WO2016196674A1 - Lutte contre les biofilms au moyen de bacillus subtilis et de proantocyanidines de vaccinium macrocarpon - Google Patents

Lutte contre les biofilms au moyen de bacillus subtilis et de proantocyanidines de vaccinium macrocarpon Download PDF

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WO2016196674A1
WO2016196674A1 PCT/US2016/035332 US2016035332W WO2016196674A1 WO 2016196674 A1 WO2016196674 A1 WO 2016196674A1 US 2016035332 W US2016035332 W US 2016035332W WO 2016196674 A1 WO2016196674 A1 WO 2016196674A1
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composition
biofilm
bacillus subtilis
carrier
surfactant
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Robert Caron
Sean DERRIG
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus

Definitions

  • the Invention relates to the control of pathogenic microorganisms by altering the ecology of sessile biofilms in food production and healthcare settings.
  • Biofilms are complex, irregular, heterogeneous hydrated structures where a consortium of microcolonies of bacteria can divide freely (Lewandowski, 2000). Inhabiting a biofilm confers significant advantages on its resident bacteria. They are protected from environmental threats such as desiccation, antibiotics (Ishida et al., 1998) and biocides (McDonnell and Russell, 1999), and form symbiotic relationships with other residents allowing exchange of nutrients, metabolites and genetic material.
  • the glycocalyx is the principal component of the extracellular matrix (ECM) - which provides the biofilm's structural integrity and is chemically similar to the bacterial capsule surrounding pathogens such as Streptococcus, Klebsiella, Pseudomonas and
  • the ECM is more diffuse and less distinct than a capsule and accounts for 50-90% of the organic carbon content of the biofilm (Flemming, Wingender and Mayer, 2000). It has been shown that B. subtilis biofilms are more nonwetting (that is, they have greater repellency to water) than Teflon® and are impervious even to 80% ethanol (Epstein et al., 2011), hence biofilms' recalcitrance.
  • the invention is directed to a biocide composition
  • a biocide composition comprising a blend of spores of Bacillus subtilis strains and proanthocyanidines from Vaccinium macrocarpon, wherein said Baccilus spores are bound to a dendritic salt and admixed into a carrier of xanthan gum, potassium sorbate, a C2-C12 alcohol, and a surfactant, and modified to a pH of 4.5 +/- 0.1 with an appropriate acid to form a suitable buffer solution at said pH.
  • a further embodiment is directed to a biocide composition comprising an effective amount of Bacillus subtilis and Vaccinium macrocarpon proanthocyanidins.
  • the composition further comprising a suitable surfactant.
  • the composition further comprising an opacifier, a buffer, and a fragrance.
  • the composition wherein the suitable surfactant is a non-ionic and amphoteric surfactant.
  • the composition comprising two or more surfactants.
  • the composition further comprising a xanthan-sorbate carrier.
  • the composition further comprising a surface tension decreasing agent.
  • the composition wherein the effective amount of the Bacillus subtilis is lxlO 8 cfu/ml.
  • the composition further comprising an effective amount of Bacillus licheniformis.
  • the use of the composition above to control environmental pathogenic bacteria in a further embodiment, the composition further comprising natural antibiotics produced systemically from the applied composition. In a further embodiment, wherein the composition further comprising lantibiotics. In a further embodiment, the use of the composition to digest organic soils. In a further embodiment, the use of the composition to disrupt biofilm formation. In a further embodiment, the use of the composition to mediate biocidal activity.
  • a composition suitable for artificially generating a benign biofilm comprising an effective amount of Bacillus subtilis, Vaccinium macrocarpon proanthocyanidins, a suitable carrier, and a surfactant.
  • the suitable carrier is a xanthan-sorbate carrier.
  • a method of disrupting an existing biofilm comprising: 1) applying a composition comprising an effective amount of Bacillus subtilis, Vaccinium macrocarpon proanthocyanidins, a suitable carrier, and a surfactant to surface for a sufficient time to transfer the Vaccinium macrocarpon proanthocyanidins to said existing biofilm, wherein said Vaccinium macrocarpon proanthocyanidins disrupt said biofilm surface by reducing the mobility of existing bacteria; and
  • a suitable carrier which is a xanthan-sorbate carrier.
  • a biocide composition comprising a blend of spores of Bacillus subtilis strains and proanthocyanidines from Vaccinium macrocarpon, wherein said Baccilus spores are bound to a dendritic salt and admixed into a carrier of xanthan gum, potassium sorbate, a C2-C12 alcohol, and a surfactant, and modified to a pH of 4.5 +/- 0.1 with an appropriate acid to form a suitable buffer solution at said pH.
  • a further embodiment is directed to a biofilm control composition
  • a biofilm control composition comprising one or more strains of Bacillus subtilis, which are prodigious producers of natural lantibiotics and other biocidal moieties, acting synergistically with a suite of biologically- active extracts; wherein, in combination with suitable surfactant and A. proanthcyanidin components, said natural lantibiotics and biocidal moieties provide a means for controlling biofilms in food production and healthcare by - inter alia - excluding pathogens as well as direct and indirect biocidal activity.
  • a further embodiment is directed to a method for reducing the occurrence of gram-negative bacteria on a surface comprising: 1) applying to a surface, a composition comprising a blend of spores of Bacillus subtilis strains and proanthocyanidines from Vaccinium macrocarpon, wherein said Baccilus spores are bound to a dendritic salt and admixed into a carrier of xanthan gum, potassium sorbate, a C2-C12 alcohol, and a surfactant, and modified to a pH of 4.5 +/- 0.1 with an appropriate acid to form a suitable buffer solution at said pH; and
  • a further embodiment is directed to a method of reducing a pathogenic biofilm on a surface comprising:
  • composition comprising a blend of spores of Bacillus subtilis strains and proanthocyanidines from Vaccinium macrocarpon, wherein said Baccilus spores are bound to a dendritic salt and admixed into a carrier of xanthan gum, potassium sorbate, a C2-C12 alcohol, and a surfactant, and modified to a pH of 4.5 +/- 0.1 with an appropriate acid to form a suitable buffer solution at said pH to a surface;
  • biofilms are impervious to traditional chemical biocides and antibiotics (Epstein et al., 2011) and so cannot be controlled by conventional biocidal chemicals.
  • Bactillus subtilis species used in the invention is not a human pathogen
  • the species utilized is a Gram-positive spore former and a facultative anaerobe (Nakano and Zuber, 1998) and is particularly well suited to this application due to its lack of pathogenicity, ubiquity in the environment (principally soil) and ease of culture.
  • biofilms offer a new paradigm for hygiene and infection control; their ubiquity and recalcitrance is only now becoming apparent and the ability of the invention to re- engineer existing biofilms and digest body fluids, fats, dirt, grease and grime as well as excluding pathogenic bacteria presents an environmentally responsible and effective solution in both food hygiene and healthcare.
  • a biofilm control composition comprises the use of one or more strains of Bacillus subtilis, which are prodigious producers of natural lantibiotics and other biocidal moieties, acting synergistically with a suite of biologically-active extracts. Together, in combination with suitable surfactant and A. proanthcyanidin components, provides a means for controlling biofilms in food production and healthcare by - inter alia - excluding pathogens as well as direct and indirect biocidal activity.
  • composition functions through several primary actions, including competitive exclusion, reduction of the native biofilm and quorum sensing, wherein present bacteria are converted from pathogenic to benign.
  • compositions generated by the Bacillus strains such as antibiotics, and other components, e.g. A.
  • the cranberry A. proanthcyanidin compounds are particularly suited in the compositions disclosed herein for the properties of disrupting of existing biofilms.
  • the proanthcyanidin compounds provide a mechanism to attack the weakened biofilm to aid in its removal and breakdown.
  • Gram-negative organisms where the unique nature of their cell wall makes them resistant to many classes of antibiotics including beta-lactams and carbapenems.
  • Gram- negative organisms are also a principal cause of food poisoning and foodborne intoxication including Campylobacter, E. coli and Salmonella. They are also highly efficient at up- regulating or acquiring mechanisms of antibiotic drug resistance, especially in the presence of antibiotic selection pressure and so are a leading cause of preventable nosocomial infections.
  • a preferred embodiment of the composition comprises a blend of Bacillus subtilis strains which produce prodigious amounts of the natural biosurfactant surfactin as well as biocidal concentrations of subtilisin and lanthionine-containing peptide antibiotics, which competitively exclude pathogenic species.
  • a-amylase enzymes by the Invention detach biofilm exopolysaccharide components; that these microbially-mediated effects on pathogens work synergistically with addition of North American cranberry (Vaccinium macrocarpon) proanthocyanidins to inhibit biofilm development and that the amylases, proteases and lipases also produced in situ make the invention a highly effective and environmentally-responsible cleaning product.
  • a further embodiment is directed to a biocide composition
  • a biocide composition comprising an effective amount of Bacillus subtilis, Bacillus licheniformis and Vaccinium macrocarpon proanthocyanidins, wherein the biocidal activity is mediated by natural enzymes and antibiotics formed in situ.
  • the in-use concentration of the Baccilus strains is about lxlO 8 cfu/ml.
  • Further embodiments may also advantageously combine Bacillus
  • environmental pathogenic bacteria are controlled using competitive exclusion through administration of the composition.
  • composition wherein the environmental pathogenic bacterial are controlled using natural antibiotics including but not limited to lantibiotics, surfactin and subtilin and wherein lantibiotics are antagonistic to Clostridium spores
  • the composition digests organic soils due to its in situ production of amylases, proteases and lipases.
  • composition disrupts biofilm formation due to its production in situ of a-amylases.
  • composition disrupts biofilm formation due to inclusion of V. macrocarpon proanthrocyanidins.
  • the composition further comprises a xanthan-sorbate carrier mix with non-ionic and amphoteric surfactants.
  • the composition further comprises a surface tension decreasing agent, for example, ethanol.
  • the xanthan-sorbate carrier is utilized to maintain the bacteria in a modified state, wherein they are ready to germinate upon contact with a food surface.
  • a surfactant is also advantageously utilized with the composition to break the surface tension of water, so that the composition will spread more easily across a wet surface. This allows the composition and the components therein to be more easily spread across and onto the surface, including into existing biofilms where the bacteria of the composition can begin to populate the surface.
  • the surfactant is a non-ionic amphoteric surfactant.
  • Further suitable surfactants include: C13 alcohol 9 mole ethoxylate, Coconut diethanol amide, C9-11 alcohol 6 mole ethoxylate, and PEG5-cocommonium methyl sulphate.
  • suitable non-ionic surfactants suitable for achieving the activity described herein.
  • An opacifier may be suitably added to the composition, for example styrene/ acrylamide copolymer 2-propenamide.
  • Other known opacifiers may be suitably used as known to one of ordinary skill in the art.
  • a preferred embodiment of the invention comprises a concentrated aqueous composition comprising Bacillus subsilis and Vaccinium macrocarpon proanthocyanidins, wherein the biocidal activity is mediated by natural enzymes and antibiotics formed in situ.
  • the composition is formulated into a stable concentrate by generating a carrier of water, ethanol, xanthan gum, sorbate, an opacifier, and a fragrance.
  • Spores of Bacillus subsilis and optionally of Bacillus licheniformis and Bacillus amyloliquefaciens are fermented or otherwise strained to induce sporulation, wherein the spores can be collected for use.
  • the spores are bound to dendritic salt and dispersed into the carrier as generated above.
  • the carrier and spore concentrate is then measured for pH and modified with HC1 to a pH of about 4.5 to 6.0.
  • the acidic pH maintains the spores in a state ready for germination, wherein the xanthan- sorb ate based carrier creates a thicker than water carrier for suspending the spores into the carrier.
  • the composition is manufactured as concentrate, such as described above, that can be suitably diluted for application to a surface.
  • a preferred diluent ration is 20: 1, with water as the diluent.
  • suitable aqueous solvents may be suitably utilized as a diluent based on the particular needs of the application.
  • Certain alcohol/water based diluents therefore may be advantageous as a mechanism to reduce surface tension in some embodiments.
  • 20: 1 is a preferred dilution ratio
  • suitable dilutions include a ratio from 100: 1 to 0.1 : 1 and all ratios in between.
  • the concentrate can also be utilized in an undiluted form.
  • compositions as described herein provide for the artificial introduction of benign biofilms and may offer a new paradigm for both hygiene and infection control.
  • the diluted composition is achieved by direct application to a surface, such as spray and wipe.
  • a surface such as spray and wipe.
  • the diluted composition can be applied via a spray mist, or poured, or applied to a cloth and applied to a surface for decontamination.
  • manual application is appropriate.
  • the manual application and mechanical wiping of the surface with a cloth or material may further advantageously remove surface contaminants through the mechanical action, as well as mechanically disrupt the biofilms present on a contaminated surface.
  • the composition, now being applied, can remove pathogenic bacterial through competitive exclusion, including consumption of water, nutrients and even the pathogenic microbes themselves.
  • the bacteria in the composition upon application of the composition to a surface, the bacteria in the composition begin generating ample amounts of the biosurfactant surfactin, as well as biocidal concentrations of subtilisin and lanthionine-containing peptide antibiotics which competitively exclude pathogenic species. Furthermore, in situ production of a- amylase enzymes work to detach biofilm exopolysaccharide components.
  • Additional mechanism for application of the composition includes spraying the composition on a surface without mechanical wiping of the surface.
  • application may be completed through a spraying or aeration system, fogger, or other similar mechanism.
  • an appropriate carrier may be further admixed into the invention to aid in application through these means.
  • Industrial scale applications may also be completed through industrial spray systems through ground or aerial vehicles, which can distribute large amounts of diluted materials onto surfaces that require biofilm control.
  • the composition may be suitably utilized in a method of controlling biofilm on a surface by applying to said surface a composition comprising the B. subtilis spores, surfactant, A. proanthcyanidin, in an appropriate carrier. Wherein the method further comprises wiping said surface clean of said composition.
  • compositions as described herein for the purposes of treating a surface to introduce a benign biofilm.
  • an appropriate use of the compositions is for reducing the occurrence of gram-negative bacteria on a surface.
  • an appropriate use of the composition is for reducing a pathogenic biofilm on a surface by applying said composition to said surface, wherein the bacteria in said composition will out compete the pathogenic bacteria, degrade the chemical bonds on the pathogenic bacteria biofilm, and through quorum sensing.
  • a composition as described herein was used in a healthcare setting with significant biofouling of washrooms. Swabs were sent for microbiological enumeration before the introduction of the composition to surfaces of several locations. The composition was administered daily for 21 days and swabs were collected again after treatment of these surfaces after 21 days. Before and after results of these tests are described in more detail below and the results shown in Tables 1-3.
  • the composition's active ingredients are a blend of B. subtilis spores at a concentration of lxlO 8 CFU ml "1 , surfactant, and cranberry A-proanthcyanidin in a suitable buffer and carrier.
  • Non-active ingredients included an opacifier, fragrance and buffer.
  • the composition can be advantageously manufactured as a concentrate and diluted for subsequent use of administration to a surface. This concentrated format of the composition was automatically diluted 20: 1 (with water) in a Venturi -based chemical proportioner to assure consistency of dilution.
  • the composition tested comprises B. subtilis strains selected from the group consisting of the following strains: 1A5, 1A28, 1A84, 1A122, 1A698, 1A699, 1A751, and 1 A752, and combinations thereof to a concentration of lxlO 8 CFU ml "1 .
  • the strains are combined with a surfactant and cranberry A-proanthcyanidin, and further comprising an opacifier, a buffer, and optionally a fragrance.
  • This concentrate can be further diluted 20: 1 in an aqueous medium and applied to a surface.
  • the B. subtilis strains are selected from the group consisting of: 1A5, 1A28, 1A84, 1A122, 1A698, 1A699, 1A751, and 1A752, and combinations thereof according to the BGSC Accession number. Further strains of B. subtilis may be advantageously utilized in certain embodiments. Furthermore, the inclusion of additional strains of B. subtilis outside of those enumerated above does not destroy the efficacy of the composition when used at a concentration of less than 50%, less than 25% of the total amount of the B. subtilis spores in the composition. Preferably, additional strains are less than 20%, less than 10%, less than 5%, or less than 1% of the total concentration of the B. subtilis strains in the composition.
  • the selected strains are prodigious producers of surfactin. Furthermore, the same strain or additional strains may be advantageously added for production of biocidal concentrations of subtilisin, lanthionine-containing peptide antibiotics and a-amylase enzymes.
  • a concentrate of the composition was generated and diluted 20: 1 in water for application to the tested surfaces.
  • streak plates from samples taken after treatment with the composition produced lower numbers of colonies, typically 10 3 to 10 4 cfu /swab.
  • Gram-positive rods (assumed to be Bacillus on the basis of colony morphology, catalase production and spore stains using a method based on that of Schaeffer and Fulton, modified by Ashby) were also present in some samples.
  • the bacterial element of the composition in a preferred embodiment is a prolific producer of antibiotics effective against a range of pathogens including S. aureus, E. coli, Helicobacter pylori (Pinchuk et al., 2001), Campylobacter (Sorokulova, Kirik and Pinchuk, 1997) and one lanthionine-containing peptide antibiotic (lantibiotic) produced, subtilin, has a broad spectrum antimicrobial activity - by forming transmembrane pores it depolarises energized bacterial cytoplasmic membranes. Using this activity might offer an alternative to current methods for controlling C diff spores: there is already evidence that using B. subtilis as a probiotic dietary supplement is effective against Clostridium diffwile- associated disease (Peys et al., 2007).
  • a preferred embodiment is a composition comprising a blend of
  • proanthcyanidin and further comprising an opacifier, fragrance and buffer which forms a concentrate that can be diluted 20: 1 in a Venturi -based chemical proportioner to assure consistency of dilution.
  • the resulting composition when applied to a surface creates transmembrane pores that deposalrses energized bacterial cytoplasmic membranes.
  • the composition provides spores that continually manufacture and administer antimicrobial compounds.
  • the composition breaks down biofilms containing bacteria and destroys these bacteria wherein they are unable to re-colonize a surface, unlike such products like alcohols or bleach based products.
  • Cetrimide agar (CET), Mannitol salt agar (MSA), Pseudomonas agar (PSA),
  • SBM Slanetz & Bartley Medium
  • TBX Tryptone bile X-glucuronide medium
  • XLD Xylose lysine desoxycholate agar
  • BMA Brilliance MRSA Agar
  • Xanthan gum and sorbate are combined into 50% final volume of water and mixed at high speed until dispersed. The mixture is chilled slowly to 4°C and left overnight for stability. The mixture is then re-heated to 18°C with a water jacket or at ambient temperature.
  • An opacifier is dissolved in a suitable volume of water, and added to the above mixture until homogeneous. The resultant mixture is again chilled slowly to 4°C and left overnight. The mixture is then re-heated to 18°C with a water jacket or at ambient temperature.
  • Alcohol C2-C12 - ethanol preferred
  • Bacterial endospores of Bactillus subtilis on dendritic salt carrier are mixed into the mixture and homogenized for 30 minutes, before adding the resultant mixture of water.
  • the resultant mixture is again chilled slowly to 4°C and left overnight.
  • the mixture is then re-heated to 18°C with a water jacket or at ambient temperature.
  • a mixture of Vaccinium macrocarpon A. proanthocyanidines is further admixed into the mixture at 18°C and homogenized for 30 minutes.
  • the resultant mixture pH is adjusted to 4.5 +/- 0.1 with an appropriate acid, e.g. HC1.
  • the pH may be adjusted to 5.5 +/- 0.1, and the pH can be adjusted with an appropriate weak acid such as citric acid or muriatic acid.
  • the acid may be appropriately chosen and added to form a buffer solution at the appropriate pH.
  • Location 1 was a section of floor below the gents' urinal area in a washroom.
  • Location 2 was a corner of a shower tray in the washroom.
  • Location 3 was in the drain of a shower tray in the washroom.
  • Bacterial numbers were then determined using the Miles & Misra drop count method (Miles, Misra and Irwin, 1938). Briefly, swabs were transferred aseptically into 9ml volumes of sterile 1 ⁇ 4 strength Ringer's solution and vortexed for 30 seconds. Samples were then serially diluted 1ml to 9ml, five times, in sterile 1 ⁇ 4 strength Ringers solution. 10 ⁇ drops of each dilution were then spotted on to over-dried nutrient agar NA plates and MAC plates before incubation at 30°C for 18 hours (NA) or 37°C for 24 hours (MAC). Plates were then examined for bacterial growth.
  • NA and MAC agar plates were incubated at 30°C (NA) or 37°C (MAC) for 48 hours before being examined for bacterial growth.
  • the number of bacterial species on each plate was estimated using a binocular microscope. Individual colony morphologies from NA plates were then re-streaked on to NA plates and incubated in the same manner in order to obtain pure cultures for further investigation.
  • Oxidase test for Gram -negative rods
  • Catalase test for Gram-positive isolates
  • ID Color Catalase reagent 1.1.3. Oxidase test (for Gram -negative rods), using oxidase test strips. 1.1.4. Catalase test (for Gram-positive isolates), using ID Color Catalase reagent.
  • MSA for Gram-positive, catalase-positive cocci
  • staphylococci and distinguishes Staphylococcus aureus.
  • SBM for Gram-positive, catalase-negative cocci
  • XLD for Gram-negative, oxidase-negative rods
  • enterobacteria such as Shigella, Salmonella etc.
  • PSA for Gram-negative, oxidase-positive rods
  • BMA for presumptive Staphylococcus aureus
  • MRSA methicillin-resistant S. aureus
  • Example 2 A further test was performed to evaluate the efficacy of B. subtilis compositions in reducing sulphite reducing bacteria (SRBs) using the B. subtilis compositions.
  • Test samples were stored at 4C for 24 hours, with recoveries of SRBs being performed at 5 minutes, 60 minutes, and 24 hours. A control using an SRB suspension containing no B. subtilis was performed to compare. Tests were duplicated on two consecutive days.
  • Bacillus subtilis inoculation level 0 min 7.2x10 E8/ml
  • Bacillus subtilis inoculation levels 0 min 6.4x10 E8/ml
  • Kearns D. et al., 2005. A master regulator for biofilm formation by Bacillus subtilis.
  • BC Blue colonies; OC, Opaque colonies; PC, Pink colonies; RC, Reddish colonies;

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Abstract

L'invention concerne une composition biocide comprenant un mélange de spores de souches de Bacillus subtilis et de proanthocyanidines de Vaccinium macrocarpon, dans laquelle lesdits spores de Bacillus sont liés à un sel dendritique et mélangés dans un support de gomme de xanthane, de sorbate de potassium, d'un alcool en C2-C12 et d'un agent tensioactif, et ajustés à un pH de 4,5 +/- 0,1 avec un acide approprié pour former une solution tampon appropriée audit pH.
PCT/US2016/035332 2015-06-01 2016-06-01 Lutte contre les biofilms au moyen de bacillus subtilis et de proantocyanidines de vaccinium macrocarpon Ceased WO2016196674A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010078660A1 (fr) * 2009-01-12 2010-07-15 The Royal Institution For The Advancement Of Learning/Mcgill University Utilisation de proanthocyanidines en tant qu'agent anti-apoptotique et agent antiadhésion bactérienne
EP2453889B1 (fr) * 2009-07-14 2015-04-08 Westfälische Wilhelms-Universität Münster Utilisation de proanthocyanidines pour la production d'une préparation anti-adhésive

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010078660A1 (fr) * 2009-01-12 2010-07-15 The Royal Institution For The Advancement Of Learning/Mcgill University Utilisation de proanthocyanidines en tant qu'agent anti-apoptotique et agent antiadhésion bactérienne
EP2453889B1 (fr) * 2009-07-14 2015-04-08 Westfälische Wilhelms-Universität Münster Utilisation de proanthocyanidines pour la production d'une préparation anti-adhésive

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KOO ET AL.: "Influence of cranberry proanthocyanidins on formation of biofilms by Streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo", CARIES RESEARCH, vol. 44, pages 116 - 126, XP055334336 *
RANE ET AL.: "Cranberry-derived proanthocyanidins prevent formation of Candida albicans biofilms in artificial urine through biofilm- and adherence-specific mechanisms", JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, vol. 69, no. 2, 10 October 2013 (2013-10-10), pages 428 - 436, XP055334332 *
ULREY ET AL.: "Cranberry proanthocyanidins have anti-biofilm properties against Pseudomonas aeruginosa", BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE, vol. 14, no. 499, 2014, pages 1 - 13, XP021208009 *

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