Inhibition of biofilm formation in Serratia marcescens by Andrographolide from Andrographis paniculata
R. Nancy Immaculate Mary1*, N. Banu2
1Research Scholar, Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies, Chennai.
2Associate Professor, Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies, Chennai.
*Corresponding Author E-mail: banunkl@yahoo.com
ABSTRACT:
Quorum sensing (QS) is well known for bacterial virulence, antibiotic resistance and biofilm production. Inhibition of quorum sensing can reduce the risk of microbial pathogenicity in all infection. In this present study the compound Andrographolide which is present in Andrographis paniculata leaf extract was assessed for its anti quorum sensing activity against acyl homoserine lactone (AHL)-dependent biofilm formation in Serratia marcescens. The Andrographolide obtained from Andrographis paniculata exhibited a concentration dependent (100 μg/ml) reduction in biofilm production in Serratia marcescens to a level of 90% and compared with the standard Andrographolide (sigma aldrich). Microtiter plate and Light microscopic analysis further confirmed the reduction in the biofilm forming ability of Serratia marcescens when treated with andrographolide. In addition the Andrographolide present in Andrographis paniculata by itself showed no growth inhibitory effect on Serratia marcescens.
KEYWORDS: Andrographis paniculata, Quorum sensing inhibition, Acyl homoserine lactone, Serratia marcescens, anti bacterial assay, Antibiofilm.
INTRODUCTION:
Several environmental stresses have been demonstrated to increase polysaccharide intercellular adhesion (PIA) synthesis and biofilm formation by the human pathogens Serratia marcescenes. In this study we characterized an adaptive response of Serratia marcescenes to Andrographis paniculata induced stress and show that it involves concomitant impairment of synthesis and biofilm formation. Serratia marcescens is gram negative bacterium and it is an opportunistic human pathogen causing several infections. Serratia marcescens causes infections including respiratory tract, urinary tract, meningitis, septicemia, pneumonia and wound infections by secreting a number of virulence factors capable of damaging human cells and tissues [2].
It shows high resistance against antibiotic. The wide range antibiotics which include b-lactam, aminoglycoside and fluoroquinolone treatment become ineffective [10]. Thus, the emergence of antibiotic resistance among bacterial pathogen necessitate for the findings of alternative strategies to antibiotic treatment. It has been known that in Serratia marcescens the quorum sensing (QS) regulates the expression of genes responsible for the production of prodigiosin pigment, virulence factors like hemolysin, proteases, chitinase, chloroperoxidase, multiple isozymes of alkaline phosphatase, the ability to swim, swarm and biofilm maturation [6,7]. Andrographis paniculata was considered to be a rich depository of bioactive compounds with antibacterial, antifungal, antiviral, antifouling, anti-HIV, antibiofilm, immunosuppressant and cytotoxic activities. Andrographis paniculata are known to produce powerful anti-biofilm and anti-inflammatory compounds [3]. Similarly, the methanol extract of Andrographis paniculata have shown a strong quorum sensing inhibitory (QSI) activity and acted as a good antagonist against the QS systems of Serratia marcescens [8]. Although, they are known for their bioactive potential, studies on its QSI properties remain scanty. Hence, the present investigation is aimed to study the effect of QSI activity of Andrographis paniculata extracts against the QS systems of Serratia marcescens.
MATERIALS AND METHODS:
Preparation of Andrographis paniculata Leaf Extracts:
Andrographis paniculata leaves used in this study were collected from well grown trees in Tamilnadu Agriculture University, Katuthotam, Thanjavur district, Tamilnadu. The leaves were washed twice with ordinary water followed by distilled water. The washed leaves were shade dried and powered using blender. 5gm of powered sample were soaked in 50 ml of methanol for overnight. The methanol phase was collected and dried at 55°C. The residues were collected and redissolved with deionized water. Finally stored at -20°C for further use.
Bacterial Strains and Their Culture Conditions:
Serratia marcescens used in this study is a clinical strain isolated from a patient with urinary tract infection at kings institute of preventive medicine, Guindy, Chennai. The bacterial strain was allowed to grow aerobically in Luria– Bertani slants. From the slants, sub culture were done and cultures were maintained in (LB) broth (Hi-Media, India) at an optimum temperature (30° C) and intensity was checked using UV spectrometer at OD 600nm before using for further analysis.
Antibacterial Assay:
Antibacterial activity of the Andrographis paniculata leaf extracts with QSI potential was performed in Muller–Hinton agar (MHA) (Hi Media, India) by the method followed by the Clinical and Laboratory Standards Institute (2006). The 100 μl of test bacterial suspensions which were expected to have cell density equivalent to 0.5 McFarland standards (approximately 1×108 CFU/ml) were uniformly spread over the surface of the MHA plate. Then, the sterile paper disks (Hi Media, India) with a diameter of 10 mm loaded with various concentrations (50–100 μg) of Andrographis paniculata extracts were placed over the plates and incubated at 30 C for 24 h and observed for growth inhibition zone[5].
Growth Curve Analysis:
One percentage of overnight culture of bacterial pathogens (0.4 OD at 600 nm) were inoculated in 250 ml Erlenmeyer flask containing 100 ml of LB broth supplemented with various concentrations (50-100 μg) of Andrographis paniculata extract. The flasks were incubated at the optimum temperature which is suitable for Serratia marcescens under 180 rpm in a rotatory shaker. The cell density was measured in UV– visible spectrophotometer at every one hour interval [4].
Biofilm Formation in 24-well MTP:
The effect of Andrographis paniculata extract on the biofilm formation on Serratia marcescens was determined by quantifying the biofilm biomass through Micro Titer Plate assay [9]. Briefly, 1% of overnight cultures with OD adjusted to 0.4 at 600 nm. The test pathogen were added into 1 ml of fresh LB medium and cultivated in the presence and absence of Andrographis paniculata extract (20,50,75,100 μg/ml) without agitation for 16 h at 30 C. After 16 h incubation, the planktonic cells in MTPs were removed by rinsing the wells. The wells were rinsed twice with sterile distilled water. The surface-adhered cells in the MTP wells were stained with 250 μl of 0.2% crystal violet (CV) solution (Hi Media, India). The solutions were left in MTP wells for 10 mins. Then the excess CV solution was removed. So, CV in the stained cells was solubilized with 1 ml of 95% ethanol. The biofilm biomass was quantified by measuring the intensity of CV solution. The intensity was measured at OD 650 nm using UV–visible spectrophotometer.
Biofilm Prevention Assay:
Biofilm prevention assay was done by adding 10 μl of overnight culture of S. marcescens in above mentioned cell density to 1 ml of LB broth in 24 well MTP containing glass slides (1×1 cm) and supplemented with and without Andrographis paniculata extracts (20,50,75,100 μg/ml). Culture set up was incubated without agitation at 30°C for 18 h [9]. After the incubation, planktonic cells and media were discarded. The adherent cells which sticked in glass slides were gently rinsed twice with deionized water (Millipore–Milli-Q) [1].
Light Microscopic Analysis:
One percentage of overnight Serratia marcescens cultures (0.4 OD at 600 nm) were added into 1 ml of fresh LB medium which containing cover glass of 1 cm2 along with and without Andrographis paniculata extracts (20,50,75,100 μg/ml). After 16 h of incubation, the cover glasses were rinsed thrice by using distilled water to remove the planktonic cells and biofilms. Then the cover glasses were stained with 0.2% CV solution. Stained cover glasses were placed on slides. The biofilm were pointed up and visible biofilms were visualized by light microscope at magnifications of 40X (Olympus CK ×41 Jenoptik Germany, ProRes C5) [9].
RESULTS AND DISCUSSION:
Biofilms are a highly dense matrix-encapsulated population which was attached to the surfaces [9]. The biofilm formation in S. marcescens is a major virulence factor which is controlled by Andrographis paniculata. Biofilm has the ability to resist host immune response. It also resists conventional antibiotics. So, the control measures are required to prevent the biofilm formation in bacterial cells [9]. In the present study, biofilm images revealed that the Andrographis paniculata extracts effectively disturb the biofilm formation as shown in light microscopic analysis. In this study Serratia marcescens was used as target pathogenic model to know anti-biofilm activity of Andrographis paniculata. The influence of methanol extract of Andrographis paniculata was assessed for its ability to inhibit biofilm formation in S. marcescens. The minimum inhibition of biofilm was 50μg/ml and maximum inhibition of biofilm was 100μg/ml. The order to analyze the antibiofilm efficiency of Andrographis paniculata extract in inhibiting biofilm formation, S. marcescens cells were allowed to grow in MTP having glass slide in presence and absence of Andrographis paniculata extract and the results were identified and visualized under a light microscope (Fig.1 and 2).
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Figure 1: Light microscopic view of Serratia marcescens treated with Andrographolide extracted from Andrographis paniculata.
Figure 2: Graphical representation of biofilm inhibition in S. marcescens using Andrographis paniculata.
CONCLUSION:
In the present study, since the Andrographis paniculata extract were made by using methanol, the possibility of anti-biofilm activity was been ruled out. Therefore, it is envisaged that the active principle exhibiting QSI activity might be an analogue of AHL molecule. However, the extract needs further purification and characterization to find out the active principle with the antibiofilm inhibitor activity. In conclusion, the reduction in QS controlled expressions and the end effect on virulence factors production without affecting the bacterial growth provided some insight into the potential of Andrographis paniculata as QS inhibitors. Therefore, it is envisaged that the QSI potential of these Andrographis paniculata could be used as anti-biofilm drug to combat with S. marcescens and also with other bacterial infections.
REFERENCES:
1. Annapoorani A, Jabar A. K. K. A, Musthafa S. K, Pandian S. K, Ravi A.V “Inhibition of Quorum sensing mediated virulence factors production in urinary pathogen Serratia marcescens PS1 by Marine sponges”. Indian J Microbiol. 2012; 52: 160-166.
2. Hejazi A, Falkiner F.R. “Serratia marcescens”. J Med Microbiol 1997; 46: 903–912.
3. Kumar R.J, Zi-rong X. “Biomedical compounds from marine Organisms”. Mar Drugs 2004; 2: 123–146.
4. Packiavathy I.A.S.V, Agilandeswari P, Musthafa K.S, Pandian S.K, Ravi A.V “Antibiofilm and quorum sensing inhibitory potential of Cuminum cyminum and its secondary metabolite methyl eugenol against Gram negative bacterial pathogens”. Food Research International 2012; 45: 85–92.
5. Packiavathy I.A.S.V, Agilandeswari P, Ramaswamy R.P, Pandian S.K, Ravi A.V. “Antiquorum sensing and antibiofilm potential of Capparis spinosa”. Archives of Medical Research. 2011; 42: 658-668.
6. Rice S.A, Koh K.S, Queck S.Y, Labbate M, Lam K.W, Kjelleberg S. “Bio film formation and sloughing in Serratia marcescens are controlled by quorum sensing and nutrient cues”. J Bacteriol 2005; 187: 3477–3485.
7. Sarah J.C, Neil R.W, Abigail K.P.H, David R.S, George P.C.S “Metabolic and regulatory engineering of Serratia marcescens mimicking phage-mediated horizontal acquisition of antibiotic biosynthesis and quorum-sensing capacities”. Microbiology. 2006; 152: 1899–1911.
8. Skindersoe M, Epstein E.P, Rasmussen T.B, Bjarnsholt T, de Rocky Nys, Givskov M. “Quorum sensing antagonism from marine organisms”. Mar Biotechnol. 2008; 10: 56–63.
9. Thenmozhi R, Nithyanand P, Rathna J, Pandian S.K. “Antibiofilm activity of coral-associated bacteria against different clinical M serotypes of Streptococcus pyogenes”. FEMS Immunol Med Microbiol. 2009; 57:284–294.
10. Traub W.H. “Antibiotic susceptibility of Serratia marcescens and Serratia liquefaciens”. Chemotherapy. 2000; 46 :315–32.
Received on 05.12.2016 Modified on 05.01.2017
Accepted on 14.01.2017 © RJPT All right reserved
Research J. Pharm. and Tech. 2017; 10(3): 789-791.
DOI: 10.5958/0974-360X.2017.00148.2