leading to antibiotic resistance as they do not pause selection pressure. Our study also revealed the anti-QS and biofilm inhibitory activity of clove oil against P. aeruginosa isolates. (Aboushleib et al 2015)

Clove essential oil – Presence of anti-QS activity in clove oil and other essential oils has indicated new anti-infective activity. The identification of anti-QS phytoconstituents is needed to assess the mechanism of action against both C. violaceum and Ps. aeruginosa. (Khan et al 2009)

Colloidal silver directly attenuates in vitro Staphylococcus aureus biofilms. (Goggin it al 2014)

Cranberry proanthocyanidins (PACs) reduced P. aeruginosa swarming motility. Cranberry PACs significantly disrupted the biofilm formation of P. aeruginosa. Proteomics analysis revealed significantly different proteins expressed following PAC treatment. In addition, we found that PACs potentiated the antibiotic activity of gentamicin in an in vivo model of infection using G. mellonella. Results suggest that A-type proanthocyanidins may be a useful therapeutic against the biofilm-mediated infections caused by P. aeruginosa and should be further tested. ((Ulrey et al 2014)

Cranberry proanthocyanidins – These findings indicate that cranberry proanthocyanidins (PACs) have excellent in vitro activity against C. albicans biofilm formation in artificial urine. We present preliminary evidence that cranberry PAC activity against C. albicans biofilm formation is due to anti-adherence properties and/or iron chelation. (Rane et al 2014)

Curcumin (Curcuma longa) – Urinary tract infection is caused primarily by the quorum sensing (QS)-dependent biofilm forming ability of uropathogens. In the present investigation, an anti-quorum sensing (anti-QS) agent curcumin from Curcuma longa (turmeric) was shown to inhibit the biofilm formation of uropathogens, such as Escherichia coli, Pseudomonas aeruginosa PAO1, Proteus mirabilis and Serratia marcescens, possibly by interfering with their QS systems. The antibiofilm potential of curcumin on uropathogens as well as its efficacy in disturbing the mature biofilms was examined under light microscope and confocal laser scanning microscope. The treatment with curcumin was also found to attenuate the QS-dependent factors, such as exopolysaccharide production, alginate production, swimming and swarming motility of uropathogens. Furthermore, it was documented that curcumin enhanced the susceptibility of a marker strain and uropathogens to conventional antibiotics. (Packiavathy et al 2014)

Curcumin, from Curcuma longa, Ajoene from Allium sativum, Iberin from Armoracia rusticana attenuate P. aeruginosa virulence by downregulating the expression of QS genes. (Sarabhi et al 2013)

European Chestnut leaf (Castanea sativa) extract – Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes), against all Staphylococcus aureus accessory gene regulator (agr) alleles. (Quave et al 2015)

Garlic (Allium sativum) is considered a rich source of many compounds, which shows antimicrobial effects. The ability of microorganisms to adhere to both biotic and abiotic surfaces and to form biofilm is responsible for a number of diseases of chronic nature, demonstrating extremely high resistance to antibiotics. Bacterial biofilms are complex communities of sessile microorganisms, embedded in an extracellular matrix and irreversibly attached to various surfaces. A. sativum L. extracts were efficient to inhibit biofilm structures and the concentration of each extract had a direct relation with the inhibitory effect. (Mohsenipour & Hassanshahian 2015)

Marjoram essential oil (EO) and Lemon EO   Marjoram EO inhibited Bacillus cereus, Pichia anomala, Pseudomonas putida and mixed-culture biofilm formation of Ps. putida and Escherichia coli and showed the best QS inhibitor effect on Chromobacterium violaceum. For B. cereus, all components showed better antibiofilm capacity than the parent EOs. Lemon EO inhibited E. coli and mixed-culture biofilms, and cinnamon was effective against the mixed forms. Scanning electron microscopy showed the loss of three-dimensional structures of biofilms. (Kerekes et al 2013)

Menthol – Our data identified menthol as a novel broad spectrum QS inhibitor. (Husain et al 2015)

Nymphaea tetragona (water lily) 50% methanol extract was demonstrated to have significant concentration-dependent inhibitory effects on quorum sensing-mediated virulence factors of bacteria with non-toxic properties, and could thus be a prospective quorum sensing inhibitor. (Hossain et al 2015)

Phyllanthus amarus (chanca piedra) – Our data suggest that P. amarus could be useful for attenuating pathogens and hence, more local traditional herbs should be screened for its anti-quorum sensing properties as their active compounds may serve as promising anti-pathogenic drugs. ((Priya et al 2013)

Piper nigrum (peppercorn), Piper betle (betle leaves) and Gnetum gnemon (belinjo leaves) – Various parts of Piper nigrum, Piper betle and Gnetum gnemon are used as food sources by Malaysians. The purpose of this study is to examine the anti-quorum sensing (anti-QS) properties of P. nigrum, P. betle and G. gnemon extracts. The hexane, chloroform and methanol extracts of these plants were assessed in bioassays involving Pseudomonas aeruginosa PA01, Escherichia coli [pSB401], E. coli [pSB1075] and Chromobacterium violaceum CV026. It was found that the extracts of these three plants have anti-QS ability. Interestingly, the hexane, chloroform and methanol extracts from P. betle showed the most potent anti-QS activity as judged by the bioassays. (Tan et al 2013)

Proanthocyanidin Extracts of the purified proanthocyanidin were prepared from dried cranberry juice. The proanthocyanidin exhibited anti-adherence property with multi-drug resistant strains of uropathogenic P-fimbriated E. coli with in vitro study. Hence proanthocyanidin may be considered as an inhibitory agent for multi-drug resistant strains of E. coli adherence to uroepithelial cells. (Gupta et al 2011) Foods high in proanthocyanidins include: ground cinnamon, dried grape seeds, sorghum, unsweetened baking chocolate, red kidney beans, hazelnuts, pecans, chokeberries and cranberries.

PropolisTogether, we present evidence that propolis contain compounds that suppress QS responses. In this regard, anti-pathogenic compounds from bee harvested propolis could be identified and isolated and thus will be valuable for the further development of therapeutics to disrupt QS signaling systems which regulate the virulome in many pathogenic bacteria. (Bulman et al 2011)

Propolis – These results suggest that Tunisian propolis ethanol extract (EEP) is able to inhibit cancer cell proliferation, cariogenic bacteria and oral biofilms formation. It could have a promising role in the future medicine and nutrition when used as antibiotic or food additive. (Kouidhi et al 2010)

Prunus armeniaca, Prunella vulgaris, Nelumbo nucifera, Panax notoginseng (root and flower), Punica granatum, Areca catechu, and Imperata cylindrica – Eight of the selected traditional Chinese medicine herbs (80%) yielded QS inhibitors: Prunus armeniaca, Prunella vulgaris, Nelumbo nucifera, Panax notoginseng (root and flower), Punica granatum, Areca catechu, and Imperata cylindrica. Compounds that interfere with QS are present in TCM herbs and these medicines may be a rich source of compounds to combat pathogenic bacteria and reduce the development of antibiotic resistance. (Koh & Tham 2011)

Quercetin – This study suggests that quercetin can act as a competitive inhibitor for signaling compound towards LasR receptor pathway and can serve as a novel QS-based antibacterial/anti-biofilm drug to manage food-borne pathogens. (Gopu et al 2015)

Resveratrol, piceatannol and oxyresveratrol – In the present study, quorum sensing inhibition activity of ten stilbenoids were tested using Chromobacterium violaceum CV026 as the bio-indicator strain and the structure-activity relationship was also investigated. Among them, resveratrol (1), piceatannol (2) and oxyresveratrol (3) showed potential anti-QS activities. (Sheng et al 2015)

Rose, geranium, lavender and rosemary, eucalyptus and citrus essential oils – Of the tested essential oils, rose, geranium, lavender and rosemary essential oils were the most potent QS inhibitors. Eucalyptus and citrus essential oils moderately reduced pigment production by Chromobacterium violaceum CV026, whereas the chamomile, orange and juniper oils were ineffective. (Szabo et al 2010)

Urtica dioica (Nettles) – The aim of this study was to assess the antibacterial and antifungal potential of some Romanian medicinal plants, arnica-Arnica montana, wormwood–Artemisia absinthium and nettle–Urtica dioica. In order to perform this antimicrobial screening, we obtained the vegetal extracts and we tested them on a series of Gram-positive and Gram-negative bacteria, and also against two fungal strains. The vegetal extracts showed antimicrobial activity preferentially directed against the planktonic fungal and bacterial growth, while the effect against biofilm formation and development was demonstrated only against S. aureus and C. albicans. Our in vitro assays indicate that the studied plant extracts are a significant source of natural alternatives to antimicrobial therapy, thus avoiding antibiotic therapy, the use of which has become excessive in recent years. (Stanciuc et al 2011)

Wheat-bran (WB) – The soluble extract of WB at 0.5% showed anti-biofilm activity, inhibiting biofilm formation and also destroying it. Similarly, the > 300 kDa fraction from WB had significant anti-biofilm activity in both in vitro assays. The WB also showed a potential to interfere with bacterial QS systems, as it was demonstrated to contain certain lactonase activity able to reduce AHL concentration in the medium. The present study reveals two additional beneficial properties of WB extract never explored before, which may be related to the presence of defence compounds in the plant extract able to interfere with microbial biofilms and also QS systems. (Gonzalez-Ortiz et al 2014)

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Compassionate Acupuncture and Healing Arts, providing craniosacral acupuncture, herbal and nutritional medicine in Durham, North Carolina. Phone number 919-309-7753.

 

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