Cis-2-Decenoic Acid Interacts with Bacterial Cell Membranes to Potentiate Additive and Synergistic Responses against Biofilm

Research Article

J Bacteriol Mycol. 2016; 3(3): 1031.

Cis-2-Decenoic Acid Interacts with Bacterial Cell Membranes to Potentiate Additive and Synergistic Responses against Biofilm

Masters EA, Harris MA and Jennings JA*

University of Memphis, Memphis, TN, USA

*Corresponding author: Jennings JA, Department of Biomedical Engineering, University of Memphis, Memphis, USA

Received: July 07, 2016; Accepted: August 08, 2016; Published: August 10, 2016


Musculoskeletal infection is a major risk in all wounds, especially orthopaedic procedures such as implants or bone grafts. Morbidity due to implant-associated infections has increased and the need for therapeutic strategies to prevent them is growing. Biofilm dispersal agent, cis-2-decenoic Acid (C2DA) may be used in conjunction with a variety of antibiotics to potentiate synergistic, additive and antagonistic responses against orthopaedic pathogens. Results demonstrate that C2DA exhibits additive and synergistic effects with tetracycline, amikacin and ceftazidime against both gram positive and gram negative bacteria. Uptake of a fluorescent probe 1-N-phenylnaphthylamine by S. aureus was doubled and uptake by P. aeruginosa was increased by over eight fold when exposed to varying concentrations of C2DA. Leakage of intracellular ATP also increased up to three times control values with P. aeruginosa and over 280 times control value with S. aureus. This suggests that the mechanism of C2DA additive and synergistic effects with antibiotics is in part due to increasing membrane permeability, allowing increased uptake of additional antimicrobials.

Keywords: Biofilm; Synergy; Infection; Antibiotic; Cis-2-decenoic acid


C2DA: Cis-2-Decenoic Acid; MRSA: Methicillin-Resistant Staphylococcus Aureus; UAMS-1: Staphylococcus Aureus; PAO1 and PA ATCC 27317: Pseudomonas Aeruginosa; TSB: Tryptic Soy Broth; CFUs: Colony Forming Units; PBS: Phosphate Buffered Saline; FICI: Fractional Inhibitory Concentration Index; MBIC: Minimum Biofilm Inhibitory Concentration; NPN: 1-N-Phenylnaphthylamine; ATP: Adenosine Triphosphate; ONPG: O-Nitrophenyl-B-DGalactopyranoside


The need for innovative therapies that prevent and treat implantassociated infections is becoming more urgent as the mortality due to such infections has increased [1,2]. Musculoskeletal infection is a major risk in all wounds, especially open fractures and surgical or orthopedic procedures such as implants or bone grafts, with a rate of infection as high as 17-21% in open fractures [3]. As this rate grows, so does the number of patients who must undergo revision procedures due to implant failure and infection treatment from otherwise successful orthopedic procedures [4]. Additionally, multi-drug resistant biofilm-based infections can increase morbidity and cost of treatment [4-7]. Biofilm formation occurs when microorganisms attach to a surface and excrete a polymeric matrix in which they can enter a state of reduced metabolic activity [6,8,9]. Specifically, orthopedic implant materials may provide advantageous surfaces for the attachment of bacteria and the formation of biofilm [7,10-12], increasing the risk of wound infection. Causing as much as 80% of infection, biofilm limits the activity of antibiotics or immune cell attack, increasing the severity of infection and making it particularly difficult to treat [13]. Many bacteria and fungi thrive in polymicrobial communities because of mutualistic and symbiotic relationships formed to promote their survival, confer antibiotic resistance, and increase virulence [14-17]. This property makes specific antibiotic therapies ineffective in eliminating the infection. If one specific microorganism is eliminated this may leave a niche for other microorganisms to fill. Methicillin-Resistant Staphylococcus Aureus (MRSA) and multidrugresistant Pseudomonas aeruginosa are identified among the most predominant infecting pathogens in musculoskeletal infections [18- 20]. The clinical impact of biofilm and musculoskeletal infection has prompted a number of strategies to combat the formation and growth of biofilm. Prophylactic antibiotics can be administered to a patient prior to the implantation of a medical device, however the efficacy and associated risk of these methods, including organ damage and increased bacterial resistance to antibiotics, has put them in question [21,22]. Local drug delivery methods through chitosan coatings have proven to eliminate infection, however these coatings must be prepared and fabricated prior to implantation [23]. A fatty acid, cis- 2-decenoic Acid (C2DA), has been shown to both disperse bacterial communities as well as inhibit biofilm growth in polymicrobial communities [13,24]. C2DA is a short chain fatty-acid with a polar head and non-polar tail. Discovered as a chemical messenger in the dispersal of Pseudomonas aeruginosa, it has been proven to signal the dispersion of mature biofilms composed of both gram-negative and gram-positive bacteria [24-27]. Additionally, there is evidence that C2DA may have additive or synergistic effects against bacterial growth and biofilm formation when paired with different antibiotics [13,25,28].

We hypothesized that C2DA increases membrane permeability, therefore antimicrobials that act within the bacterial cell, such as those with protein translation mechanisms, will be more likely to portray synergistic responses in conjunction with C2DA. If this hypothesis is true, C2DA will increase the amount of antimicrobial that crosses the bacterial cell membrane and affects the cell. In continuation, antimicrobials that act at the bacterial cell membrane are likely to portray additive responses in conjunction with C2DA as their effects are simply combined at the surface of the cell membrane.

The aim of this study is to investigate the mechanism of action of a biofilm inhibitor, cis-2-decenoic acid, and determine the if the combinatorial effects of C2DA with various antimicrobials could be predicted based off of the mechanism of action of the antimicrobial. This hypothesis was tested using previously developed membrane permeabilization assays [29,30] and combinations of C2DA with the antimicrobials vancomycin, cefazolin, linezolid, tetracycline, chlorhexidine, ceftazidime, daptomycin, amikacin and ciprofloxacin. These antimicrobials have various mechanisms of action, some that act within the bacterial cell body and some that act at the bacterial membrane [31-36]. Synergistic combinations were determined using checkerboard inhibition assays, and the results were studied for trends between antibiotic mechanism of action and synergy. Synergistic combinations of C2DA and antimicrobials, when coupled with an appropriate local drug delivery device, may be able to reduce the rate of orthopedic infection, reducing healthcare costs and improving patient outcome.


Growth of bacterial stock

The bacterial cultures used in this study were Staphylococcus aureus (UAMS-1) and Pseudomonas aeruginosa (PAO1 and PA ATCC 27317), which are common gram-positive and gram-negative orthopaedic pathogens respectively. Before each experiment, bacterial culture was diluted from stock. Overnight growth of cultures in tryptic soy broth (TSB) were diluted to obtain 105 colony forming units (CFUs)/mL by diluting 1:50 and 1:200 for Staphylococcus aureus and Pseudomonas aeruginosa respectively.

In vitro biofilm inhibition assays

Synergy studies of different antimicrobials combined with C2DA were performed to determine whether C2DA interacts with different antimicrobials to potentiate additive, synergistic, or even antagonistic responses against biofilm and bacterial growth. Interactions between antimicrobials and C2DA were determined using checkerboard assays using varying concentrations of C2DA and the antimicrobials vancomycin, cefazolin, linezolid, tetracycline, chlorhexidine, ceftazidime, daptomycin, amikacin and ciprofloxacin.

An array of sterile tubes containing 1.75mL TSB, 100μL of varying C2DA dilutions, and 100μL of varying antibiotic dilutions were prepared to obtain test concentrations of antimicrobials. Then 100μl of combined mixture was added to wells of 96 well plates in triplicate in a checkerboard array with increasing concentrations of C2DA along one axis and increasing concentrations of antimicrobial on the other axis. Each well was then inoculated with 50μL of diluted bacterial culture for a total inoculum of 10³ CFUs/mL. Inoculated and non-inoculated TSB only controls were also included on each plate. Final test concentrations of C2DA were 1000, 500, 250, 125, 62.5, 31.3 and 0 μg/ml. Due to the varying minimum biofilm inhibitory concentrations (MBIC) of the antimicrobials used in this study, the testing concentrations for each antimicrobial had to be individually adjusted to obtain a starting concentration of approximately double the MBIC. The starting antimicrobial concentration, two serial twofold dilutions and a PBS control were tested for each antimicrobial. After incubating overnight at 37°C, the turbidity of each well was measured at 540nm in a plate reader spectrophotometer (Biotek Synergy H1, Winooski VT) to assess initial planktonic bacterial growth. Planktonic bacteria were then removed from the plates by cautiously aspirating all liquid from each well, while leaving the biofilm on the bottom surface undisturbed. The plates were washed gently 3 times with 150μl Phosphate Buffered Saline (PBS) to remove any remaining planktonic bacteria. The remaining biofilm was heat fixed in an oven at 60°C for an hour. Once fixed, the biofilm was stained with 150μl crystal violet. Crystal violet stain was gently rinsed from the plates with water, removing excess stain that had not been taken up by biofilm, before photographing biofilm growth illuminated with a backlight. Finally, 150μL of de-staining solution, composed of 10% methanol and 7.5% acetic acid in water, was added to each well to dissolve the bound crystal violet before measuring the absorbance of each well in a spectrophotometer at 560 nm. Absorbance was proportional to the amount of biofilm present in each well.

The response of each antimicrobial when combined with C2DA was quantified using the Fractional Inhibitory Concentration Index (FICI). To determine FICI, the ratio of MBIC for the antimicrobial alone/ MBIC in combination with C2DA was added to the ratio of MBIC for C2DA alone/MBIC of C2DA when combined with antimicrobial (Equation 1) [37]. The various MBIC values in these studies were determined as the lowest concentration of antimicrobial to completely inhibit growth of biofilm on the bottom surface of the well plate, as determined by the concentration at which staining values were <10% of positive controls [38]. The effect of synergistic antimicrobials together will be greater than the sum of their parts, causing the FICI to be less than one. Antagonistic combinations will reduce the effectiveness of each antimicrobial and lead to a FICI greater than 2. Those antimicrobials where no effects in improving or reducing effectiveness are considered indifferent and will have FICI values of approximately 2, since MIC concentrations are the same singly or in combination. In accordance with similar studies [39, 40], we considered FICI values below 0.5 to be synergistic responses, values below 1 additive, and values above 2 to be antagonistic. The FICI was calculated separately for each strain of bacteria.

Membrane permeabilization assays

NPN uptake assay: To examine the effects of C2DA as a membrane permeabilizer against common gram-positive and gramnegative pathogens, bacterial membrane permeability was measured using a 1-N-phenylnaphtylamine (NPN) uptake assay [30]. This assay uses hydrophobic probe, NPN, which fluoresces strongly in a phospholipid environment where the membrane is damaged but only weakly in an aqueous environment in the extracellular fluid outside the membrane. In the presence of a membrane permeabilizer, the damaged bacterial membrane will allow more NPN to enter the membrane and become exposed to the phospholipid environment, observed as increased fluorescence. This study used antibiotic polymyxin B as a positive control membrane permeabilizer, as it is known to act at by disrupting the bacterial cell membrane [41, 42]. Additionally, the same concentrations of antimicrobials and noninoculated buffer were used as controls.

PA01 and UAMS-1 bacterial cultures were prepared using previously described methods and then washed 3 times with 5 mM HEPES buffer (pH = 7.4) by spinning down cells at 10,000 rpm for 5 min and re-suspending in buffer. NPN working solution was prepared freshly for each experiment by diluting NPN to a concentration of 0.5mM in acetone. Next, 23μL of HEPES buffer was added to a black 100μL, 96 well plate along with 2μL of NPN solution and increasing concentrations of either C2DA or polymyxin B. The 96 well plate was inoculated with 50μL of either PA01 or UAMS-1 bacterial culture while including the same number of wells with no bacteria added to normalize the NPN fluorescence. Concentrations used for polymyxin B ranged from 0.1μg/mL to 6.4μg/mL using 2X serial dilutions, and 2X dilutions from 15.6-1000 μg/ml for C2DA. PBS and 50% EtOH were used as blank controls for polymyxin B and C2DA, respectively. The permeabilization of membranes was monitored over the course of 30minutes by measuring fluorescence intensity at 460nm with excitation at 355nm (Biotek Synergy H1, Winooski VT). Results are displayed as an NPN uptake ratio, which was calculated as the ratio of highest fluorescence in bacteria and antibiotic samples to the highest fluorescence in bacteria samples in buffer only.

ATP leakage assay: When bacterial membranes become more permeable or when they burst, intracellular contents such as adenosine triphosphate (ATP) will be released into the surrounding medium. Methods to measure ATP in the supernatant after exposure to C2DA were adapted from procedures described by Higgins et al [29]. Overnight cultures of Staphylococcus aureus (UAMS-1) and Pseudomonas aeruginosa (PA ATCC 27317) were prepared in TSB in 50 ml centrifuge tubes. Bacterial cultures were centrifuged at 3000rpm for 10minutes. TSB was removed and the bacteria were resuspended in 5mM sterile HEPES buffer at an optical density A600 = 1 for UAMS-1 and A600 = 0.5 for PA ATCC 27317. A 30mg/ml stock of cis-2-decenoic Acid (C2DA) in 50% ethanol was prepared. Two four-fold dilutions of C2DA stock were prepared, including plain 50% ethanol as a non-C2DA control. 1.9ml bacterial suspension and 67μL of the appropriate C2DA test solution were added to 5 ml test tubes (n=3 per group). A control group received 1.9 ml HEPES without bacteria and 67μl of the appropriate test solution. Final C2DA concentrations were 1000, 250, 62.5, and 0μg/ml. The tubes were incubated at 37°C for 30 minutes, then centrifuged at 3000rpm for 5minutes. The supernatant from each tube was plated in triplicate in an opaque white 96 well plate. An equal amount of Cell Titer Glo (Promega, Madison, WI) was added to each sample well. The plate was allowed to sit for 10 minutes at room temperature, then luminescence was read using a Biotek Synergy H1 plate reader.

Statistical analysis

Using SigmaPlot 12.5 (Systat, Chicago, IL, USA), one-way Kruskal-Wallis ANOVA with Dunnett’s post-hoc analysis was performed to determine statistical differences between groups and controls for NPN uptake and ATP leakage. Each sample was tested in triplicate.


In vitro biofilm inhibition assays

In brief, tetracycline, linezolid, and chlorhexidine were synergistic against S. aureus (Figure 1), while amikacin, ceftazidime, and ciprofloxacin produced synergistic effects against P. aeruginosa (Table 1). Vancomycin, daptomycin, cefazolin, amikacin and ceftazidime were additive with S. aureus, while tetracycline was additive with P. aeruginosa. Chlorhexidine is the only antimicrobial with a mechanism of action at the cell membrane to produce a synergistic response with C2DA. Additionally, chlorhexidine is the only antimicrobial to potentiate an antagonistic response against bacterial growth, producing a FICI of 4.0 against P. aeruginosa. All antibiotics not active against gram-negative microorganisms, produced indifferent interactions with C2DA against gram-negative bacteria.