The first twelve authors should be regarded as joint First Authors.
Lu Yu, Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China. E-mail: firstname.lastname@example.org
Aims: To investigate the antimicrobial efficacy of an alkaloid, harmaline alone and in combination with chlorhexidine digluconate (CHG) against clinical isolates of Staphylococcus aureus (S. aureus) grown in planktonic and biofilm cultures.
Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for each micro-organism grown in suspension and in biofilm using microbroth dilution method. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between harmaline and CHG, and the some of results were verified by confocal laser scanning microscopy.
Results: Harmaline and CHG showed effective antimicrobial activity against suspensions and biofilm cultures of S. aureus, respectively. As determined by fractional inhibitory concentration index (FICI), synergistic antimicrobial effects between harmaline and CHG were observed in nine and 11 of the 13 S. aureus strains when in suspension and in biofilm, respectively. FICI values were from 0·375 to 1·25 when in suspension and from 0·25 to 1·25 when in biofilm.
Conclusions: Synergistic activity of harmaline and CHG against clinical isolates of S. aureus (in suspension and in biofilm) was observed in vitro.
Significance and Impact of the Study: This study might provide alternative methods to overcome the problem of drug-resistance of S. aureus both in suspension and in biofilm.
The Gram-positive bacterium Staphylococcus aureus (S. aureus) is a significant community-acquired and nosocomially acquired pathogen that can cause both local and systemic infections in humans, including skin infections, arthritis, pneumonia, meningitis, endocarditis, osteomyelitis and toxic shock syndrome (Lowy 1998). Bacteria including S. aureus are able to grow biofilms that adhere to almost every surface. Biofilms are complex bacterial communities embedded in a self-produced glycocalyx slime that protects the cells from environmental and antimicrobial threats (Parra-Ruiz et al. 2010). Previous study has shown that antimicrobial MICs of bacteria, which were embedded in biofilms, were 10 to 1000 times higher than those in a planktonic state (Ceri et al. 1999). Therefore, new agents are needed for the treatment of S. aureus biofilm.
Chlorhexidine is one of the most widely used antimicrobials within clinical practice for skin antisepsis. It is currently recommended within the Evidence-Based Practice in Infection Control (EPIC) (Pratt et al. 2007) and Healthcare Infection Control Practices Advisory Committee (HICPAC) (O’Grady et al. 2002) guidelines. Chlorhexidine digluconate (CHG) possesses broad bactericidal activity against many Gram-negative and Gram-positive bacteria, such as S. aureus (Hope and Wilson 2004). Despite the proven antimicrobial activity of CHG, micro-organisms can reside in the deeper layers of the skin to survive current recommendations for antisepsis (Karpanen et al. 2008a,b); one of the reasons may be that the in vivo antimicrobial activity of CHG alone is bacteriostatic (Beighton et al. 1991). Thus, new therapeutic strategies are necessary. Improvements in the efficacy of antisepsis drug may be achieved by using combination therapy.
Plants and other natural materials may prove to be valuable sources of new antimicrobials or antimicrobial synergist (Guo et al. 2009; Ge et al. 2010a,b). Some of them have been known to possess broad-spectrum antimicrobial activity against resistant bacterial strains (Li et al. 2011). Noteworthily, harmaline (Fig. 1), an alkaloid, is a major component of Peganum harmala seed extracts, which have been frequently reported to possess antibacterial potential through in vitro studies (Arshad et al. 2008). However, the antibacterial activity of harmaline against both planktonic and biofilm cultures of S. aureus strains is currently unknown.
In this study, the microbroth dilution method and the checkerboard assay were employed to investigate the antimicrobial efficacy of harmaline alone and in combination with CHG against the clinically isolated S. aureus grown in planktonic and biofilm cultures.
Materials and methods
Congo Red agar for demonstrating slime production in the test strains S. aureus was prepared by mixing 0·08 g of Congo Red (Hopkins and Williams Ltd, Essex, UK), 5 g of sucrose (Fisher Scientific, Leicestershire, UK) and 1 g of agar No. 1 (Oxoid, Basingstoke, UK) with 98 ml of brain heart infusion (Oxoid) and sterilized according to the manufacturer’s recommendations. Mueller-Hinton agar (MHA) and Mueller-Hinton broth (MHB) (Oxoid) were also prepared and sterilized in line with the manufacturer’s recommendations. Phosphate-buffered saline (PBS), aqueous CHG (20% in water) and harmaline (≥97%) were purchased from Sigma-Aldrich (Dorset, UK), and glucose was purchased from Fisher Scientific. Cell culture plate, polystyrene, flat bottom with low evaporation lid, sterile, tissue culture-treated 96-well microtitre plates were from Nest Biotech Ltd (Wuxi, China).
Clinically isolated S. aureus were obtained from the First Hospital of Jilin University from blood samples of infected patients. The quality control (QC) strain ATCC 29213 was obtained from the China Medical Culture Collection Center (CMCC). The S. aureus strains were stored on MicroBank beads (Pro-Lab Diagnostics, Cheshire, UK) at −70°C until required.
Preparation of antimicrobial agents
Harmaline dissolved in deionized water to obtain a stock solution of 20480 μg ml−1 under sterile conditions and stored at −70°C until used. Aqueous CHG was diluted with MHB to obtain a stock solution of 512 μg ml−1.
Preparation of Staphylococcus aureus inoculum for suspension assay
The strains were plated on MHA and incubated at 37°C overnight. Colonies from the plates were resuspended in MHB, and using optical density (OD) at 660 nm, the concentration of micro-organism in each overnight suspension was determined using previously established OD/concentration standard curves. The suspensions were further diluted with MHB to obtain inocula containing 1 × 106 CFU ml−1.
Determination of MICs and MBCs of aqueous CHG and harmaline for Staphylococcus aureus in suspension
The MICs of aqueous CHG, as well of harmaline, were determined as described previously by the broth microdilution method adapted from previous studies and in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2009). Serial twofold dilutions of antimicrobial agents were prepared in MHB to obtain the required concentrations (CHG, 0·125–128 μg ml−1; Harmaline, 0·5–512 μg ml−1). The assay was performed in 96-well microtitre plates, and a positive control, which contained inoculated broth without drugs, was included in every plate. Each well contained 100 μl of test antimicrobial and 100 μl of S. aureus suspension diluted in MHB, containing 1 × 105 CFU. Plates were incubated in air for 24 h at 37°C. The MIC was defined as the lowest concentration of the drug that inhibited the growth of the test micro-organism by >90% (Oo et al. 2010). MBCs were determined by removing the total volume (200 μl) from each of the clear wells into duplicate plates and mixing with 20 ml of cooled molten MHA, which was then allowed to set. The MBC was identified as the lowest concentration plated to show no microbial growth. The assay was performed in triplicate.
Chequerboard assay to assess the antimicrobial activity of CHG in combination with harmaline against Staphylococcus aureus in suspension
The antimicrobial activity of aqueous CHG in combination with harmaline was assessed in a suspension assay by the chequerboard method (Shin and Lim 2004). Serial twofold dilutions of the antimicrobial compounds were prepared in MHB that the final concentrations of both drugs ranged from 1/16 to 4 times the MIC for harmaline and from 1/256 to 4 times the MIC for CHG. 50 μl of each CHG solution was added to the rows of a 96-well microtitre plate in diminishing concentrations, and 50 μl of the harmaline was added to the columns in diminishing concentrations. The wells were then inoculated with 100 μl of S. aureus suspension containing 1 × 105 CFU. Columns 12 served as controls containing MHB and inoculum alone, and antimicrobial compounds separately with the inoculum. After inoculation and agitation, the microplates were incubated in air at 37°C for 24 h, and the MIC of each agent alone and in combination was determined as described previously. To assess the synergistic or antagonistic activity of antimicrobial combinations, the FIC and FIC index (FICI) were determined using the following formulae:
The interpretation of the FICI was as follows: FICI ≤ 0·5, demonstrated synergy; 0·5 < FICI ≤ 4, indicated indifference; FICI > 4, showed antagonism. The assay was performed in duplicate microtitre plates (Karpanen et al. 2008a,b).
Establishment of Staphylococcus aureus biofilms
For each clinically isolated S. aureus, the OD of the overnight suspension was determined at 660 nm and diluted in MHB to obtain the final concentration of 1× 105 CFU ml−1. The ability of clinical S. aureus strains to produce slime was confirmed by culturing the bacteria on Congo Red agar (Freeman et al. 1989). To confirm biofilm production by each test micro-organism was achieved by applying Alcian Blue stain to each of the wells (Shea 1971). Bacterial biofilms were prepared by aliquotting 200 μl of the bacterial suspension diluted in 2% glucose-supplemented MHB containing 1 × 105 CFU ml−1 into the wells 96-well microtitre plates (Karpanen et al. 2008a,b). Microtitre plates were then incubated in air for 48 h at 37°C.
Determination of MICs and MBCs of CHG and Harmaline for Staphylococcus aureus in biofilm
An ultrasonic-plate method was employed to study the MICs and MBCs of CHG and harmaline for S. aureus in biofilm. After incubation, the microtitre plates containing S. aureus biofilms were washed once with sterile PBS to remove any unbound bacteria. Serial double dilutions of antimicrobial agents were prepared in MHB to obtain the required concentrations (CHG, 0·125–128 μg ml−1; Harmaline, 0·5–512 μg ml−1). To triplicate wells, two hundred microlitres of each antimicrobial agent was added to each microtitre plate well in decreasing concentrations across the rows. Columns 12 served as controls containing the biofilm and saline. Following incubation in air at 37°C for 24 h, the antimicrobial agents were removed and the wells were washed again with PBS. PBS (250 μl) was then added to each well, and the plate sonicated at 50 Hz in a water bath for 30 min at room temperature. Biofilms were recovered from the wells by a scrape and wash procedure (Adams et al. 2005), and the entire contents of the well mixed with 20 ml of cooled molten MHA. After 24 h of incubation in air at 37°C for 24 h, the MIC was determined as the lowest concentration to show growth below or equal to that of the control (biofilm in saline). The MBC was identified as the lowest concentration demonstrating no bacterial growth.
Chequerboard assay to assess the antimicrobial activity of CHG in combination with harmaline against Staphylococcus aureus in biofilm
Biofilms were grown in 96-well microtitre plates then gently washed once with PBS to remove any unbound bacteria. Serial twofold dilutions of the antimicrobial compounds were prepared in MHB that the final concentrations of both drugs ranged from 1/16 to 4 times the MIC for harmaline and from 1/256 to 4 times the MIC for CHG. 100 μl of each CHG solution was added to the rows of a 96-well microtitre plate in diminishing concentrations, and 100 μl of the harmaline was added to the columns in diminishing concentrations. Following incubation in air at 37°C for 24 h, the antimicrobial agents were removed and the wells were washed again with PBS. The MIC, and the FIC and FICI values were determined as described previously. The assay was performed in duplicate microtitre plates.
Confocal laser scanning microscopy (CLSM)
Images of biofilm were collected for treatment with harmaline and CHG alone, and combination as well as the growth control. Aliquots (2 ml) of TSB-diluted overnight culture were used to grow biofilm on coverslides in six-well dishes for 24 h after centrifugation. The coverslides were then washed carefully with PBS, moved to a new plate and treated for 24 h with harmaline and CHG alone and combination. The coverslides were washed again with PBS and stained with a LIVE/DEAD BacLight Bacterial Viability kit (Invitrogen Molecular Probes, Eugene, OR, USA) following the manufacturer’s instructions. CLSM images were collected using an Olympus FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan) with a × 60 objective lens. For detection of SYTO 9 (green, alive), we used 488 nm excitation and 520 nm emission filter settings. For PI detection (red, dead), we used the 543 nm excitation and 572 nm emission filter settings. Image analyses and export were performed in a Fluoview ver. 220.127.116.11 (Olympus).
The MICs and MBCs of aqueous CHG and harmaline for Staphylococcus aureus in suspension and biofilm
In our study, 12 clinical isolates of S. aureus and one quality control strain ATCC 29213 were selected. CHG and harmaline demonstrated antimicrobial activity against suspensions and biofilms of S. aureus (Table 1). MICs of CHG and harmaline against micro-organisms grown in suspension ranged from 0·5 to 2 and 32 to 256 μg ml−1, while MBCs of CHG and harmaline were 8–32 and 128–512 μg ml−1, respectively. Of the clinical isolates tested, CHG had biofilm MICs ranging from 8 to 32 μg ml−1, while the biofilm MICs of harmaline ranged from 256 to 512 μg ml−1. CHG showed a 8- to 32-fold increase in bactericidal concentrations than bacteriostatic concentrations against suspensions; however, the planktonic MBC values of harmaline were two to fourfold higher than their MIC values for the same strain. MIC values of CHG and harmaline were 8- to 32-fold and 2- to 8-fold higher for S. aureus growing in biofilm compared with cells in suspension, respectively. Unfortunately, CHG and harmaline both had higher biofilm MBCs values (≥512 μg ml−1).
Table 1. MICs and MBCs of CHG and Harmaline for suspension and biofilm cultures of Staphylococcus aureus
The antimicrobial activity of CHG in combination with harmaline against Staphylococcus aureus in suspension and in biofilm
In the checkerboard assay, to analyse the interaction of combinations of CHG and harmaline against S. aureus in suspension and in biofilm, we used a nonparametric approach FICI, based on the Loewe additivity (LA) theory. The results of antimicrobial activity of CHG combined with harmaline against S. aureus in suspension were shown in Table 2. By the FICI method, nine of the 13 S. aureus strains showed synergistic interactions in suspension, with FICI values ranging from 0·375 to 0·5; four S. aureus strains showed indifference interactions in suspension, with FICI values ranging from 0·563 to 1·25.
Table 2. Antimicrobial activities of CHG combined with Harmaline against Staphylococcus aureus growing in suspension
MIC of CHG (μg ml−1) in combination/alone
FIC of CHG (average)
MIC of harmaline (μg ml−1) in combination/alone
FIC of harmaline (average)
Fractional inhibitory concentration index (average)
The interaction between CHG and harmaline showed synergy in 11 of the 13 S. aureus strains when in biofilm according to the FICI method, with the FICI values ranged from 0·25 to 0·5. Indifference was demonstrated with the combination of CHG and harmaline against S. aureus 3629 and S. aureus 3218, with FICI values of 0·75 and 1·25, respectively (Table 3). We did not find antagonistic interactions between CHG and harmaline.
Table 3. Antimicrobial activities of CHG combined with Harmaline against Staphylococcus aureus growing in biofilm
MIC of CHG (μg ml−1) in combination/alone
FIC of CHG (average)
MIC of harmaline (μg ml−1) in combination/alone
FIC of harmaline (average)
Fractional inhibitory concentration index (average)
CLSM of bacterial cell survival in biofilms exposed to CHG alone and in combination with harmaline
The effect of CHG alone and in combination with harmaline on pre-existing biofilms was also studied by using CLSM (Fig. 2). After treatment for 24 h, control group was chiefly comprised of living bacterial cells (Fig. 2e). Compared to the control group, treatment with 4× MIC of the harmaline killed a significant portion of the bacterial population and reduced the number of bacteria present in the biofilm (Fig. 2c). Similarly, 4× MIC of the CHG killed a portion of the bacterial population, without fractured or chipped cells (Fig. 2a). In contrast, the combination of harmaline with CHG killed the vast majority of the biofilm bacteria, with few survivors at the surface (Fig. 2f).
In this study, we employed the checkerboard microdilution method for an initial analysis of CHG-harmaline interactions. We found that harmaline alone showed relatively weak antistaphylococcal activity against S. aureus strains in suspension and in biofilm, whereas when combined with CHG, it demonstrated synergistic antimicrobial activity against most clinically isolated S. aureus and one standard susceptive S. aureus strain when growing both in suspension and in biofilm. Previous reports have showed eucalyptus oil and CHG have good synergistic ability against S. aureus biofilm with FICI of 0·156–0·375 (Karpanen et al. 2008a,b; Hendry et al. 2009), which was similar to our results. Peganum harmala seed extracts have been frequently reported to possess antibacterial potential through in vitro studies (Arshad et al. 2008). Harmaline and harmine are two major beta-carboline alkaloids in the seed extracts of Peganum harmala (Sobhani et al. 2002). However, there is very limited information about the effect of harmaline on S. aureus in biofilm cultures before our research.
Importantly, the positive interactions in checkerboard microdilution were verified by the CLSM test. CLSM imaging of harmaline-treated biofilms showed not only that biofilm bacteria are effectively killed by harmaline, but also that harmaline at higher concentration (8× MIC) is able to detach biofilms (Fig. 2d). Similarly, high concentration of CHG (16× MIC) could efficiently removed the bacteria from the biofilm; however, it could hardly kill the remaining bacteria (Fig. 2b). Especially, CHG combined harmaline resulted in both bacterial detachment and death.
Biofilms, as has been reported in previous studies (Johansen et al. 1997; Saginur et al. 2006), exhibit increased resistance to antimicrobial agents compared with their planktonic counterparts. This makes biofilms particularly disturbing in clinical settings, while their presence often creates chronic polymer-associated infections (Lindsay and Holden 2004). Results from this study indicate that MICs of CHG and harmaline were 8- to 32-fold and 2- to 8-fold higher for S. aureus strains growing in biofilm compared with cells in suspension, respectively. At the same time, CHG and harmaline had higher biofilm MBCs values (≥512 μg ml−1). Thus, the results of this study concur with previous researches.
In the present studies, synergistic activity was observed for harmaline in combination with CHG against all S. aureus tested grown in suspension with the exception of S. aureus 1987, S. aureus 3800, S. aureus 1980 and S. aureus 3629, no antagonism between harmaline and CHG was observed. Moreover, harmaline caused a two to eightfold decreased in the MICs of CHG against clinically isolated S. aureus. While growing in biofilm, antimicrobial synergy of CHG and harmaline was also observed in 11 of the 13 S. aureus strains, only isolates S. aureus 3629 and S. aureus 3218 showed indifference. When used together, the amount of CHG and harmaline required to achieve the growth inhibition were both reduced significantly. To our knowledge, this is the first report of synergism between harmaline and CHG.
CHG is widely used as a skin antiseptic within the clinical setting (Pratt et al. 2007). On the contrary, subinhibitory concentrations of CHG may increase a biofilm mode of growth of staphylococci (Houari and Di Martino 2007), which may reduce the efficacy of skin antisepsis if we employ levels of antiseptic inappropriately. CHG has hydrophilic and hydrophobic properties, and CHG is thought to act on the plasma membranes (Karpanen et al. 2008a,b). Harmaline, the β-carboline alkaloid, possessed high lipophilic properties (Di Giorgio et al. 2004) that have been shown to exert a wide range of pharmacological properties including inhibition of Na+/Ca2+ exchanger (Suleiman and Reeves 1987), effects on plasma or mitochondria membrane potentials (Di Giorgio et al. 2004), and antioxidant and hydroxyl radical-scavenging properties (Berrougui et al. 2006; Moura et al. 2007), while harmaline also exhibit differential affinities for the NHE (Na+/H+ exchanger) isoforms (Blaustein and Lederer 1999). Furthermore, harmaline can interact with several enzymes and neurotransmitters including topoisomerase I, and monoamine oxidase-A (Sobhani et al. 2002; Herraiz et al. 2010). Recent research reported that the negatively charged extracellular matrix hinders cationic CHG diffusion in the biofilm, which changes the physicochemical properties of the extracellular matrix and its tertiary structure (Hope and Wilson 2004). However, the underlying mechanism of CHG/harmaline synergy is necessary to be explored by further research.
In conclusion, the present study provides evidence that harmaline shows potent synergistic antimicrobial effects when combined with CHG against clinical and standard S. aureus strains grown in planktonic and biofilm cultures, although harmaline shows relatively weak antimicrobial activity when used alone. The synergistic activity between CHG and in harmaline combination may therefore be of benefit in the clinical setting. At the same time, this new finding of combination treatment with CHG and harmaline might provide an alternative approach to overcome resistance to biofilm formation in S. aureus. However, the clinical significance should be evaluated.
Financial supports for this work came from Fund of the State Key Laboratory for Molecular Virology and Genetic Engineering (grant no. 2011003), the National Nature Science Foundation of China (no. 30871889), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) (no. 200801831051), the Fund for Science and Technology Development of the Jilin Province, China (no. 200705233), and the Fundamental Research Funds for the Central Universities.