Cis-2-decenoic Acid Research Articles

Virtual Screening Uncovers DspS Activators That Disperse Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa is the predominant bacterium found in many chronic biofilm infections. Over the past few decades, biofilm-related infections have posed a significant challenge to medical practice due to the increasing emergence of multidrug resistance. Cis-2-decenoic acid (CDA), a small molecule found in P. aeruginosa, has been shown to disperse biofilms formed by various bacteria and to work in synergy with common antibiotics. Despite that, the binding mechanism between CDA and the predicted cyclases/histidine kinases associated sensory extracellular (CHASE) domain of sensor protein DspS remains unknown in the absence of a crystallized protein structure. Moreover, the therapeutic potential of CDA is limited by its susceptibility to oxidative degradation and isomerization. In this work, we propose a structural model for the DspS CHASE domain. The resulting model displays an overall topology reminiscent of the sensor protein PcrK in Xanthomonas campestris. Through molecular dynamics simulations, a stable potential binding site for CDA was further identified. Virtual screening against the predicted site of DspS CHASE using our developed pipeline discovered two promising compounds, compounds 2 and 9, capable of dislodging 7-day P. aeruginosa biofilms at 50 μM without affecting bacterial growth. These compounds also enhanced the effects of ciprofloxacin against P. aeruginosa, reduced the survival of dispersed cells, and increased the expression of matrix-degrading enzyme genes pelA, pslG, and eddA. This study provides insights into CDA recognition by DspS and represents the first large-scale effort to uncover first-in-class DspS activators. At the same time, this work also underscores the effectiveness of a computational-aided drug discovery process in finding new activators, even without a known protein structure.

Low-biofouling membrane bioreactor: Effects of cis-2-Decenoic acid addition on EPS and biofouling mitigation

Biofouling is inevitable in the membrane process, particularly in membrane bioreactors (MBR) combined with activated sludge processes. Regulating microbial signaling systems with diffusible signal factors such as cis-2-Decenoic acid (CDA) can control biofilm formation without microbial death or growth inhibition. This study assessed the effectiveness of CDA in controlling biofouling in membrane bioreactors (MBRs), essential for wastewater treatment. By modulating microbial signaling, CDA mitigated biofilm formation without hindering microbial growth. Analysis using Confocal Laser Scanning Microscopy (CLSM) revealed structural alterations in the biofilm, reducing biomass and thickness upon CDA application. Moreover, examination of extracellular polymeric substances (EPS) highlighted a decrease in total EPS, particularly effective polysaccharides. In addition, the possibility of shifting from high molecular weight EPS to low molecular weight EPS was revealed through the change in dispersion activity. The 56% extension of MBR operational lifespan resulting from the reduction in EPS is anticipated to offer potential cost savings and improved performance. Despite these results, further investigation is crucial to validate any potential environmental risks associated with CDA and to comprehend its long-term effects at various conditions.

Chitosan Membranes Stabilized with Varying Acyl Lengths Release Cis-2-Decenoic Acid and Bupivacaine at Controlled Rates and Inhibit Pathogenic Biofilm.

Adherence of complex bacterial biofilm communities to burned tissue creates a challenge for treatment, with infection causing 51% of burn victim deaths. This study evaluated the release of therapeutics from wound care biomaterials and their antimicrobial activity against pathogens Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Electrospun chitosan membranes (ESCMs) were fabricated and acylated with chain lengths ranging from 6-10 carbons then loaded with 0.15 mg of anti-biofilm agent, cis-2-decenoic acid (C2DA), and 0.5 mg of local anesthetic, bupivacaine. Combinations of therapeutics released from modified ESCMs at a cumulative amount of 45-70% of bupivacaine and less than 20% of C2DA. Results from bacterial studies suggest that this combination reduced biofilm 10-fold for S. aureus, 2-fold for Acinetobacter baumannii, and 2-3-fold for Pseudomonas aeruginosa by 24 hours. Additionally, dual loaded groups reduced planktonic Staphylococcus aureus ~4-fold by 24 hours as well as Acinetobacter baumannii ~3-fold by 48 hours. The combination of therapeutics used has a significant role in biofilm prevention for selected strains via direct contact or diffusion in aqueous solutions.

Cis-2-Decenoic Acid and Bupivacaine Delivered from Electrospun Chitosan Membranes Increase Cytokine Production in Dermal and Inflammatory Cell Lines.

Wound dressings serve to protect tissue from contamination, alleviate pain, and facilitate wound healing. The biopolymer chitosan is an exemplary choice in wound dressing material as it is biocompatible and has intrinsic antibacterial properties. Infection can be further prevented by loading dressings with cis-2-decenoic acid (C2DA), a non-antibiotic antimicrobial agent, as well as bupivacaine (BUP), a local anesthetic that also has antibacterial capabilities. This study utilized a series of assays to elucidate the responses of dermal cells to decanoic anhydride-modified electrospun chitosan membranes (DA-ESCMs) loaded with C2DA and/or BUP. Cytocompatibility studies determined the toxic loading ranges for C2DA, BUP, and combinations, revealing that higher concentrations (0.3 mg of C2DA and 1.0 mg of BUP) significantly decreased the viability of fibroblasts and keratinocytes. These high concentrations also inhibited collagen production by fibroblasts, with lower loading concentrations promoting collagen deposition. These findings provide insight into preliminary cellular responses to DA-ESCMs and can guide future research on their clinical application as wound dressings.

Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

Due to antibiotic tolerance of microbes within a biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for short-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid (T2DA), which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. Titanium coupons were drop coated with 0.75mg of DSF in ethanol and dried. Surface characteristics and presence of DSF were confirmed with Fourier Transform infrared spectroscopy (FTIR), x-ray photoelectron spectroscopy (XPS), and water contact angle. Antimicrobial assays on 12-well plates analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-hour incubation, but only sustained through 72 hours for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-hour time point, but not through 72 hours for E. coli. Although approximately 60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. In this study we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

2-Heptylcyclopropane-1-Carboxylic Acid Disperses and Inhibits Bacterial Biofilms.

Fatty-acid signaling molecules can inhibit biofilm formation, signal dispersal events, and revert dormant cells within biofilms to a metabolically active state. We synthesized 2-heptylcyclopropane-1-carboxylic acid (2CP), an analog of cis-2-decenoic acid (C2DA), which contains a cyclopropanated bond that may lock the signaling factor in an active state and prevent isomerization to its least active trans-configuration (T2DA). 2CP was compared to C2DA and T2DA for ability to disperse biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa. 2CP at 125 μg/ml dispersed approximately 100% of S. aureus cells compared to 25% for C2DA; both 2CP and C2DA had significantly less S. aureus biofilm remaining compared to T2DA, which achieved no significant dispersal. 2CP at 125 μg/ml dispersed approximately 60% of P. aeruginosa biofilms, whereas C2DA and T2DA at the same concentration dispersed 40%. When combined with antibiotics tobramycin, tetracycline, or levofloxacin, 2CP decreased the minimum concentration required for biofilm inhibition and eradication, demonstrating synergistic and additive responses for certain combinations. Furthermore, 2CP supported fibroblast viability above 80% for concentrations below 1 mg/ml. This study demonstrates that 2CP shows similar or improved efficacy in biofilm dispersion, inhibition, and eradication compared to C2DA and T2DA and thus may be promising for use in preventing infection for healthcare applications.

In vitro evaluation of loaded chitosan membranes for pain relief and infection prevention.

Wounds resulting from surgeries, implantation of medical devices, and musculoskeletal trauma result in pain and can also result in infection of damaged tissue. Up to 80% of these infections are due to biofilm formation either on the surface of implanted devices or on surrounding wounded tissue. Bacteria within a biofilm have intrinsic growth and development characteristics that allow them to withstand up to 1,000 times the minimum inhibitory concentration of antibiotics, demonstrating the need for new therapeutics to prevent and treat these infections. Cis-2-decenoic acid (C2DA) is known to disperse preformed biofilms and can prevent biofilm formation entirely for some strains of bacteria. Additionally, local anesthetics like bupivacaine have been shown to have antimicrobial effects against multiple bacterial strains. This study sought to evaluate hexanoic acid-treated electrospun chitosan membranes (HA-ESCM) as wound dressings that release C2DA and bupivacaine to simultaneously prevent infection and alleviate pain associated with musculoskeletal trauma. Release profiles of both therapeutics were evaluated, and membranes were tested in vitro against Methicillin-resistant Staphylococcus aureus (MRSA) to determine efficacy in preventing biofilm infection and bacterial growth. Results indicate that membranes release both therapeutics for 72 hr, and release profile can be tailored by loading concentration. Membranes were effective in preventing biofilm growth but were toxic to fibroblasts when loaded with 2.5 or 5mg of bupivacaine.

Tetradecanoic Acids With Anti-Virulence Properties Increase the Pathogenicity of Pseudomonas aeruginosa in a Murine Cutaneous Infection Model.

Blocking virulence is a promising alternative to counteract Pseudomonas aeruginosa infections. In this regard, the phenomenon of cell-cell communication by quorum sensing (QS) is an important anti-virulence target. In this field, fatty acids (FA) have gained notoriety for their role as autoinducers, as well as anti-virulence molecules in vitro, like some saturated FA (SAFA). In this study, we analyzed the anti-virulence activity of SAFA with 12 to18 carbon atoms and compared their effect with the putative autoinducer cis-2-decenoic acid (CDA). The effect of SAFA on six QS-regulated virulence factors and on the secretion of the exoenzyme ExoU was evaluated. In addition, a murine cutaneous infection model was used to determine their influence on the establishment and damage caused by P. aeruginosa PA14. Dodecanoic (lauric, C12:0) and tetradecanoic (myristic, C14:0) acids (SAFA C12-14) reduced the production of pyocyanin by 35–58% at 40 and 1,000 µM, while CDA inhibited it 62% at a 3.1 µM concentration. Moreover, the SAFA C12-14 reduced swarming by 90% without affecting biofilm formation. In contrast, CDA reduced the biofilm by 57% at 3 µM but did not affect swarming. Furthermore, lauric and myristic acids abolished ExoU secretion at 100 and 50 µM respectively, while CDA reduced it by ≈ 92% at 100 µM. Remarkably, the coadministration of myristic acid (200 and 1,000 µM) with P. aeruginosa PA14 induced greater damage and reduced survival of the animals up to 50%, whereas CDA to 500 µM reduced the damage without affecting the viability of the PA14 strain. Hence, our results show that SAFA C12-14 and CDA have a role in regulation of P. aeruginosa virulence, although their inhibition/activation molecular mechanisms are different in complex environments such as in vivo systems.

Dual Antibiotic and Diffusible Signal Factor Combination Nanoliposomes for Combating Staphylococcus epidermidis Biofilm.

Purpose: Microbial biofilms are one of the main causes of persistent human infections. Encapsulation of an antibiotic and a biofilm dispersal agent within a nano-carrier has been recognized as a novel approach to combat the problem of biofilm-related infections. Here, we develop the nanoliposomal formulation for delivery of vancomycin in combination with cis-2- decenoic acid (C2DA), to Staphylococcus epidermidis biofilm. The effects of the formulations were studied at two stages: biofilm growth inhabitation and biofilm eradication. Methods: Liposomal formulations were prepared by the solvent evaporation dehydration-rehydration method and were evaluated for size, zeta potential, and encapsulation efficacy. The ability of different agents in free and encapsulated forms were assessed to evaluate the anti-biofilm activities. Results: Vancomycin and C2DA were successfully co-encapsulated in the same nanoliposome (liposomal combination). The zeta potential values of the liposomal formulations of vancomycin, C2DA, and the liposomal combination were 37.2, 40.2, 51.5 mV, and the mean sizes of these liposomal formulations were 167.8±1.5, 215.5±8.8, 235.5±0.01, respectively. Encapsulation efficacy of C2DA was 65% and about 40% for vancomycin. The results indicated that liposomal combination exerted strong anti-biofilm activities, slightly exceeding those observed by the free form of a combination of vancomycin and C2DA, but higher than either agent used alone in their free forms. The anti-biofilm activity of formulations followed concentration and time-dependent manner. Conclusion: The combination of vancomycin and C2DA could inhibit biofilm formation. Employing the liposomal combination is a considerable method to remove bacterial biofilm.

Rifampin and Cis-2-Decenoic Acid Co-entrapment in Solid Lipid Nanoparticles as an Efficient Nano-system with Potent Anti-biofilm Activities

This study was performed to explore the physicochemical and anti-biofilm properties of rifampin (Rif) and cis-2-decenoic acid (C2DA) co-entrapped in solid lipid nanoparticles, Rif-C2DA-SLN, against staphylococcal biofilm. The formulation was prepared by high-shear homogenization and ultrasound methods and characterized for size, zeta potentials, encapsulation efficacy, drug lipid interaction studies (DSC), shape morphology (TEM), stability studies, and in vitro anti-biofilm activity against Staphylococcus aureus and S. epidermidis biofilm. The zeta potentials, particle sizes, and encapsulation efficacy of final formulations were 19.0 ± 7.64 mV, 127.2 ± 2.8 nm, and approximately 69% (Rif) and 46% (C2DA), respectively. SLN formulations were stable for 12 months. DSC studies showed no chemical interaction between drugs and lipids. SLN formulations showed better in vitro anti-biofilm activity than free forms especially in the stage of biofilm formation. Nanoparticles were not able to remove the formed biofilm. Simultaneous entrapment of Rif and C2DA by SLN is reported for the first time, and Rif-C2DA-SLN can be applied as an efficient nanoparticulate system with potential anti-biofilm activities. These data suggest that the combined strategies for delivery of both C2DA and Rif by nanoparticulate systems would pave the way toward developing the strategies to combat biofilms.

From normal to competo-allosteric regulation: insights into the binding pattern dynamics of DSPI protein of Pseudomonas aeruginosa

DSPI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, is anticipated to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule present in the superbug Pseudomonas aeruginosa. The current study not only adapts a broad-spectrum strategy for the lucid design of small molecule modulators but also provides novel allosteric inhibitors for DSPI, to investigate its function and potential as a therapeutic target. Docking analysis revealed that the compound 10252273, bound to the specific allosteric site, interacted with Glu118, unique amino acid residue of the active binding pocket, hence indicates the presence of a competitive allosteric site. The current study thus identifies and characterizes inhibitors by targeting the normal binding site and also reports the presence of the competo-allosteric site in the same binding tunnel as the normal site. Molecular docking studies proposed two chemical compounds that share a benzamide-benzimidazole (BB) backbone as potent inhibitors that can obstruct the mechanism of DSPI by targeting both the normal and proposed allosteric binding sites. MD simulations further revealed the disruption of the normal binding site due to the displacement of critical residues Cys146 and Glu118. The rearrangement of H-bond pattern, pi-pi interactions, and strong hydrophobic interactions were observed at both the binding sites. The allosteric pocket inhibitor exhibited improved binding energy than the normal site inhibitor based on MMGBSA and MMPBSA analysis. With subsequent characterization, the current study reveals the allosteric binding site and provides insights into the drug binding mechanism of DSPI.Communicated by Ramaswamy H. Sarma

Synergistic Anti-Borreliae Efficacy of a Composition of Naturally-occurring Compounds: an In vitro Study

Background: Borrelia sp, which is a pathogenic agent of Lyme diseases in mammals, has become an increasing problem worldwide due to the emergence of persistence. In this study we investigated whether a defined composition of naturally occurring substances could display a broad and synergistic action in vitro against both active and persistent forms of Borrelia spp.Methods: A formulation of six plant-derived compounds combined at their 1/32-1/2 MIC values was tested in vitro against two species of Borrelia recognized as causative agents of Lyme disease in North America and Europe.Results: The results showed that a composition of baicalein, luteolin, rosmarinic acid, monolaurin, cis-2 decenoic acid, and iodine at their 1/8 MIC values has significant synergistic effect against the active and persisting latent forms. This composition revealed anti-oxidative properties affecting Borrelia’s membrane but not DNA. Finally, we observed its inhibitory effect on the release of IL-1α, IL-1β, and IL-6 by human CD14+ monocytes stimulated with live Borrelia sp.Conclusion: These results suggest that such a formulation of compounds might be considered and further explored for its significant pleotropic anti-Borreliae efficacy. Additional in vivo and human studies are warranted to validate this possibility.

Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis

DspI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, was proposed to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule in the pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study provided a structural basis for the dehydration reaction mechanism of DspI during CDA synthesis. Structural analysis reveals that Glu126, Glu146, Cys127, Cys131 and Cys154 are important for its enzymatic function. Moreover, we show that the deletion of dspI results in a remarkable decreased in the pyoverdine production, flagella-dependent swarming motility, and biofilm dispersion as well as attenuated virulence in P. aeruginosa PA14. This study thus unravels the mechanism of DspI in diffusible signal factor (DSF) CDA biosynthesis, providing vital information for developing inhibitors that interfere with DSF associated pathogenicity in P. aeruginosa.

Understanding the fundamental mechanisms of biofilms development and dispersal: BIAM (Biofilm Intensity and Architecture Measurement), a new tool for studying biofilms as a function of their architecture and fluorescence intensity

Confocal laser scanning microscopy (CLSM) is one of the most relevant technologies for studying biofilms in situ. Several tools have been developed to investigate and quantify the architecture of biofilms. However, an approach to quantify correctly the evolution of intensity of a fluorescent signal as a function of the structural parameters of a biofilm is still lacking. Here we present a tool developed in the ImageJ open source software that can be used to extract both structural and fluorescence intensity from CLSM data: BIAM (Biofilm Intensity and Architecture Measurement). This is of utmost significance when studying the fundamental mechanisms of biofilm growth, differentiation and development or when aiming to understand the effect of external molecules on biofilm phenotypes. In order to provide an example of the potential of such a tool in this study we focused on biofilm dispersion. cis-2-Decenoic acid (CDA) is a molecule known to induce biofilm dispersion of multiple bacterial species. The mechanisms by which CDA induces dispersion are still poorly understood. To investigate the effects of CDA on biofilms, we used a reporter strain of Escherichia coli (E. coli) that expresses the GFPmut2 protein under control of the rrnBP1 promoter. Experiments were done in flow cells and image acquisition was made with CLSM. Analysis carried out using the new tool, BIAM, indicates that CDA affects the fluorescence intensity of the biofilm structures as well as biofilm architectures. Indeed, our results demonstrate that CDA removes more than 35% of biofilm biovolume and suggest that it results in an increase of the biofilm's mean fluorescence intensity (MFI) by more than 26% compared to the control biofilm in the absence of CDA.

Phosphatidylcholine Coatings Deliver Local Antimicrobials and Reduce Infection in a Murine Model: A Preliminary Study.

Phosphatidylcholine coatings have been shown to elute antibiotics for several days. A recently developed biofilm inhibitor, cis-2-decenoic acid (C2DA), has been shown to exhibit synergistic activity with several common antibiotics. This study aims to evaluate the effectiveness of C2DA and amikacin dual drug delivery from a phosphatidylcholine coating. (1) What are the in vitro elution profiles of amikacin and C2DA from phosphatidylcholine-coated coupons in incubated phosphate-buffered saline? (2) Does the presence of C2DA in eluate samples lower the amount of amikacin needed for bacterial inhibition in overnight bacterial turbidity assays? (3) Does addition of amikacin and C2DA result in decreased colony-forming units (CFUs) on wire implants and bone when compared with phosphatidylcholine coatings alone in a mouse model of periprosthetic joint infection? Effects of loading concentrations were assessed during 7-day in vitro elution studies for coatings containing all mixtures of 0%, 5%, 15%, and 25% wt of amikacin and C2DA (n = 4) through quantitative high-performance liquid chromatography concentration determination and plotting concentration eluted over time. Antimicrobial activity was assessed by overnight turbidity testing of elution study samples against Staphylococcus aureus or Pseudomonas aeruginosa. In vivo efficacy was assessed using phosphatidylcholine-coated wire implants in a murine (mouse) model of infection (n = 3). Wire implants were coated with phosphatidylcholine containing no antimicrobials, amikacin alone, C2DA alone, or amikacin and C2DA and then inserted into the intramedullary femur of each mouse and inoculated with S aureus. The number of viable bacterial colonies on the implant surface and in the surrounding bone was determined after 1 week with the goal of achieving complete bacterial clearance. Total viable CFU count and proportion of samples achieving complete clearance were compared between groups. Elution samples showed a burst response of amikacin and C2DA for 1 to 2 days with C2DA release continuing at low levels through Day 4. All tested eluate samples inhibited P aeruginosa. Samples from coatings containing 25% amikacin or 15% amikacin and any amount of C2DA were able to inhibit S aureus formation, but all coatings with 5% amikacin or 15% amikacin but no C2DA were not inhibitory. All in vivo treatment groups achieved complete bacterial clearance on the wire implant, and the C2DA alone and amikacin alone coatings cleared all CFUs in bone (pin: phosphatidylcholine only one of three; amikacin three of three, C2DA three of three, amikacin + C2DA three of three, p = 0.04 [Fisher's exact test]; bone: coating only: zero of three; amikacin: three of three; C2DA; three of three; C2DA + amikacin: one of three; p = 0.03 [Fisher's exact test]). Phosphatidylcholine coatings elute antimicrobials in vitro under infinite sink conditions for up to 4 days in phosphate-buffered saline and were able to reduce bacterial colonies in a preliminary in vivo model. Turbidity testing with eluate samples containing varying amounts of C2DA and amikacin agrees with previous studies showing synergy between them. Used as an adjunctive to systemic therapy, C2DA-loaded phosphatidylcholine coatings have potential value as a prophylactic infection prevention measure. Future studies may include different antibiotics, animal studies with larger sample sizes and more controls, and advanced coating delivery methods.

Measuring and modeling elution of Cis 2-decenoic acid from phosphatidylcholine coatings

Measuring and modeling elution of Cis 2-decenoic acid from phosphatidylcholine coatings

Low-concentration diffusible molecules affect the formation of biofilms by mixed marine communities

AbstractBiofilm formation is a major concern in any venture in the marine environment and often precedes the establishment of fouling by macro-organisms. In this study, the effects of three known cell-to-cell signalling molecules, nitric oxide (NO), cis-2-decenoic acid (CDA) and patulin, on the formation of marine biofilms were investigated. Each of the molecules has been shown to affect biofilms and this is the first study to investigate their effect on mixed communities of marine biofilm-forming micro-organisms. Studies of the biomass of those biofilms grown in the presence of the molecules showed that all three reduced biofilm formation by marine communities, with both NO and patulin reducing biofilm formation by more than 90% at the highest concentrations studied. However, colony counts revealed that the effect of patulin is likely due to toxicity. Analyses of the biofilm communities were also carried out using DGGE to determine whether there was any variation in the effects of each molecule on differ...

CORR Insights(®): D-amino acid inhibits biofilm but not new bone formation in an ovine model.

C omplex musculoskeletal injuries are complicated by infections in as many 30% of traumatized patients [2], depending on the type of injury and the timing of the interventions. Biomaterials often are necessary components to restore function and promote healing in injuries where muscle, nerve, and bone damage present challenges to healing. While biomaterials serve critical functions in repairing defects, they also increase the risk of infection. This is in part due to the promotion of biofilm formation on the surface of implants, and in particular, the presence of persister cells (dormant cells that survive exposure to antimicrobials) within biofilm. Biofilm microorganisms and persister cells have altered metabolic function that make them resistant to traditional antibiotic therapy and require antimicrobial concentrations several orders of magnitude higher for complete eradication than those necessary for killing planktonic counterparts [9]. Basic science studies of biofilm and persister-cell biology have led to the discovery of a number of molecules that specifically target these phenotypes. D-amino acids (D-AAs), cis 2decenoic acid, and farnesol are among these recently discovered biofilm inhibitors that have been shown to prevent biofilm formation, disperse existing biofilm, and revert persister cells to a more active and antimicrobial-susceptible state [4, 7, 8]. While in vitro results using D-AAs are encouraging, clinical use of these biofilm inhibitors remains investigational. Dose considerations and compatibility with bone and soft tissue must be established for safety. Since systemic delivery of D-AAs and other biofilm inhibitors may be ineffective, local delivery carriers for D-AAs are an advantageous strategy for clinical use.

A combination of cis-2-decenoic acid and chlorhexidine removes dental plaque

ObjectivesTo investigate the ability of cis-2-decenoic acid (C2DA) to induce dispersion in single-species biofilms formed by Streptococcus mutans and Candida albicans, as well as to remove their bacterial-fungal dual-species biofilms when combined with low concentrations of chlorhexidine (CHX). MethodsFor biofilm dispersal bioassays, single-species biofilms of S. mutans and C. albicans were grown on the inside surface of petri dishes, using a semi-batch culture method in which the medium was replaced every 24h for 5 days. Biofilms were then treated with very low concentrations of C2DA (100 and 310nM) for 1h to release cells into the bulk liquid and to evaluate dispersed cell number by measuring the optical density (OD). To assess the ability of C2DA combined CHX treatments to remove tested microorganisms’ dual-species biofilms, they were grown on saliva-coated hydroxyapatite (sHA) discs for 48h and then were treated with three different concentrations of CHX (0.08%, 0.06% and 0.04%) alone or in combination with indicated concentrations of C2DA for 1min twice daily for 3 subsequent days. Biofilms were then either subjected to the field emission scanning electron microscopy (FESEM) analysis or harvested and colony forming units (CFUs) were counted after plating on agar. ResultsTreatment of pre-established biofilms with 310nM C2DA caused an approximately two-fold increase in the number of planktonic cells in both cultures. A combination of 310nM C2DA and 0.04% CHX resulted in significant removal (p-value <0.05) of dual-species biofilms from sHA discs surface. ConclusionsAnti-biofilm characteristic of C2DA boosts the action of CHX even at low concentrations.

Osteocompatibility of biofilm inhibitors.

The demand for infection prevention therapies has led to the discovery of several biofilm inhibitors. These inhibiting signals are released by bacteria, fungi, or marine organisms to signal biofilm dispersal or disruption in Gram-positive, Gram-negative, and fungal microorganisms. The purpose of this study was to test the biocompatibility of five different naturally-produced biofilm chemical dispersal and inhibition signals with osteoblast-like cells: D-amino acids (D-AA), lysostaphin (LS), farnesol, cis-2-decenoic acid (C2DA), and desformyl flustrabromine (dFBr). In this preliminary study, compatibility of these anti-biofilm agents with differentiating osteoblasts was examined over a 21 days period at levels above and below concentrations active against bacterial biofilm. Anti-biofilm compounds listed above were serially diluted in osteogenic media and added to cultures of MC3T3 cells. Cell viability and cytotoxicity, after exposure to each anti-biofilm agent, were measured using a DNA assay. Differentiation characteristics of osteoblasts were determined qualitatively by observing staining of mineral deposits and quantitatively with an alkaline phosphatase assay. D-AA, LS, and C2DA were all biocompatible within the reported biofilm inhibitory concentration ranges and supported osteoblast differentiation. Farnesol and dFBr induced cytotoxic responses within the reported biofilm inhibitory concentration range and low doses of dFBr were found to inhibit osteoblast differentiation. At high concentrations, such as those that may be present after local delivery, many of these biofilm inhibitors can have effects on cellular viability and osteoblast function. Concentrations at which negative effects on osteoblasts occur should serve as upper limits for delivery to orthopaedic trauma sites and guide development of these potential therapeutics for orthopaedics.