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ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 1  |  Page : 27-32  

Absence of antibacterial, anti-candida, and anti-dengue activities of a surfactin isolated from Bacillus subtilis


1 Laboratory of Medical Microbiology, Central-West Campus Dona Lindu, Federal University of São João del-Rei, Divinopolis, MG, Brazil
2 Laboratory of Biotechnological Processes and Macromolecules Purification, Central-West Campus Dona Lindu, Federal University of São João del-Rei, Divinopolis, MG, Brazil
3 Laboratory of Pharmacology of Pain and Inflammation, Central-West Campus Dona Lindu, Federal University of São João del-Rei, Divinopolis, MG, Brazil

Date of Web Publication21-Aug-2018

Correspondence Address:
Jaqueline Maria Siqueira Ferreira
Laboratory of Medical Microbiology, Central-West Campus Dona Lindu, Federal University of Sao Joao del-Rei, Sebastiao Goncalves Coelho Street, 400, Divinopolis, Minas Gerais
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpnr.JPNR_11_17

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   Abstract 


Background: Biosurfactants are biological compounds that possess many pharmacological proprieties. Among them, surfactin is one of the most active against pathogens of medical interest, such as fungi, bacteria, and enveloped viruses. Objectives: In this study, we aim to evaluate the antibacterial anti-Candida, and anti-dengue potential of a surfactin biosynthesized by Bacillus subtilis ATCC 6633. In addition, the fractional inhibitory concentration (FIC) was calculated to determine the behavior of this biosurfactant in association with antibacterial and antifungal drugs of clinical use. Materials and Methods: B. subtilis ATCC 6633 culture was maintained on nutrient agar plates, and biosurfactant production was carried out in mineral salt medium with 2% glucose. The isolated and purified surfactin was used for determination of antibacterial and antifungal effects by the broth microdilution method. A checkerboard assay was performed to determine the potential synergic effect of surfactin with gentamicin, penicillin, and nystatin. Finally, the activity against Dengue virus (DENV) was evaluated through quantification of cell viability after viral infection. Results: Although it had low cytotoxicity (CC50 >400 μg/mL), surfactin was inactive against Gram-negative and Gram-positive bacteria, Candida species, and DENV at the highest concentration tested (500 μg/mL). According to FIC values, none of the antimicrobials tested showed a synergistic association with surfactin. Conclusions: Surfactin produced by B. subtilis ATCC 6633 is not a promising antimicrobial agent, and its combination with clinically available antibiotics does not lead to a synergic effect.

Keywords: Antimicrobial activity, broth microdilution assay, cellular viability, surfactin, synergic effect


How to cite this article:
Lima WG, Parreira AG, Alves Nascimento LA, Leonel CA, Andrade JT, Campos Palumbo JM, Soares AC, Granjeiro PA, Siqueira Ferreira JM. Absence of antibacterial, anti-candida, and anti-dengue activities of a surfactin isolated from Bacillus subtilis. J Pharm Negative Results 2018;9:27-32

How to cite this URL:
Lima WG, Parreira AG, Alves Nascimento LA, Leonel CA, Andrade JT, Campos Palumbo JM, Soares AC, Granjeiro PA, Siqueira Ferreira JM. Absence of antibacterial, anti-candida, and anti-dengue activities of a surfactin isolated from Bacillus subtilis. J Pharm Negative Results [serial online] 2018 [cited 2018 Dec 18];9:27-32. Available from: http://www.pnrjournal.com/text.asp?2018/9/1/27/239505




   Introduction Top


Biosurfactants are amphipathic molecules synthesized by microorganisms with capacity of modulating the growth of other organisms.[1] Therefore, medical applications of these compounds as antimicrobial agents have been studied extensively in recent decades. Among these biomolecules, surfactin is noted for its high antibacterial, antifungal, and antiviral activities.[3],[4],[5],[6] Characterized by an anionic cyclic lipoheptapeptide, surfactin contains two negative charges (on Glu and Asp) linked to a β-hydroxy fatty acid of 12–16 carbons, which is directly associated with the type of antimicrobial effect and its potency.[1],[2] In nature, it is produced as a result of secondary metabolism of Bacillus spp. species and plays an important role in chemical ecology of this bacterium.[2]

The rapid emergence of infections caused by multidrug-resistant (MDR) bacteria is limiting the available therapeutic options.[7] Opposing the emergence of these infections, we can see a clear negligence by major pharmaceutical companies on investing in research and development for new antibacterial agents.[8] In this context, several studies point out that surfactin, by inducing osmotic lysis through damage in the cell membranes of these pathogens, may be a potential candidate for fighting antibacterial resistance.[9] In fact, there are considerable advantages of an antibiotic targeting the cytoplasmic membrane. Clinical drugs with action in the membrane, such as daptomycin and polymyxin, possess the highest activity against MDR and nonmultiplying bacteria, showing rapid and potent microbicidal action.[10],[11] Therefore, resistance mechanisms against an antibiotic that targets the membrane are more difficult to establish, considering that modifications in this cellular structure may reduce bacterial fitness. Interestingly, it has been shown that the deleterious effects of compounds with action on the cell membrane may favor the incorporation of other active agents.[12] These facts highlight the investigation of the synergistic potential of biosurfactants when associated with different antibiotics.

Furthermore, many studies have described the antifungal activity of different lipopeptides isolated from bacteria of the genus Bacillus. Surfactin showed activity mainly against filamentous fungi such as Aspergillus flavus,[6]Phomopsis phaseoli,[13]Rhizoctonia spp.,[14] and Pythium spp.[14] However, most studies evaluated the biological potential of surfactin against phytopathogenic fungi,[13],[14] presenting little evidence against species of medical interest. In this context, yeasts of the genus Candida are the most important etiological agents in the epidemiology of mycoses, appearing in the last decades among the main causes of nosocomial infections.[15] Despite main Candida species isolated as pathogens are sensitive to conventional therapies, strains resistant to first- and second-generation antifungal drugs are becoming more frequent.[16] This resistance has been associated with therapeutic failure and a consequent increase in morbidity and mortality rates. Such facts make the search for new anti-Candida agents necessary.

In addition to its effects against bacteria and fungi, surfactin has demonstrated positive results in antiviral assays, especially against enveloped viruses of the family Togaviridae, Herpesviridae, Rhabdoviridae, and Retroviridae.[4],[5] Dengue virus (DENV) is an enveloped arbovirus belonging to the Flaviviridae family and the Flavivirus genus, which affects approximately 400 million people per year.[17] Severe dengue fever, although it represents fewer than 1% of cases, kills more than 20,000 people yearly, and only palliative measures are available for these cases.[17] Thus, the antiviral potential of surfactin against enveloped viruses aligned with the clinical importance of dengue motivates studies to evaluate the antiviral potential of this biosurfactant against DENV.

Due to the increase of bacterial and fungal resistance to conventional therapies and the medical importance of arboviruses such as dengue, it is necessary to search for new effective therapeutic agents directed against these pathogens. Thus, this study aimed to evaluate the antibacterial, anti- Candida, and anti-dengue activities of a surfactin obtained from Bacillus subtilis ATCC 6633. In addition, synergistic potential of the interaction of this biosurfactant with clinically available antimicrobial agents was also determined.


   Materials and Methods Top


Surfactin production

Media and cultivation conditions

B. subtilis ATCC 6633 was employed to surfactant production. Initially, a culture was grown in nutrient broth (Himedia, Brazil) for 24 h on a rotary shaker at 200 rpm and 30°C. This was used to prepare the inoculum which was adjusted to optical density (OD) of 0.85 at 600 nm (approximately 106 UFC/mL). In the subsequent step, 2% (w/v) of glucose in mineral salt medium was used for biosurfactant production. The basic mineral salt medium was composed of NH4 NO3 (0.05 M), KH2 PO4 (0.03 M), Na2 HPO4 (0.04 M), MgSO4(8.0 × 10−4 M), CaCl2 (7.0 × 10−6 M), FeSO4 (4.0 × 10−6 M), yeast extract (5 g/L), and peptone (0.7 g/L). After addition of the inoculum, the flasks containing the medium were incubated on a rotary shaker at 200 rpm for 5 days. Reduction in surface tension was observed every day for 5 days. Surface tension was measured by the Du Noüy ring method using a surface tension balance – interfacial tensiometer with a 4 cm platinum ring.[18]

Surfactin isolation, purification, and identification

Surfactants were collected by adjusting the pH to 2.0 with 6.0 M HCl, overnight incubation at 4°C, and centrifugation at 9000 g for 10 min. Supernatants were discarded and the tubes incubated at 45°C until particulate material were visible. Surfactants were subsequently purified by extraction with organic solvent (dichloromethane) and water (1:1) using a rotary evaporator. The solid residue was triturated, weighed, and stored frozen for subsequent analysis. The samples were dispersed over the surface of KBr crystals and subjected to Fourier transform infrared spectroscopy (FT-IR) analysis (Perkin Elmer Spectrum One model), generating spectra between 500 and 4000/cm at a resolution of 4/cm.[19] Spectra were compared with that of the surfactin standard (Sigma Chemical Co. St. Louis, MO, USA). The concentration of surfactin was determined by HPLC using a Shimadzu gas chromatograph 10Ai (Osaka, Japan) coupled to an SPD-10Ai UV-VIS detector and a C18 reverse-phase column (Merck, Germany).[19] The system was operated in isocratic mode with 3.8 mM trifluoroacetic acid (Sigma, USA) and acetonitrile (Vetec Chemical, Rio de Janeiro, RJ, Brazil) (1:4 v/v) injected at 1 mL/min. Samples (10 μL) were injected and the absorbance of the effluent monitored at 205 nm. The concentration of surfactants was calculated using a standard curve generated with known concentrations of surfactin (Sigma Chemical Co., St. Louis, MO, USA). Surfactant extracts used for HPLC analysis were obtained by acid precipitation as described above.

Microorganisms

The microorganisms employed in the study were originated from the American Type Culture Collection (ATCC) and were kindly provided by the Reference Microorganisms Laboratory of the Oswaldo Cruz Foundation (FIOCRUZ, Brazil). Antibacterial tests were conducted with twelve strains, which included (i) seven glucose-fermenting Gram-negative bacilli (Enterobacteriaceae): Klebsiella pneumoniae ATCC 43816, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 23355, Serratia marcescens ATCC 14756, Salmonella enterica subsp. enterica serovar Typhi ATCC 19430, Shigella flexneri ATCC 12022, and Proteus mirabilis ATCC 15290; (ii) two non-glucose-fermenting Gram-negative bacilli: Pseudomonas aeruginosa ATCC 15442 and Acinetobacter baumannii ATCC 19606; and (iii) three Gram-positive bacteria: Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 15313, and Streptococcus agalactiae ATCC 13813. For antifungal evaluation, four Candida species were used: Candida albicans ATCC 14053, Candida krusei ATCC 34135, Candida tropicalis ATCC 28707, and Candida glabrata ATCC 2001.

Antibacterial assay

To evaluate the antibacterial activity against Gram-positive and Gram-negative bacteria, the broth microdilution method described by document M07-A9 of the Clinical and Laboratory Standards Institute (CLSI)[20] was employed, with minor modifications. Surfactin solution was prepared in sterile water by sonication and subsequently diluted in microplates using Mueller-Hinton broth (Kasvi, Brazil) to a concentration range of 1–500 μg/mL. Bacterial inoculum (100 μL) at 105 CFU/mL, prepared from an overnight culture in the appropriate medium, was added to each well and the plates were incubated at 37 ± 2°C for 18 h. The minimum inhibitory concentration (MIC) was determined as the lowest concentration where no visible growth was observed. For positive controls, ciprofloxacin (Multilab, Brazil) was used in glucose-fermenting Gram-negative bacilli, chloramphenicol (Sigma-Aldrich, Brazil) in non-glucose-fermenting Gram-negative bacilli, and amoxicillin (Germed, Brazil) in Gram-positive species, with both antibiotics in the same surfactin concentration range (1–500 μg/mL). The assay was performed in triplicate, and the results were expressed as the mean of three experiments.

Antifungal assay

To evaluate antifungal activity against Candida spp., the broth microdilution method described by document M27-A2 of CLSI [21] was employed, with some modifications. Surfactin solution was prepared by sonication in sterile water and subsequently diluted in microplates containing Sabouraud dextrose broth (Acumedia, Brazil) in the concentration range of 1–500 μg/mL. Subsequently, 100 μL of Candida inoculum with 103 CFU/mL, prepared from a 48 h culture in the appropriate medium, was added to each well. The plates were incubated at 35 ± 2°C for 48 h, and the MIC was determined visually as described above. In positive controls, ketoconazole (Pharma Nostra, Brazil) and nystatin (Pharma Nostra, Brazil) were used, with both antifungals in the same concentration range as that of surfactin (1–500 μg/mL). The assay was performed in triplicate and the results were expressed as the mean of three experiments.

Checkerboard assay

Synergistic effects were assessed by the checkerboard test.[22] Samples of surfactin prediluted in water were serially diluted in concentrations ranging from 3.9 to 250 μg/mL. Subsequently, antibiotic solutions in the same concentration range were combined in a 1:1 ratio to evaluate the antimicrobial effects resulting from the interaction of antibiotics with surfactin. Gentamicin (Inlab, Brazil) was employed in association with surfactin against Gram-negatives (K. pneumoniae, E. coli, and A. baumannii), amoxicillin (Germed, Brazil) against Gram-positives (S. aureus and L. monocytogenes), and nystatin (Pharma Nostra, Brazil) was combined with surfactin against Candida species (C. albicans, C. tropicalis, and C. glabrata).

The fractional inhibitory concentration index (FIC index) is the sum of the FICs of each of the drugs, which in turn is defined as the MIC of each drug when it is used in combination, divided by the MIC of the drug when it is used alone. An FIC index of 0.5 or less has traditionally been defined as synergism. An FIC index between 0.5 and 2.0 is defined as additive and between 2.0 and 4.0 as indifferent. An FIC index more than 4.0 is defined as antagonism.[22] All experiments were repeated independently three times.

Cytotoxicity assay

The concentration that was cytotoxic to 50% of cells in culture (CC50) was evaluated by the colorimetric method 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as previously described.[23] Briefly, baby hamster kidney cells (BHK-21) (ATCC CCL-10, USA) were seeded into each well of 96 well plates (4 × 104 cells/well) in Dulbecco's Modified Eagle's Minimum Medium (DMEM) (Cultilab, Brazil) with 5% fetal bovine serum (FBS) and 0.3% penicillin-streptomycin-amphotericin solution (10,000 U/mL + 10 mg/mL + 2 mg/mL, Sigma-Aldrich, USA). Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. The next day, different concentrations of the compounds were added (3.15–400 mg/mL) to each well and incubated for 48 h at 37°C. After this period, MTT solution (5 mg/mL in phosphate-buffered solution [PBS]) (Sigma-Aldrich, Brazil) was added to each well. The OD at 540 nm was determined on a microtiter plate reader. Cell viability was estimated based on the reduction of the MTT salt from the concentration-effect curves by linear regression analysis.[23] All assays were carried out in triplicate, and the results were expressed as the mean of three independent experiments.

Antiviral assay

Antiviral assays were performed using BHK-21, which were seeded into each well of a 96 well flat bottom plate (4 × 104 cells/well), in DMEM supplemented with 5% FSB and 0.3% penicillin-streptomycin-amphotericin solution (10,000 U/mL + 10 mg/mL + 2 mg/mL, Sigma-Aldrich, USA).[24],[25] After a confluent monolayer formed, the medium was replaced by 100 μL of DMEM (1.0 μg/mL of trypsin, without FBS) with DENV-2 at a multiplicity of infection of 0.1.[24] Immediately, 100 μl of DMEM (1.0 μg/mL of trypsin, without FBS) was added, which contained serial dilutions of surfactin (3.125-200 μg/mL). The starting concentration was the previously determined by CC50. The plate was incubated for 5 days at 37°C in an atmosphere of 5.0% CO2 with high humidity. After 5 days, MTT solution (5 mg/mL in 1x PBS) was added.[26] The effective antiviral concentration (EC50) was expressed as the concentration that protected 50% of the treated cells. The percentage of viral protection was calculated as described by Hidari et al.[24] DENV-2 employed in this study was kindly donated by Erna Geessien Kroon of Universidade Federal de Minas Gerais, Brazil, and used for antiviral assays.


   Results and Discussion Top


Natural products have already been highlighted as the main source of agents with antimicrobial activity, and secondary metabolites of species of the genus Bacillus spp. are featured for this effect.[1],[27],[28] Polymyxin B and colistin, obtained from Bacillus polymyxa, are some examples of highly successful compounds in the medical clinic produced by bacteria of this genus.[11],[29] Therefore, studies aiming to explore the biological potential of other classes of Bacillus metabolites should be encouraged. In this context, biosurfactants such as surfactin, which have already shown potent antibacterial, antifungal, and antiviral activity in previous studies, stand out.[3],[4],[5],[6] Taking this into account, the present work was conducted to evaluate the antibacterial, anti-Candida, and anti-dengue effects of a surfactin isolated from B. subtilis ATCC 6633.

The production of biosurfactants was carried out by B. subtilis ATCC 6633 in mineral salt medium with 2% glucose and confirmed by surface tension readings. The surface tension of the culture was 30.2 mN/m, while the surface tension of the control (tap water) was 60.0 mN/m. The FT-IR spectra of samples were similar to those of the surfactin standard [Figure 1] and those described by other authors (e.g., Joshi et al., 2008), which confirmed the production of surfactin. The surfactin concentration was 23.9 mg/L, and this value was significantly different from the control value (P < 0.05, Student's t-test). Surfactin concentration was obtained by the sum of the areas of the six characteristic surfactin peaks [Figure 2].
Figure 1: Fourier-transform infrared spectroscopy spectra of surfactant obtained by Bacillus subtilis ATCC 6633 (A) and isolate control (B and C) spectrum of surfactin (Sigma, USA)

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Figure 2: Chromatograms of surfactin produced by Bacillus subtilis ATCC 6633 (A), isolate control (B), and spectrum of surfactin (C). The chromatograms illustrate the results obtained for one of three replicates of each case

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Biological tests have shown that surfactin is inactive against different species of pathogenic bacteria [Table 1]. For the Enterobacteriaceae (K. pneumoniae, E. coli, E. cloacae, S. marcescens, S. flexneri, P. mirabilis, and S. enterica) evaluated in this study, ciprofloxacin was highly active (MIC range: <0.001–0.016 μg/mL). Surfactin in the concentration range employed (1–500 μg/mL) did not inhibit the growth of any of these species. Similarly, growth of the non-glucose-fermenting Gram-negative (A. baumannii and P. aeruginosa) and Gram-positive (S. aureus, S. agalactiae, and L. monocytogenes) bacteria tested was not noticeably inhibited by surfactin at the highest concentration (500 μg/mL) evaluated. However, the chloramphenicol and amoxicillin showed high activity, with MIC ranges of 3.9–250 μg/mL and 0.2–>250 μg/mL, respectively. These results show that surfactin produced by B. subtilis ATCC 6633 does not show significant antibacterial activity under the experimental conditions employed. In agreement with our work, Loiseau et al. (2015)[3] revealed that a surfactin produced by B. subtilis AM1 was inactive (MIC >256 μg/mL) against the Gram-positive bacteria Enterococcus faecalis, L. monocytogenes, S. aureus, Listeria ivanovii and B. subtilis, as well as against the Gram-negative K. pneumoniae, P. mirabilis, E. cloacae, P. aeruginosa, Aeromonas hydrophila, S. enterica, Flavobacterium breve, E. coli, and Pseudomonas syringae. In contrast, the same study showed a potent anti-Legionella[3] activity with this lipopeptide, suggesting that its antibacterial action might be genus-specific.
Table 1: Minimum inhibitory concentration of surfactin against pathogenic bacteria and fungi

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MICs results for anti-Candida activity are summarized in [Table 1]. Compared with ketoconazole (MIC range 7.8–125 μg/mL) and nystatin (1.95–7.8 μg/mL), Candida species were insensitive to the fungistatic effects of surfactin. In accordance with this study, C14 and C15 surfactin isolated from different strains of Bacillus spp. were inactive against C. albicans (MIC >100 μg/mL).[25] Despite the inactivity of surfactin, we evaluated its synergistic potential when combined with clinical antimicrobials [Table 2]. Another studies have shown that although inactive against C. albicans when tested alone, surfactin showed a potent synergistic effect when combined with ketoconazole; in some cases, the MIC may be reduced by more than 10-fold.[30] However, we did not find any evidence of a synergistic effect when surfactin was combined with an aminoglycoside (gentamicin) against E. coli (additive effect), K. pneumoniae (indifferent effect), and A. baumannii (indifferent effect); with a beta-lactam (amoxicillin) against S. aureus (additive effect), S. agalactiae (additive effect), and L. monocytogenes (indifferent effect); and with the antifungal nystatin against C. albicans (indifferent effect), C. glabrata (indifferent effect), and C. tropicalis (additive effect) [Table 2].
Table 2: Effect of the combination of surfactin with penicillin, gentamicin, or nystatin against pathogenic microorganisms

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It is well known that surfactin can induce structural changes in the envelope of several viruses, resulting in virucidal activity.[4],[5] However, no previous study has evaluated the antiviral potential of this biosurfactant against Flavivirus, an important genus of enveloped virus. Therefore, we selected DENV because of the absence of available therapies for its treatment as well as its medical importance. In the antiviral assay, the EC50 was higher than the highest concentration tested (100 μg/mL), revealing that this compound had no anti-dengue activity. The cytopathic effect, for example, was pronounced even in cells infected with DENV-2 that received surfactin (data not shown).

Despite the absence of biological activity against the pathogens evaluated in this study, surfactin showed low cytotoxicity. The CC50 was higher than the highest concentration tested (400 μg/mL) against BHK-21 renal cells, suggesting low nephrotoxicity.


   Conclusion Top


We established the lack of biologic activity of a surfactin produced by B. subtilis ATCC 6633 against several species of bacterial and Candida. Moreover, we showed for the first time the inactivity of surfactin against DENV. Thus, this work removes the need for any future study of these biosurfactants against the species employed, as well as the evaluation of its synergic potential with aminoglycosides (gentamicin), beta-lactams (amoxicillin), and nystatin.

Financial support and sponsorship

This study was financially supported by Brazilian Council for Development of Science and Technology (CNPq) and Minas Gerais Foundation for the Support of Science (FAPEMIG). W.G.L. thank FAPEMIG for having received a postgraduate scholarship.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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