Antonie van Leeuwenhoek DOI 10.1007/s10482-014-0324-z
ORIGINAL PAPER
Usnic acid, a lichen secondary metabolite inhibits Group A Streptococcus biofilms Paramasivam Nithyanand • Raja Mohmed Beema Shafreen Subramanian Muthamil • Shunmugiah Karutha Pandian
•
Received: 30 June 2014 / Accepted: 29 October 2014 Ó Springer International Publishing Switzerland 2014
Abstract Group A Streptococci (GAS) are involved in a number of life threatening diseases and biofilm formation by these pathogens are considered as an important virulence determinant as it mediates antibiotic resistance among them. In the present study, we have explored the ability of (?)-usnic acid, a lichen secondary metabolite, as an antibiofilm agent against four serotypes of Streptococcus pyogenes causing pharyngitis. Usnic acid inhibited the biofilms of M serotypes M56, st38, M89 efficiently and the biofilm of M74 to a lesser extent. Confocal imaging of the treated samples showed that usnic acid reduced the biomass of the biofilms when compared to that of the control. Fourier Transfer Infrared (FT-IR) spectroscopy indicated that usnic acid reduced the cellular components (proteins and fatty acids) of the biofilms. Interestingly, the FT-IR spectrum further revealed that
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0324-z) contains supplementary material, which is available to authorized users. P. Nithyanand (&) School of Chemical & Biotechnology, Centre for Research on Infectious Diseases (CRID), SASTRA University, Tirumalaisamudram, Thanjavur 613401, Tamil Nadu, India e-mail: [email protected] R. M. Beema Shafreen S. Muthamil S. Karutha Pandian Department of Biotechnology, Alagappa University, Science campus, Karaikudi 630 004, Tamil Nadu, India
usnic acid probably acted upon the fatty acids of the biofilms as evident from the disappearance of a peak at 2,455–2,100 cm-1 when compared to the control only in serotypes M56, st38 and M89 but not in M74. The present study shows, for the first time, that usnic acid can act as an effective antibiofilm agent against GAS. Keywords S. pyogenes Biofilms Usnic acid Lichen secondary metabolite GAS
Introduction Streptococcus pyogenes (Group A Streptococcus or GAS) has established itself as a devastating human pathogen as it is responsible for causing a number of complications ranging from superficial infections to several deleterious diseases such as pharyngitis, impetigo and necrotizing fasciitis, and can also lead to rheumatic heart disease (Cunningham 2000). Worldwide there has been a resurgence in rheumatic heart disease (Menon et al. 2004) and in India alone the prevalence of rheumatic fever and rheumatic heart disease varies from 0.3 to 5.4 per 1,000 children (Padmavati 2001). Rheumatic fever is often a sequelae to pharyngitis and the severity of the disease is more prevalent among children from rural backgrounds (Menon et al. 2004). Of late, GAS biofilms have received more attention and it is increasingly accepted that biofilms may play an important role in the
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pathogenesis of S. pyogenes (Lembke et al. 2006; Thenmozhi et al. 2011). Biofilms of S. pyogenes are formed as a result of an orchestrated process called quorum sensing wherein certain peptide signals induce the quorum sensing pathways of S. pyogenes to enhance biofilm formation (Chang et al. 2011). This phenomenon of biofilm formation by pathogens is considered as a strategy to adapt to the changing selective pressures (for example, due to antibiotic administration) of the host. In addition, the formation of a thick exopolysaccharide (EPS) layer and a mesh work of extracellular DNA in the biofilm can prevent antimicrobial agents from reaching the bacterial cell envelope (Pammi et al. 2013). The disturbing observation that one in three tonsillectomies can be colonized with GAS biofilms (Roberts et al. 2012) in itself showcases the deleterious affects of GAS biofilms. Like other biofilms of pathogens, GAS biofilms also have been clinically related to antibiotic treatment failure (Baldassarri et al. 2006) which has prompted several researchers to explore various natural resources such as medicinal plants (Limsuwan et al. 2008; Samoilova et al. 2014), plant derived flavanols (Green et al. 2012), manuka honey (Maddocks et al. 2012), mangrove plants (Musthafa et al. 2013), marine bacteria (Thenmozhi et al. 2009; Nithyanand et al. 2010) and phage encoded endolysins (Shen et al. 2013) as antibiofilm agents against GAS biofilms. Traditionally, lichens have been widely used in folk medicine to treat several bacterial and fungal ailments (Ingo`lfsdo`ttir 2002) and the secondary metabolites of lichens (SMLs) have proven to possess several important pharmacological properties such as antitumour and anti-bacterial activities (Grube et al. 2009; Backorova et al. 2012). These metabolites arise from the secondary metabolism of the fungal component that shares a symbiotic association with lichens along with its algal or cyanobacterial counterpart (Grube et al. 2009). Predominantly, lichens are found in moist areas near an aquatic environment and the role of SMLs is envisaged to protect these lichens from any invading pathogens (Pompilio et al. 2013). Of all the SMLs, usnic acid, which is a component of the polyketide pathway in several lichens (Pompilio et al. 2013), has been the most explored for its antibiofilm ability and it has shown to possess a broad spectrum activity against the biofilms of clinical and environmental isolates. Usnic acid has shown to disrupt
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mature Staphylococcus aureus biofilms isolated from cystic fibrosis patients (Pompilio et al. 2013) and it also inhibited S. aureus biofilms formed on various polymer surface-based medical implants and bone cement coupons (Francolini et al. 2004; Kim et al. 2011). Further, usnic acid also prevented the initial attachment of certain marine bacteria involved in biofouling (Salta et al. 2013) and is has also been shown to interfere in the quorum sensing mechanism of Pseudomonas aeruginosa (Francolini et al. 2004). Although usnic acid has shown to exhibit antibiofilm activity against S. aureus, there are no reports regarding its antibiofilm activity against S. pyogenes involved in any kind of disease. The present study shows, for the first time, the antibiofilm activity of (?)-usnic acid against various biofilm forming serotypes of S. pyogenes isolated from children with pharyngitis (Thenmozhi et al. 2011).
Materials and methods M serotypes of S. pyogenes and usnic acid All S. pyogenes strains used in this study were isolated from throat swabs collected from children of the age group 5–15 years with pharyngitis (Thenmozhi et al. 2011). Four strains namely SP5 (M serotype M56), SP7 (M serotype st38), SP9 (M serotype M89) and SP31 (M serotype M74), which formed substantial biofilms were selected for this study (Thenmozhi et al. 2011) and all the isolates were grown in Todd Hewitt Broth (THB) (Himedia Laboratories, India). (?)Usnic acid was purchased from Sigma Aldrich (Product no. 329967). Bactericidal activity of usnic acid Anti-bacterial activity of usnic acid against various strains of S. pyogenes was tested by agar well diffusion assay in Mueller–Hinton Agar (MHA) (Himedia Laboratories, India). Briefly, overnight cultures of the S. pyogenes (1 9 106 CFU/ml) were uniformly spread over the surface of the agar plate. Usnic acid was dissolved in DMSO (1 mg/ml). Then 50–200 lg/ ml of usnic acid was loaded into the wells and the plates were incubated at 37 °C and the zone of inhibition was measured after 24 h. DMSO alone was used as control. The Minimal Inhibitory concentration
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(MIC) of usnic acid was performed as per CLSI 2006 guidelines (Shafreen et al. 2011). Antibiofilm activity of usnic acid Usnic acid with concentrations ranging from 10–200 lg/ml was added in THB containing bacterial suspensions of 106 CFU/ml. The plates were incubated for 24 h at 37 °C. After incubation, biofilms were stained with 0.4 % crystal violet. The lowest concentration of usnic acid that showed visible biofilm inhibition was determined as the biofilm inhibitory concentration (BIC) for each serotype (Nithyanand et al. 2010). DMSO alone was used as control. Aggregation assay The four serotypes of S. pyogenes strains were grown overnight in THB. The bacteria were harvested by centrifugation at 4,0009g for 10 min, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS to an optical density at 600 nm (OD600) of approximately 0.6. Nine hundred microliters of bacterial suspension and usnic acid at the BIC of each serotype were mixed in a test tube, vortexed, and transferred to cuvettes. Bacterial suspension in PBS and without the addition of usnic acid served as a control. After equilibrating the cuvettes for 5 min at room temperature, the OD600 of the samples was recorded at 10 min intervals at 37 °C for 120 min in a spectrophotometer. Percent aggregation (percent decrease in OD600) was calculated as (OD600 at 0 min - OD600 at 120 min)/(OD600 at 0 min) 9 100. All assays were performed in triplicates (Ahn et al. 2008). Confocal laser scanning microscopy (CLSM) CLSM was used to determine the three-dimensional architecture, thickness and morphology of biofilms formed by all four strains of S. pyogenes with and without the addition of usnic acid at their BIC as described by Thenmozhi et al. (2011). DMSO alone was used as control. Briefly, the biofilms of the four S. pyogenes strains were grown on glass slides and stained with 0.02 % acridine orange (Sigma, St Louis, USA) for 5 min at room temperature in the dark. The rinsed and stained slides were observed under a CLSM microsope (Zeiss LSM710 meta, Germany). The
488 nm line of an Argon laser was used for excitation. Biofilm images were observed with a long-distance 639 objective. Images were captured and processed by using Zeiss LSM Image Examiner Version 4.2.0.121. Features such as total biomass, maximum thickness and surface coverage area were assessed using COMSTAT software (kindly gifted by Dr. Claus Sternberg, Technical University of Denmark) (Heydron et al. 2000). Usnic acid was also further evaluated for its potential to disrupt mature biofilms. Biofilms of S. pyogenes were allowed to develop on cover glasses for 12 h as mentioned above. The mature biofilms were then treated with usnic acid at the BIC of each strain for 5 h and observed under a light microscope after crystal violet staining. Fourier transfer infrared (FT-IR) analysis of S. pyogenes biofilms The BIC of usnic acid was added to cell suspensions of 106 CFU/ml of the S. pyogenes strains and incubated overnight. DMSO alone was used as control. Both treated and the control cells were washed three times with MilliQ water. Twenty microliters of each bacterial sample was applied to KBr coated disks and analyzed by FT-IR (NicoletTM iSTM5, Thermo Scientific, U.S.A) spectroscopy. Sixty four scans were taken with 4 cm-1 resolution. All IR spectra were obtained over the range of 4,000–400 cm-1 and analyzed using OMNIC software as specified by the manufacturer (NicoletTM iSTM5, Thermo Scientific, U.S.A). Statistical analysis Statistical comparisons among control and the treated samples were performed by using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). All the experiments were performed in triplicates and the comparative statistical difference was performed using independent t test (where p was \0.05).
Results Bactericidal activity of usnic acid Among various concentrations of usnic acid tested, only higher concentrations (150–200 lg/ml) exhibited anti-bacterial activity against all of the strains of
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Fig. 1 Percentage inhibition of biofilm formation of usnic acid at its biofilm inhibitory concentration (BIC) against various strains of S. pyogenes. a Sp 5 b Sp 7 c Sp 9 and d Sp 31. Mean values of triplicate independent experiments and SD are shown.
Dunnett’s test demonstrates significant difference between the tests and the control (P \ 0.05). Asterisk indicates P \ 0.0001. All the P values are calculated with reference to the lowest concentration used in each experiment
S. pyogenes used in this study, with inhibition zones of 16–18 mm (Fig. S1a–d).
demonstrated against five additional M serotypes of S. pyogenes namely ES199, SF370, SP11, SP24, SP30. From the results it was observed that 30 lg/ml of usnic acid showed maximum antibiofilm activity against all the serotypes (Fig. S2a, b).
Biofilm inhibitory potential of usnic acid Various concentrations of usnic acid (from 10 to 70 lg/ ml) showed varying levels of biofilm inhibitory activity against all four strains of S. pyogenes. About 61 % of biofilm inhibition was noticed for strain SP5 at a very low concentration of 10 lg/ml. An increase in concentration resulted in reduced antibiofilm activity (Fig. 1a). However, higher concentrations of usnic acid ranging from 55–70 lg/ml were required to inhibit the biofilms of strains SP7, SP9 and SP31 (Fig. 1b–d). At 55 lg/ml, about 31 and 48 % of inhibition was noticed in strains SP7 and SP9 respectively. From the obtained results it was observed that usnic acid showed very low biofilm inhibitory activity (15 %) against the biofilm of strain SP31. The maximum biofilm inhibition was observed at concentrations ranging from 55–70 lg/ml (Fig. 1). The antibiofilm activity of usnic acid was also
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Usnic acid prevents bacterial aggregation The aggregation inhibitory potential of usnic acid was performed with 10 lg/ml usnic acid. From the results it was observed that the inhibition potential varied for each strain. On an average, usnic acid was able to prevent aggregation for up to 60 min for all the strains. For strains SP9 and SP31, usnic acid was able to completely inhibit aggregation as there was only minimal aggregation (SP9) or no aggregation (SP31) for the maximum time duration of 105 min (Fig. 2). CLSM imaging of S. pyogenes biofilms The antibiofilm activity at the BIC of usnic acid of each strain was observed using CLSM. Pronounced
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Fig. 2 Prevention of cell aggregation by usnic acid (10 lg/ml) against various strains of S. pyogenes. Control—aggregation of S. pyogenes cells without the addition of usnic acid, US—aggregation of S. pyogenes cells after the addition of usnic acid
antibiofilm activity was observed for all the four strains of S. pyogenes when compared with the control as there was a decrease in the surface area of the biofilms treated with usnic acid (Fig. 3a–d). CLSM provides three dimensional and non-invasive images of biofilms and the COMSTAT software helps in quantifying the biofilms of the generated three dimensional images (Heydorn et al. 2000). COMSTAT analysis parameters showed that there was a notable decrease in the biomass of the biofilms (directly proportional to biofilm thickness) of all the four strains of S. pyogenes when treated with usnic acid (Table 1). As expected, there was an increase in the surface to bio-volume ratio of the treated sample when compared to its control (Table 1). Usnic acid was also found to be effective on matured biofilms of SP5, SP7, SP9 and SP31. The light microscopic images indicated major disruption in the biofilm architecture of all the four serotypes (Fig. 4).
1,700–1,600 cm-1, which shows amide linkage from proteins and peptides; and 1,500–1,000 cm-1, which indicates mixed signals of proteins and fatty acids (Gowrishankar et al. 2012). In addition to the above mentioned variations, a very interesting observation was noted in the FT-IR spectrum in the region of 2,455–2,100 cm-1 (represented by dashed lines) (Fig. 5a–d). There was a reduction in the intensity of the peak in the treated cells of strain of SP5 (Fig. 5a). The same peak was totally absent in treated spectra of strains SP7 and SP9 (Fig. 5b–c), but was not altered in the strain SP31 spectrum (Fig. 4d). We interpret this data as showing that this peak corresponds to the fatty acids of the biofilm and that usnic acid acted on these fatty acids present in the biofilm. Overall, the FT-IR profile of the treated cells showed alterations in the regions of mixed protein signals, lipids, fatty acids and showed less deviations in the region corresponding to the peptide bonds in proteins (amide I and II). Cell dehydration was observed in almost all the strains treated with usnic acid.
FT-IR analysis of S. pyogenes biofilms The FT-IR spectra revealed variations between the control cells and cells treated with usnic acid (Fig. 5a– d). On comparison of the FT-IR spectra, maximum variations were observed in the regions of 3,700–3,100 cm-1 indicating hydration of bacterial cells; the 3,050–2,750 cm-1 region, which indicates the fatty acids in bacterial cell membrane;
Discussion The emergence of drug resistance among GAS biofilms and the surge of biofilm forming fluoroquinolone and macrolide resistant S. pyogenes strains (Balaji et al. 2013) of late, has driven the need to explore alternative sources for antibiofilm agents
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Antonie van Leeuwenhoek Fig. 3 Confocal Laser Scanning Microscopic (CLSM) image showing the antibiofilm activity of usnic aicd at their BICs against various serotypes of S. pyogenes. a Sp 5 control; b Sp 5 treated (10 lg/ml); c Sp 7 control; d Sp 7 treated (55 lg/ml); e Sp 9 control; f Sp 9 treated (55 lg/ml); g Sp 31 control; h Sp 31 treated (55 lg/ml)
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Antonie van Leeuwenhoek Table 1 COMSTAT analysis of S. pyogenes strains COMSTAT PARAMETERS
SP5
SP5_USa
SP7
SP7_USa
SP9
SP9_USa
SP31
SP31_USa
Biomass (lm3/lm2)
68.83
19.85b
47.47
14.52b
37.93
8.42b
32.95
17.22b
66.45
b
35.83
b
31.12
16.26b
Maximum thickness (lm) Surface to biovolume ratio (lm2/lm3)
0.0192
18.98
0.0527
45.11 0.0234
13.7
0.0712
a
Indicates strains treated with usnic acid at their BIC
b
Statistical significance (p \ 0.05) when compared with the untreated control
against GAS. In the present study, we have explored the ability of usnic acid to act as an antibiofilm agent against GAS biofilms. Based on the results obtained it was observed that the antibiofilm activity of usnic acid varied against different M serotypes of S. pyogenes which were all reported to be substantial biofilm formers (Thenmozhi et al. 2011). It was observed that a very low concentration of usnic acid (10 lg/ml) was sufficient to inhibit the biofilm of strain SP5 (serotype M56). However, an increased concentration (55 lg/ ml) was required to inhibit the biofilms of strains belonging to other serotypes namely, SP7 (st38), SP9 (M89) and SP31 (M74). The variation in the antibiofilm activity among various serotypes might be attributed to the varying amount of EPS layer present in each strain. Similarly to other reported biofilm inhibitors such as the flavanol morin (Green et al. 2012) and manuka honey (Maddocks et al. 2012), usnic acid also showed a good reduction in biofilm biomass (Table 1). Bacterial aggregation plays a very important role in adherence to host receptors as well as other bacteria during biofilm development (Maddocks et al. 2012). Since usnic acid very effectively prevented the aggregation of all the four strains, it was able to disrupt the biofilms of S. pyogenes. FT-IR analysis revealed that usnic acid affects various cellular components present in S. pyogenes biofilms. The FT-IR spectra showed an evident reduction of peaks in the signal regions of proteins (1,800–1,500 cm-1) and polysaccharide (1,200–900 cm-1) for the treated cells. Some recent studies have shown that the extra cellular layer of the biofilms of various pathogens are made up of amyloid like protein fibers and the degradation of these amyloid like protein fibers leads to the disassembly of biofilms (Romero et al. 2013). Usnic acid might possibly share a similar mechanism in the disassembly of biofilms. The reduction of the peaks in the polysaccharide region might be attributed to the well known observation that biofilms are
b
0.0287
8.13
0.1211
0.0327
0.06
typically made up of a thick EPS layer (Pammi et al. 2013) and usnic acid may cause degradation of this EPS layer. This result was in concordance with the confocal images and the COMSTAT analysis, which showed a reduction in the biofilm biomass (Table 1). The reduction of peak height or lack of a peak in the FT-IR spectrum in the region of 2,500–2,100 cm-1 in the treated S. pyogenes cells indicate that usnic acid may interfere with the fatty acid components of the biofilm. A possible mechanism that can be envisaged is usnic acid could act on one of the enzymes involved in the FAS2 biosynthesis pathway, as a recent study reports about the actions of several SMLs that inhibited beta-ketoacyl-ACP reductase (FabG) a key enzyme involved in FAS2 in several Gram-positive pathogens and in the malarial parasite Plasmodium (Lauinger et al. 2013). The observation that there was no significant change in the peak in the above mentioned region for strain SP 31 may be attributed to the fact that usnic acid did not have a marked antibiofilm activity for that particular strain. This result is also in concordance with the antibiofilm inhibition assay, where there was only 15 % biofilm inhibition of strain SP 31. SMLs have shown to possess antibiofilm activity against several Gram-positive and Gram-negative pathogens. Usnic acid and atranorin extracted from Chilean lichens showed good antibiofilm activity against MSSA and MRSA isolated from cystic fibrosis patients (Pompilio et al. 2013). Furthermore, US also disrupted matured biofilms of all the four S. pyogenes strains (Fig. 4) which means that usnic acid could break down or disrupt the thick EPS layer, which most antibiotics fail to do. This is in concordance with a previous study which showed that usnic acid disrupted preformed biofilms of S. aureus (Pompilio et al. 2013). The fact that usnic acid inhibits the biofilms of both Gram-positive and Gram-negative bacteria regardless of the nature of bacteria (pathogen or environmental
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Antonie van Leeuwenhoek Fig. 4 Light Microscopic image showing the antibiofilm activity of usnic aicd at their BICs against preformed (matured) biofilms of various serotypes of S. pyogenes. a Sp 5 control; b Sp 5 treated (10 lg/ml); c Sp 7 control; d Sp 7 treated (55 lg/ml); e Sp 9 control; f Sp 9 treated (55 lg/ml); g Sp 31 control; h Sp 31 treated (55 lg/ml)
isolate) suggests that lichens, which are surface associated communities, may produce these secondary metabolites as a defense against the biofilms of invading bacteria, which may explain their inherent
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resistance against pathogens (Grube et al. 2009). As usnic acid tends to exhibit low cytotoxicity at low concentrations (Pompilio et al. 2013), it augurs well for its therapeutic usage.
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Fig. 5 FT-IR spectrum of S. pyogenes strains treated with and without usnic acid a Sp 5 control; b Sp 5 treated (10 lg/ml); c Sp 7 control; d Sp 7 treated (55 lg/ml); e Sp 9 control; f Sp 9 treated (55 lg/ml); g Sp 31 control; h Sp 31 treated (55 lg/ml). The regions showing maximum variations were taken for analysis: (1) 3,500–3,100 cm-1: hydration of bacterial cells; (2)
3,050–2,700 cm-1: fatty acids in the bacterial cell membrane; (3) 1,700–1,600 cm-1: amide linkage from proteins and peptides; (4) 1,500–1,000 cm-1: mixed region, proteins and fatty acids. Dashed lines represent a unique region where there is a disappearance of a peak in the treated spectrum when compared to the control (untreated)
Conclusion
References
Novel natural and chemical compounds that disrupt the biofilm of pathogens are gaining importance. Since substantial differences in the inhibitory effect of usnic acid against several different M-serotypes were observed, further investigations need to be carried out wherein usnic acid or SMLs in general can be considered as lead compounds against biofilms generated by the most prevalent M-types in India, and against highly invasive M-types, since these serotypes represent a major threat with respect to development of invasive systemic disease (Kostenko et al. 2010; Shen et al. 2013).
Ahn SJ, Ahn SJ, Wen ZT, Brady LJ, Burne RA (2008) Characteristics of biofilm formation by streptococcus mutans in the presence of saliva. Infect Immun 76:4259–4268 Backorova M, Jendzelovsky R, Kello M, Backor M, Mikes J, Fedorocko P (2012) Lichen secondary metabolites are responsible for induction of apoptosis in HT-29 and A2780 human cancer cell lines. Toxicol Vitro 26:462–468 Balaji K, Thenmozhi R, Pandian SK (2013) Effect of subinhibitory concentrations of fluoroquinolones on biofilm production by clinical isolates of Streptococcus pyogenes. Indian J Med Res 137:963–971 Baldassarri L, Creti R, Recchia S, Imperi M, Facinelli B, Giovanetti E et al (2006) Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation. J Clin Microbiol 44:2721–2727 Chang JC, LaSarre B, Jimenez JC, Aggarwal C, Federle MJ (2011) Two group A streptococcal peptide pheromones act through opposing Rgg regulators to control biofilm development. PLoS Pathog 7(8):e1002190 Cunningham MW (2000) Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13:470–511 Francolini I, Norris P, Piozzi A, Donelli G, Stoodley P (2004) Usnic acid, a natural antimicrobial agent able to inhibit bacterial biofilm formation on polymer surfaces. Antimicrob Agents Chemother 48:4360–4365
Acknowledgments This work was supported by the Prof. T. R. Rajagopalan Research Fund of SASTRA University, Thanjavur. Authors express their sincere thanks to the Thoracic Science Department, Government Rajaji Hospital, Madurai for providing the throat swab samples for the study after the due approval and clearance of The Ethical Committee of the Government Rajaji Hospital, Madurai (Ref. No. 21156/E4/1/05 dated December 12, 2005). Conflict of interest
The authors declare no conflict of interest.
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Antonie van Leeuwenhoek Gowrishankar S, Duncun Mosioma N, Pandian SK (2012) Coral associated bacteria as a promising antibiofilm agent against methicillin—resistant and susceptible Staphylococcus aureus biofilms. Evid Based Complement Altern Med 862374:16 Green AE, Rowlands RS, Cooper RA, Maddocks SE (2012) The effect of the flavonol morin on adhesion and aggregation of Streptococcus pyogenes. FEMS Microbiol Lett 333:54–58 Grube M, Cardinale M, de Castro JV, Mu¨ller H Jr, Berg G (2009) Species-specific structural and functional diversity of bacterial communities in lichen symbioses. ISME J 3:1105–1115 Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersboll BK et al (2000) Quantification of biofilm structures by the novel computer program COMSTAT. Microbiol 146:2395–2407 Ingo`lfsdo`ttir K (2002) Usnic acid. Phytochemistry 61:729–736 Kim S, Greenleaf R, Miller MC, Satish L, Kathju S, Ehrlich G et al (2011) Mechanical effects, antimicrobial efficacy and cytotoxicity of usnic acid as a biofilm prophylaxis in PMMA. J Mater Sci Mater Med 22:2773–2780 Kostenko V, Lyczak J, Turner K, Martinuzzi RJ (2010) Impact of silver-containing wound dressings on bacterial biofilm viability and susceptibility to antibiotics during prolonged treatment. Antimicrob Agents Chemother 54:5120–5131 Lauinger IL, Vivas L, Perozzo R, Stairiker C, Tarun A, Zloh M et al (2013) Potential of lichen secondary metabolites against plasmodium liver stage parasites with FAS-II as the potential target. J Nat Prod 76:1064–1070 Lembke C, Podbielski A, Hidalgo-Grass C, Jonas L, Hanski E, Kreikemeyer B (2006) Characterization of biofilm formation by clinically relevant serotypes of group A streptococci. Appl Environ Microbiol 72:2864–2875 Limsuwan S, Voravuthikunchai SP (2008) Boesenbergia pandurata (Roxb.) Schltr., Eleutherine americana Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. as antibiofilm producing and antiquorum sensing in Streptococcus pyogenes. FEMS Immunol Med Microbiol 53:429–436 Maddocks SE, Lopez MS, Rowlands RS, Cooper RA (2012) Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins. Microbiology 158:781–790 Menon T, Shanmugasundaram S, Kumar MP, Kumar CP (2004) Group A streptococcal infections of the pharynx in a rural population in south India. Indian J Med Res 119:171–173 Musthafa KS, Sahu SK, Ravi AV, Kathiresan K (2013) Antiquorum sensing potential of the mangrove Rhizophora annamalayana. World J Microbiol Biotechnol 29:1851–1858
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Nithyanand P, Thenmozhi R, Rathna J, Pandian SK (2010) Inhibition of Streptococcus pyogenes biofilm formation by coralassociated actinomycetes. Curr Microbiol 60:454–460 Padmavati S (2001) Rheumatic fever and rheumatic heart disease in India at the turn of the century. Indian Heart J 53:35–37 Pammi M, Liang R, Hicks J, Mistretta TA, Versalovic J (2013) Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans. BMC Microbiol 13:257 Pompilio A, Pomponio S, Di Vincenzo V, Crocetta V, Nicoletti M, Piovano M et al (2013) Antimicrobial and antibiofilm activity of secondary metabolites of lichens against methicillin-resistant Staphylococcus aureus strains from cystic fibrosis patients. Future Microbiol 8:281–292 Roberts AL, Connolly KL, Kirse DJ, Evans AK, Poehling KA, Peters TR et al (2012) Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage. BMC Pediatr 12:3 Romero D, Sanabria-Valentı´n E, Vlamakis H, Kolter R (2013) Biofilm inhibitors that target amyloid proteins. Chem Biol 20:102–110 Salta M, Wharton JA, Dennington SP, Stoodley P, Stokes KR (2013) Antibiofilm performance of three natural products against initial bacterial attachment. Int J Mol Sci 14:21757–21780 Samoilova Z, Muzyka N, Lepekhina E, Oktyabrsky O, Smirnova G (2014) Medicinal plant extracts can variously modify biofilm formation in Escherichia coli. Antonie Van Leeuwenhoek 105:709–722 Shafreen RM, Srinivasan S, Manisankar P, Pandian SK (2011) Biofilm formation by Streptococcus pyogenes: modulation of exopolysaccharide by fluoroquinolone derivatives. J Biosci Bioeng 112:345–350 Shen Y, Ko¨ller T, Kreikemeyer B, Nelson DC (2013) Rapid degradation of Streptococcus pyogenes biofilms by PlyC, a bacteriophage-encoded endolysin. J Antimicrob Chemother 68:1818–1824 Thenmozhi R, Nithyanand P, Rathna J, Pandian SK (2009) Antibiofilm activity of coral-associated bacteria against different clinical M serotypes of Streptococcus pyogenes. FEMS Immunol Med Microbiol 57:284–294 Thenmozhi R, Balaji K, Kumar R, Rao TS, Pandian SK (2011) Characterization of biofilms in different clinical M serotypes of Streptococcus pyogenes. J Basic Microbiol 51:196–204
ORIGINAL PAPER
Usnic acid, a lichen secondary metabolite inhibits Group A Streptococcus biofilms Paramasivam Nithyanand • Raja Mohmed Beema Shafreen Subramanian Muthamil • Shunmugiah Karutha Pandian
•
Received: 30 June 2014 / Accepted: 29 October 2014 Ó Springer International Publishing Switzerland 2014
Abstract Group A Streptococci (GAS) are involved in a number of life threatening diseases and biofilm formation by these pathogens are considered as an important virulence determinant as it mediates antibiotic resistance among them. In the present study, we have explored the ability of (?)-usnic acid, a lichen secondary metabolite, as an antibiofilm agent against four serotypes of Streptococcus pyogenes causing pharyngitis. Usnic acid inhibited the biofilms of M serotypes M56, st38, M89 efficiently and the biofilm of M74 to a lesser extent. Confocal imaging of the treated samples showed that usnic acid reduced the biomass of the biofilms when compared to that of the control. Fourier Transfer Infrared (FT-IR) spectroscopy indicated that usnic acid reduced the cellular components (proteins and fatty acids) of the biofilms. Interestingly, the FT-IR spectrum further revealed that
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0324-z) contains supplementary material, which is available to authorized users. P. Nithyanand (&) School of Chemical & Biotechnology, Centre for Research on Infectious Diseases (CRID), SASTRA University, Tirumalaisamudram, Thanjavur 613401, Tamil Nadu, India e-mail: [email protected] R. M. Beema Shafreen S. Muthamil S. Karutha Pandian Department of Biotechnology, Alagappa University, Science campus, Karaikudi 630 004, Tamil Nadu, India
usnic acid probably acted upon the fatty acids of the biofilms as evident from the disappearance of a peak at 2,455–2,100 cm-1 when compared to the control only in serotypes M56, st38 and M89 but not in M74. The present study shows, for the first time, that usnic acid can act as an effective antibiofilm agent against GAS. Keywords S. pyogenes Biofilms Usnic acid Lichen secondary metabolite GAS
Introduction Streptococcus pyogenes (Group A Streptococcus or GAS) has established itself as a devastating human pathogen as it is responsible for causing a number of complications ranging from superficial infections to several deleterious diseases such as pharyngitis, impetigo and necrotizing fasciitis, and can also lead to rheumatic heart disease (Cunningham 2000). Worldwide there has been a resurgence in rheumatic heart disease (Menon et al. 2004) and in India alone the prevalence of rheumatic fever and rheumatic heart disease varies from 0.3 to 5.4 per 1,000 children (Padmavati 2001). Rheumatic fever is often a sequelae to pharyngitis and the severity of the disease is more prevalent among children from rural backgrounds (Menon et al. 2004). Of late, GAS biofilms have received more attention and it is increasingly accepted that biofilms may play an important role in the
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pathogenesis of S. pyogenes (Lembke et al. 2006; Thenmozhi et al. 2011). Biofilms of S. pyogenes are formed as a result of an orchestrated process called quorum sensing wherein certain peptide signals induce the quorum sensing pathways of S. pyogenes to enhance biofilm formation (Chang et al. 2011). This phenomenon of biofilm formation by pathogens is considered as a strategy to adapt to the changing selective pressures (for example, due to antibiotic administration) of the host. In addition, the formation of a thick exopolysaccharide (EPS) layer and a mesh work of extracellular DNA in the biofilm can prevent antimicrobial agents from reaching the bacterial cell envelope (Pammi et al. 2013). The disturbing observation that one in three tonsillectomies can be colonized with GAS biofilms (Roberts et al. 2012) in itself showcases the deleterious affects of GAS biofilms. Like other biofilms of pathogens, GAS biofilms also have been clinically related to antibiotic treatment failure (Baldassarri et al. 2006) which has prompted several researchers to explore various natural resources such as medicinal plants (Limsuwan et al. 2008; Samoilova et al. 2014), plant derived flavanols (Green et al. 2012), manuka honey (Maddocks et al. 2012), mangrove plants (Musthafa et al. 2013), marine bacteria (Thenmozhi et al. 2009; Nithyanand et al. 2010) and phage encoded endolysins (Shen et al. 2013) as antibiofilm agents against GAS biofilms. Traditionally, lichens have been widely used in folk medicine to treat several bacterial and fungal ailments (Ingo`lfsdo`ttir 2002) and the secondary metabolites of lichens (SMLs) have proven to possess several important pharmacological properties such as antitumour and anti-bacterial activities (Grube et al. 2009; Backorova et al. 2012). These metabolites arise from the secondary metabolism of the fungal component that shares a symbiotic association with lichens along with its algal or cyanobacterial counterpart (Grube et al. 2009). Predominantly, lichens are found in moist areas near an aquatic environment and the role of SMLs is envisaged to protect these lichens from any invading pathogens (Pompilio et al. 2013). Of all the SMLs, usnic acid, which is a component of the polyketide pathway in several lichens (Pompilio et al. 2013), has been the most explored for its antibiofilm ability and it has shown to possess a broad spectrum activity against the biofilms of clinical and environmental isolates. Usnic acid has shown to disrupt
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mature Staphylococcus aureus biofilms isolated from cystic fibrosis patients (Pompilio et al. 2013) and it also inhibited S. aureus biofilms formed on various polymer surface-based medical implants and bone cement coupons (Francolini et al. 2004; Kim et al. 2011). Further, usnic acid also prevented the initial attachment of certain marine bacteria involved in biofouling (Salta et al. 2013) and is has also been shown to interfere in the quorum sensing mechanism of Pseudomonas aeruginosa (Francolini et al. 2004). Although usnic acid has shown to exhibit antibiofilm activity against S. aureus, there are no reports regarding its antibiofilm activity against S. pyogenes involved in any kind of disease. The present study shows, for the first time, the antibiofilm activity of (?)-usnic acid against various biofilm forming serotypes of S. pyogenes isolated from children with pharyngitis (Thenmozhi et al. 2011).
Materials and methods M serotypes of S. pyogenes and usnic acid All S. pyogenes strains used in this study were isolated from throat swabs collected from children of the age group 5–15 years with pharyngitis (Thenmozhi et al. 2011). Four strains namely SP5 (M serotype M56), SP7 (M serotype st38), SP9 (M serotype M89) and SP31 (M serotype M74), which formed substantial biofilms were selected for this study (Thenmozhi et al. 2011) and all the isolates were grown in Todd Hewitt Broth (THB) (Himedia Laboratories, India). (?)Usnic acid was purchased from Sigma Aldrich (Product no. 329967). Bactericidal activity of usnic acid Anti-bacterial activity of usnic acid against various strains of S. pyogenes was tested by agar well diffusion assay in Mueller–Hinton Agar (MHA) (Himedia Laboratories, India). Briefly, overnight cultures of the S. pyogenes (1 9 106 CFU/ml) were uniformly spread over the surface of the agar plate. Usnic acid was dissolved in DMSO (1 mg/ml). Then 50–200 lg/ ml of usnic acid was loaded into the wells and the plates were incubated at 37 °C and the zone of inhibition was measured after 24 h. DMSO alone was used as control. The Minimal Inhibitory concentration
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(MIC) of usnic acid was performed as per CLSI 2006 guidelines (Shafreen et al. 2011). Antibiofilm activity of usnic acid Usnic acid with concentrations ranging from 10–200 lg/ml was added in THB containing bacterial suspensions of 106 CFU/ml. The plates were incubated for 24 h at 37 °C. After incubation, biofilms were stained with 0.4 % crystal violet. The lowest concentration of usnic acid that showed visible biofilm inhibition was determined as the biofilm inhibitory concentration (BIC) for each serotype (Nithyanand et al. 2010). DMSO alone was used as control. Aggregation assay The four serotypes of S. pyogenes strains were grown overnight in THB. The bacteria were harvested by centrifugation at 4,0009g for 10 min, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS to an optical density at 600 nm (OD600) of approximately 0.6. Nine hundred microliters of bacterial suspension and usnic acid at the BIC of each serotype were mixed in a test tube, vortexed, and transferred to cuvettes. Bacterial suspension in PBS and without the addition of usnic acid served as a control. After equilibrating the cuvettes for 5 min at room temperature, the OD600 of the samples was recorded at 10 min intervals at 37 °C for 120 min in a spectrophotometer. Percent aggregation (percent decrease in OD600) was calculated as (OD600 at 0 min - OD600 at 120 min)/(OD600 at 0 min) 9 100. All assays were performed in triplicates (Ahn et al. 2008). Confocal laser scanning microscopy (CLSM) CLSM was used to determine the three-dimensional architecture, thickness and morphology of biofilms formed by all four strains of S. pyogenes with and without the addition of usnic acid at their BIC as described by Thenmozhi et al. (2011). DMSO alone was used as control. Briefly, the biofilms of the four S. pyogenes strains were grown on glass slides and stained with 0.02 % acridine orange (Sigma, St Louis, USA) for 5 min at room temperature in the dark. The rinsed and stained slides were observed under a CLSM microsope (Zeiss LSM710 meta, Germany). The
488 nm line of an Argon laser was used for excitation. Biofilm images were observed with a long-distance 639 objective. Images were captured and processed by using Zeiss LSM Image Examiner Version 4.2.0.121. Features such as total biomass, maximum thickness and surface coverage area were assessed using COMSTAT software (kindly gifted by Dr. Claus Sternberg, Technical University of Denmark) (Heydron et al. 2000). Usnic acid was also further evaluated for its potential to disrupt mature biofilms. Biofilms of S. pyogenes were allowed to develop on cover glasses for 12 h as mentioned above. The mature biofilms were then treated with usnic acid at the BIC of each strain for 5 h and observed under a light microscope after crystal violet staining. Fourier transfer infrared (FT-IR) analysis of S. pyogenes biofilms The BIC of usnic acid was added to cell suspensions of 106 CFU/ml of the S. pyogenes strains and incubated overnight. DMSO alone was used as control. Both treated and the control cells were washed three times with MilliQ water. Twenty microliters of each bacterial sample was applied to KBr coated disks and analyzed by FT-IR (NicoletTM iSTM5, Thermo Scientific, U.S.A) spectroscopy. Sixty four scans were taken with 4 cm-1 resolution. All IR spectra were obtained over the range of 4,000–400 cm-1 and analyzed using OMNIC software as specified by the manufacturer (NicoletTM iSTM5, Thermo Scientific, U.S.A). Statistical analysis Statistical comparisons among control and the treated samples were performed by using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). All the experiments were performed in triplicates and the comparative statistical difference was performed using independent t test (where p was \0.05).
Results Bactericidal activity of usnic acid Among various concentrations of usnic acid tested, only higher concentrations (150–200 lg/ml) exhibited anti-bacterial activity against all of the strains of
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Fig. 1 Percentage inhibition of biofilm formation of usnic acid at its biofilm inhibitory concentration (BIC) against various strains of S. pyogenes. a Sp 5 b Sp 7 c Sp 9 and d Sp 31. Mean values of triplicate independent experiments and SD are shown.
Dunnett’s test demonstrates significant difference between the tests and the control (P \ 0.05). Asterisk indicates P \ 0.0001. All the P values are calculated with reference to the lowest concentration used in each experiment
S. pyogenes used in this study, with inhibition zones of 16–18 mm (Fig. S1a–d).
demonstrated against five additional M serotypes of S. pyogenes namely ES199, SF370, SP11, SP24, SP30. From the results it was observed that 30 lg/ml of usnic acid showed maximum antibiofilm activity against all the serotypes (Fig. S2a, b).
Biofilm inhibitory potential of usnic acid Various concentrations of usnic acid (from 10 to 70 lg/ ml) showed varying levels of biofilm inhibitory activity against all four strains of S. pyogenes. About 61 % of biofilm inhibition was noticed for strain SP5 at a very low concentration of 10 lg/ml. An increase in concentration resulted in reduced antibiofilm activity (Fig. 1a). However, higher concentrations of usnic acid ranging from 55–70 lg/ml were required to inhibit the biofilms of strains SP7, SP9 and SP31 (Fig. 1b–d). At 55 lg/ml, about 31 and 48 % of inhibition was noticed in strains SP7 and SP9 respectively. From the obtained results it was observed that usnic acid showed very low biofilm inhibitory activity (15 %) against the biofilm of strain SP31. The maximum biofilm inhibition was observed at concentrations ranging from 55–70 lg/ml (Fig. 1). The antibiofilm activity of usnic acid was also
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Usnic acid prevents bacterial aggregation The aggregation inhibitory potential of usnic acid was performed with 10 lg/ml usnic acid. From the results it was observed that the inhibition potential varied for each strain. On an average, usnic acid was able to prevent aggregation for up to 60 min for all the strains. For strains SP9 and SP31, usnic acid was able to completely inhibit aggregation as there was only minimal aggregation (SP9) or no aggregation (SP31) for the maximum time duration of 105 min (Fig. 2). CLSM imaging of S. pyogenes biofilms The antibiofilm activity at the BIC of usnic acid of each strain was observed using CLSM. Pronounced
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Fig. 2 Prevention of cell aggregation by usnic acid (10 lg/ml) against various strains of S. pyogenes. Control—aggregation of S. pyogenes cells without the addition of usnic acid, US—aggregation of S. pyogenes cells after the addition of usnic acid
antibiofilm activity was observed for all the four strains of S. pyogenes when compared with the control as there was a decrease in the surface area of the biofilms treated with usnic acid (Fig. 3a–d). CLSM provides three dimensional and non-invasive images of biofilms and the COMSTAT software helps in quantifying the biofilms of the generated three dimensional images (Heydorn et al. 2000). COMSTAT analysis parameters showed that there was a notable decrease in the biomass of the biofilms (directly proportional to biofilm thickness) of all the four strains of S. pyogenes when treated with usnic acid (Table 1). As expected, there was an increase in the surface to bio-volume ratio of the treated sample when compared to its control (Table 1). Usnic acid was also found to be effective on matured biofilms of SP5, SP7, SP9 and SP31. The light microscopic images indicated major disruption in the biofilm architecture of all the four serotypes (Fig. 4).
1,700–1,600 cm-1, which shows amide linkage from proteins and peptides; and 1,500–1,000 cm-1, which indicates mixed signals of proteins and fatty acids (Gowrishankar et al. 2012). In addition to the above mentioned variations, a very interesting observation was noted in the FT-IR spectrum in the region of 2,455–2,100 cm-1 (represented by dashed lines) (Fig. 5a–d). There was a reduction in the intensity of the peak in the treated cells of strain of SP5 (Fig. 5a). The same peak was totally absent in treated spectra of strains SP7 and SP9 (Fig. 5b–c), but was not altered in the strain SP31 spectrum (Fig. 4d). We interpret this data as showing that this peak corresponds to the fatty acids of the biofilm and that usnic acid acted on these fatty acids present in the biofilm. Overall, the FT-IR profile of the treated cells showed alterations in the regions of mixed protein signals, lipids, fatty acids and showed less deviations in the region corresponding to the peptide bonds in proteins (amide I and II). Cell dehydration was observed in almost all the strains treated with usnic acid.
FT-IR analysis of S. pyogenes biofilms The FT-IR spectra revealed variations between the control cells and cells treated with usnic acid (Fig. 5a– d). On comparison of the FT-IR spectra, maximum variations were observed in the regions of 3,700–3,100 cm-1 indicating hydration of bacterial cells; the 3,050–2,750 cm-1 region, which indicates the fatty acids in bacterial cell membrane;
Discussion The emergence of drug resistance among GAS biofilms and the surge of biofilm forming fluoroquinolone and macrolide resistant S. pyogenes strains (Balaji et al. 2013) of late, has driven the need to explore alternative sources for antibiofilm agents
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Antonie van Leeuwenhoek Fig. 3 Confocal Laser Scanning Microscopic (CLSM) image showing the antibiofilm activity of usnic aicd at their BICs against various serotypes of S. pyogenes. a Sp 5 control; b Sp 5 treated (10 lg/ml); c Sp 7 control; d Sp 7 treated (55 lg/ml); e Sp 9 control; f Sp 9 treated (55 lg/ml); g Sp 31 control; h Sp 31 treated (55 lg/ml)
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Antonie van Leeuwenhoek Table 1 COMSTAT analysis of S. pyogenes strains COMSTAT PARAMETERS
SP5
SP5_USa
SP7
SP7_USa
SP9
SP9_USa
SP31
SP31_USa
Biomass (lm3/lm2)
68.83
19.85b
47.47
14.52b
37.93
8.42b
32.95
17.22b
66.45
b
35.83
b
31.12
16.26b
Maximum thickness (lm) Surface to biovolume ratio (lm2/lm3)
0.0192
18.98
0.0527
45.11 0.0234
13.7
0.0712
a
Indicates strains treated with usnic acid at their BIC
b
Statistical significance (p \ 0.05) when compared with the untreated control
against GAS. In the present study, we have explored the ability of usnic acid to act as an antibiofilm agent against GAS biofilms. Based on the results obtained it was observed that the antibiofilm activity of usnic acid varied against different M serotypes of S. pyogenes which were all reported to be substantial biofilm formers (Thenmozhi et al. 2011). It was observed that a very low concentration of usnic acid (10 lg/ml) was sufficient to inhibit the biofilm of strain SP5 (serotype M56). However, an increased concentration (55 lg/ ml) was required to inhibit the biofilms of strains belonging to other serotypes namely, SP7 (st38), SP9 (M89) and SP31 (M74). The variation in the antibiofilm activity among various serotypes might be attributed to the varying amount of EPS layer present in each strain. Similarly to other reported biofilm inhibitors such as the flavanol morin (Green et al. 2012) and manuka honey (Maddocks et al. 2012), usnic acid also showed a good reduction in biofilm biomass (Table 1). Bacterial aggregation plays a very important role in adherence to host receptors as well as other bacteria during biofilm development (Maddocks et al. 2012). Since usnic acid very effectively prevented the aggregation of all the four strains, it was able to disrupt the biofilms of S. pyogenes. FT-IR analysis revealed that usnic acid affects various cellular components present in S. pyogenes biofilms. The FT-IR spectra showed an evident reduction of peaks in the signal regions of proteins (1,800–1,500 cm-1) and polysaccharide (1,200–900 cm-1) for the treated cells. Some recent studies have shown that the extra cellular layer of the biofilms of various pathogens are made up of amyloid like protein fibers and the degradation of these amyloid like protein fibers leads to the disassembly of biofilms (Romero et al. 2013). Usnic acid might possibly share a similar mechanism in the disassembly of biofilms. The reduction of the peaks in the polysaccharide region might be attributed to the well known observation that biofilms are
b
0.0287
8.13
0.1211
0.0327
0.06
typically made up of a thick EPS layer (Pammi et al. 2013) and usnic acid may cause degradation of this EPS layer. This result was in concordance with the confocal images and the COMSTAT analysis, which showed a reduction in the biofilm biomass (Table 1). The reduction of peak height or lack of a peak in the FT-IR spectrum in the region of 2,500–2,100 cm-1 in the treated S. pyogenes cells indicate that usnic acid may interfere with the fatty acid components of the biofilm. A possible mechanism that can be envisaged is usnic acid could act on one of the enzymes involved in the FAS2 biosynthesis pathway, as a recent study reports about the actions of several SMLs that inhibited beta-ketoacyl-ACP reductase (FabG) a key enzyme involved in FAS2 in several Gram-positive pathogens and in the malarial parasite Plasmodium (Lauinger et al. 2013). The observation that there was no significant change in the peak in the above mentioned region for strain SP 31 may be attributed to the fact that usnic acid did not have a marked antibiofilm activity for that particular strain. This result is also in concordance with the antibiofilm inhibition assay, where there was only 15 % biofilm inhibition of strain SP 31. SMLs have shown to possess antibiofilm activity against several Gram-positive and Gram-negative pathogens. Usnic acid and atranorin extracted from Chilean lichens showed good antibiofilm activity against MSSA and MRSA isolated from cystic fibrosis patients (Pompilio et al. 2013). Furthermore, US also disrupted matured biofilms of all the four S. pyogenes strains (Fig. 4) which means that usnic acid could break down or disrupt the thick EPS layer, which most antibiotics fail to do. This is in concordance with a previous study which showed that usnic acid disrupted preformed biofilms of S. aureus (Pompilio et al. 2013). The fact that usnic acid inhibits the biofilms of both Gram-positive and Gram-negative bacteria regardless of the nature of bacteria (pathogen or environmental
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Antonie van Leeuwenhoek Fig. 4 Light Microscopic image showing the antibiofilm activity of usnic aicd at their BICs against preformed (matured) biofilms of various serotypes of S. pyogenes. a Sp 5 control; b Sp 5 treated (10 lg/ml); c Sp 7 control; d Sp 7 treated (55 lg/ml); e Sp 9 control; f Sp 9 treated (55 lg/ml); g Sp 31 control; h Sp 31 treated (55 lg/ml)
isolate) suggests that lichens, which are surface associated communities, may produce these secondary metabolites as a defense against the biofilms of invading bacteria, which may explain their inherent
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resistance against pathogens (Grube et al. 2009). As usnic acid tends to exhibit low cytotoxicity at low concentrations (Pompilio et al. 2013), it augurs well for its therapeutic usage.
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Fig. 5 FT-IR spectrum of S. pyogenes strains treated with and without usnic acid a Sp 5 control; b Sp 5 treated (10 lg/ml); c Sp 7 control; d Sp 7 treated (55 lg/ml); e Sp 9 control; f Sp 9 treated (55 lg/ml); g Sp 31 control; h Sp 31 treated (55 lg/ml). The regions showing maximum variations were taken for analysis: (1) 3,500–3,100 cm-1: hydration of bacterial cells; (2)
3,050–2,700 cm-1: fatty acids in the bacterial cell membrane; (3) 1,700–1,600 cm-1: amide linkage from proteins and peptides; (4) 1,500–1,000 cm-1: mixed region, proteins and fatty acids. Dashed lines represent a unique region where there is a disappearance of a peak in the treated spectrum when compared to the control (untreated)
Conclusion
References
Novel natural and chemical compounds that disrupt the biofilm of pathogens are gaining importance. Since substantial differences in the inhibitory effect of usnic acid against several different M-serotypes were observed, further investigations need to be carried out wherein usnic acid or SMLs in general can be considered as lead compounds against biofilms generated by the most prevalent M-types in India, and against highly invasive M-types, since these serotypes represent a major threat with respect to development of invasive systemic disease (Kostenko et al. 2010; Shen et al. 2013).
Ahn SJ, Ahn SJ, Wen ZT, Brady LJ, Burne RA (2008) Characteristics of biofilm formation by streptococcus mutans in the presence of saliva. Infect Immun 76:4259–4268 Backorova M, Jendzelovsky R, Kello M, Backor M, Mikes J, Fedorocko P (2012) Lichen secondary metabolites are responsible for induction of apoptosis in HT-29 and A2780 human cancer cell lines. Toxicol Vitro 26:462–468 Balaji K, Thenmozhi R, Pandian SK (2013) Effect of subinhibitory concentrations of fluoroquinolones on biofilm production by clinical isolates of Streptococcus pyogenes. Indian J Med Res 137:963–971 Baldassarri L, Creti R, Recchia S, Imperi M, Facinelli B, Giovanetti E et al (2006) Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation. J Clin Microbiol 44:2721–2727 Chang JC, LaSarre B, Jimenez JC, Aggarwal C, Federle MJ (2011) Two group A streptococcal peptide pheromones act through opposing Rgg regulators to control biofilm development. PLoS Pathog 7(8):e1002190 Cunningham MW (2000) Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13:470–511 Francolini I, Norris P, Piozzi A, Donelli G, Stoodley P (2004) Usnic acid, a natural antimicrobial agent able to inhibit bacterial biofilm formation on polymer surfaces. Antimicrob Agents Chemother 48:4360–4365
Acknowledgments This work was supported by the Prof. T. R. Rajagopalan Research Fund of SASTRA University, Thanjavur. Authors express their sincere thanks to the Thoracic Science Department, Government Rajaji Hospital, Madurai for providing the throat swab samples for the study after the due approval and clearance of The Ethical Committee of the Government Rajaji Hospital, Madurai (Ref. No. 21156/E4/1/05 dated December 12, 2005). Conflict of interest
The authors declare no conflict of interest.
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Antonie van Leeuwenhoek Gowrishankar S, Duncun Mosioma N, Pandian SK (2012) Coral associated bacteria as a promising antibiofilm agent against methicillin—resistant and susceptible Staphylococcus aureus biofilms. Evid Based Complement Altern Med 862374:16 Green AE, Rowlands RS, Cooper RA, Maddocks SE (2012) The effect of the flavonol morin on adhesion and aggregation of Streptococcus pyogenes. FEMS Microbiol Lett 333:54–58 Grube M, Cardinale M, de Castro JV, Mu¨ller H Jr, Berg G (2009) Species-specific structural and functional diversity of bacterial communities in lichen symbioses. ISME J 3:1105–1115 Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersboll BK et al (2000) Quantification of biofilm structures by the novel computer program COMSTAT. Microbiol 146:2395–2407 Ingo`lfsdo`ttir K (2002) Usnic acid. Phytochemistry 61:729–736 Kim S, Greenleaf R, Miller MC, Satish L, Kathju S, Ehrlich G et al (2011) Mechanical effects, antimicrobial efficacy and cytotoxicity of usnic acid as a biofilm prophylaxis in PMMA. J Mater Sci Mater Med 22:2773–2780 Kostenko V, Lyczak J, Turner K, Martinuzzi RJ (2010) Impact of silver-containing wound dressings on bacterial biofilm viability and susceptibility to antibiotics during prolonged treatment. Antimicrob Agents Chemother 54:5120–5131 Lauinger IL, Vivas L, Perozzo R, Stairiker C, Tarun A, Zloh M et al (2013) Potential of lichen secondary metabolites against plasmodium liver stage parasites with FAS-II as the potential target. J Nat Prod 76:1064–1070 Lembke C, Podbielski A, Hidalgo-Grass C, Jonas L, Hanski E, Kreikemeyer B (2006) Characterization of biofilm formation by clinically relevant serotypes of group A streptococci. Appl Environ Microbiol 72:2864–2875 Limsuwan S, Voravuthikunchai SP (2008) Boesenbergia pandurata (Roxb.) Schltr., Eleutherine americana Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. as antibiofilm producing and antiquorum sensing in Streptococcus pyogenes. FEMS Immunol Med Microbiol 53:429–436 Maddocks SE, Lopez MS, Rowlands RS, Cooper RA (2012) Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins. Microbiology 158:781–790 Menon T, Shanmugasundaram S, Kumar MP, Kumar CP (2004) Group A streptococcal infections of the pharynx in a rural population in south India. Indian J Med Res 119:171–173 Musthafa KS, Sahu SK, Ravi AV, Kathiresan K (2013) Antiquorum sensing potential of the mangrove Rhizophora annamalayana. World J Microbiol Biotechnol 29:1851–1858
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Nithyanand P, Thenmozhi R, Rathna J, Pandian SK (2010) Inhibition of Streptococcus pyogenes biofilm formation by coralassociated actinomycetes. Curr Microbiol 60:454–460 Padmavati S (2001) Rheumatic fever and rheumatic heart disease in India at the turn of the century. Indian Heart J 53:35–37 Pammi M, Liang R, Hicks J, Mistretta TA, Versalovic J (2013) Biofilm extracellular DNA enhances mixed species biofilms of Staphylococcus epidermidis and Candida albicans. BMC Microbiol 13:257 Pompilio A, Pomponio S, Di Vincenzo V, Crocetta V, Nicoletti M, Piovano M et al (2013) Antimicrobial and antibiofilm activity of secondary metabolites of lichens against methicillin-resistant Staphylococcus aureus strains from cystic fibrosis patients. Future Microbiol 8:281–292 Roberts AL, Connolly KL, Kirse DJ, Evans AK, Poehling KA, Peters TR et al (2012) Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage. BMC Pediatr 12:3 Romero D, Sanabria-Valentı´n E, Vlamakis H, Kolter R (2013) Biofilm inhibitors that target amyloid proteins. Chem Biol 20:102–110 Salta M, Wharton JA, Dennington SP, Stoodley P, Stokes KR (2013) Antibiofilm performance of three natural products against initial bacterial attachment. Int J Mol Sci 14:21757–21780 Samoilova Z, Muzyka N, Lepekhina E, Oktyabrsky O, Smirnova G (2014) Medicinal plant extracts can variously modify biofilm formation in Escherichia coli. Antonie Van Leeuwenhoek 105:709–722 Shafreen RM, Srinivasan S, Manisankar P, Pandian SK (2011) Biofilm formation by Streptococcus pyogenes: modulation of exopolysaccharide by fluoroquinolone derivatives. J Biosci Bioeng 112:345–350 Shen Y, Ko¨ller T, Kreikemeyer B, Nelson DC (2013) Rapid degradation of Streptococcus pyogenes biofilms by PlyC, a bacteriophage-encoded endolysin. J Antimicrob Chemother 68:1818–1824 Thenmozhi R, Nithyanand P, Rathna J, Pandian SK (2009) Antibiofilm activity of coral-associated bacteria against different clinical M serotypes of Streptococcus pyogenes. FEMS Immunol Med Microbiol 57:284–294 Thenmozhi R, Balaji K, Kumar R, Rao TS, Pandian SK (2011) Characterization of biofilms in different clinical M serotypes of Streptococcus pyogenes. J Basic Microbiol 51:196–204
