Antibiofilm activity of Vetiveria zizanioides root extract against methicillin-resistant Staphylococcus aureus

Accepted Manuscript Antibiofilm activity of Vetiveria zizanioides root extract against methicillin-resistant Staphylococcus aureus Arunachalam Kannappan, Shanmugaraj Gowrishankar, Ramanathan Srinivasan, Shunmugiah Karutha Pandian, Arumugam Veera Ravi PII:

S0882-4010(16)30186-3

DOI:

10.1016/j.micpath.2017.07.016

Reference:

YMPAT 2354

To appear in:

Microbial Pathogenesis

Received Date: 11 April 2016 Revised Date:

13 September 2016

Accepted Date: 10 July 2017

Please cite this article as: Kannappan A, Gowrishankar S, Srinivasan R, Pandian SK, Ravi AV, Antibiofilm activity of Vetiveria zizanioides root extract against methicillin-resistant Staphylococcus aureus, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.07.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Antibiofilm activity of Vetiveria zizanioides root extract against methicillin-resistant

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Staphylococcus aureus

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Arunachalam Kannappan, Shanmugaraj Gowrishankar, Ramanathan Srinivasan, Shunmugiah

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Karutha Pandian and Arumugam Veera Ravi*

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Department of Biotechnology, Science Campus, Alagappa University, Karaikudi - 630004,

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Tamil Nadu, India

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*Corresponding Author

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Present address:

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Associate Professor, Department of Biotechnology, Science Campus,

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Alagappa University, Karaikudi - 630003, Tamil Nadu, India.

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Tel: +91 4565 225215; Fax: +91 4565 225205.

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E-mail: [email protected]

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Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is a leading human pathogen

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responsible for causing chronic clinical manifestation worldwide. In addition to antibiotic

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resistance genes viz. mecA and vanA, biofilm formation plays a prominent role in the

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pathogenicity of S. aureus by enhancing its resistance to existing antibiotics. Considering the

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role of folk medicinal plants in the betterment of human health from the waves of multidrug

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resistant bacterial infections, the present study was intended to explore the effect of Vetiveria

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zizanioides root on the biofilm formation of MRSA and its clinical counterparts. V. zizanioides

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root extract (VREX) showed a concentration-dependent reduction in biofilm formation without

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hampering the cellular viability of the tested strains. Micrographs of scanning electron

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microscopy (SEM) and confocal laser scanning microscopy (CLSM) portrayed the devastating

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impact of VREX on biofilm formation. In addition to antibiofilm activity, VREX suppresses the

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production of biofilm related phenotypes such as exopolysaccharide, slime and α-hemolysin

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toxin. Furthermore, variation in FT-IR spectra evidenced the difference in cellular factors of

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untreated and VREX treated samples. Result of mature biofilm disruption assay and down

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regulation of genes like fnbA, fnbB, clfA suggested that VREX targets these adhesin genes

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responsible for initial adherence. GC-MS analysis revealed the presence of sesquiterpenes as a

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major constituent in VREX. Thus, the data of present study strengthen the ethnobotanical value

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of V. zizanioides and concludes that VREX contain bioactive molecules that have beneficial

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effect over the biofilm formation of MRSA and its clinical isolates.

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Keywords: Antibiofilm; Clinical isolates; FT-IR; MRSA; V. zizanioides.

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Abbreviations: VREX – Vetiveria zizanioides root extract; MRSA - methicillin-resistant

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Staphylococcus aureus; SEM - scanning electron microscopy; CLSM - confocal laser scanning

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microscopy; XTT - 2,3-Bis(2-methoxy-4-nitro- 5-sulfophenyl)-2H-tetrazolium-5-carboxanilide;

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MSCRAMMs - Microbial Surface Components Recognize Adhesive Matrix Molecules.

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1. Introduction Antibiotic treatment has been employed largely to diminish bacterial infections since their

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discovery. However, the inappropriate usage and chronic overuse of antibiotics have exerted

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selection pressure for the development of resistance in pathogens, which significantly increases

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the rate of mortality and morbidity in nosocomial settings. As a consequence, the U.S Center for

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Disease Control and Prevention (CDC) and the U.S. National Institutes of Health (NIH) have

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alarmed the emergence of multiple drug resistant (MDR) pathogens as a global concern [1, 2].

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Staphylococcus aureus, a commensal of human skin and mucous membrane, is a

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predominant opportunistic human pathogen responsible for the outbreak of several infections

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ranging from skin and soft tissue infections like boils, cellulitis, carbuncles, pimples, abscesses

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to severe infections like bacteremia, toxic shock syndrome, osteomyelitis, meningitis, etc. [3]. S.

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aureus has long been recognized as an important bacterial threat in most of the developing and

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developed countries. Emergence of methicillin-resistant (MRSA) strains and recent appearance

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of vancomycin intermediate (VISA) and vancomycin-resistant (VRSA) strains holds S. aureus as

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one among the top three pathogens of clinical interest [4, 5]. Regardless of the antibiotic resistant

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genes in S. aureus, the formation of biofilms is indeed a prime virulence trait that enhances the

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pathogenicity of this organism by augmenting the secreted virulence factors and expanding

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resistance against antibiotics. This has attributed its competency to play a crucial role in causing

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persistent and chronic infections especially in implanted materials [6].

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The foremost phase in biofilm developmental stage in S. aureus is the initial attachment

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with host cell surface, which is primly conferred by Microbial Surface Components Recognize

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Adhesive Matrix Molecules (MSCRAMMs). These MSCRAMMs includes elastin binding

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protein (ebpS), collagen binding protein (cna), fibronectin binding proteins (fnbA and fnbB), 4

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fibrinogen binding protein (fib) and clumping factors (clfA and clfB) [7]. Even though, biofilm

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formation in S. aureus is governed by multiple factors, polysaccharide intercellular adhesins

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(PIA) plays a vital role in S. aureus biofilm formation which is encoded by ica operon. However,

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global regulators in S. aureus also have their impact on biofilm formation viz. accessory gene

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regulator (agr) and the Staphylococcal accessory regulator (sarA) [8, 9].

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Since biofilm infections are hard to eradicate owing to their innate antibiotic resistance,

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biofilm mediated infections account for 80% of medical infections [10]. Considering the clinical

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complications implicated by the emergence of biofilm mediated MDR, finding new treatment

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strategies that specifically target the biofilm formation and virulence factors production rather

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than the cellular viability could be more promising. Whilst several strategies and approaches

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have been proposed for treating staphylococcal biofilm infections [11], medicinal plants used in

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folklore are one among the alternatives, which are being explored presently [12, 13]. Till date,

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researchers are exploring medicinal plants for their antibacterial usefulness. Instead of focusing

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the potential of antimicrobial effectiveness, changing the attention towards anti-virulent

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potentials by using the antimicrobials at sub-lethal concentration may unveil the real purpose for

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using folklore medicinal plants in treating infections.

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Vetiveria zizanioides (L.) Nash, a perennial long tufted grass, which belongs to the family

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Poaceae is native of India and well recognized in southern India. It is commonly called as Khus,

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vetiver, vala, etc in different Indian languages. Importance of this plant, especially root, in the

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field of perfumery and medicine is highly recognized since the ancient days [14]. The root of this

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plant is traditionally known for its antiseptic properties and is being used to treat skin infections

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such as acne, boils, burns, weeping sores, etc [14]. With the available knowledge from folklore,

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this plant has recently been reported for its antioxidant, insecticidal, anticancer, antiseptic, anti-

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inflammatory, antifungal, cicatrisant, vulnerary properties, etc [15, 16, 17]. However, inhibitory

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efficacy of V. zizanioides on bacterial biofilm formation and virulence factors productions

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remains unexplored. Hence, the present study was undertaken to explore the in vitro potential of

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Vetiveria zizanioides root extract (VREX) against the biofilm formation and biofilm related

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phenotypes production in MRSA ATCC 33591 and its clinical isolates.

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2.

Materials and methods

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2.1

Plant materials and extract preparation

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The plant V. zizanioides was collected from the local Ayurvedic farm in Karaikudi, India.

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The plant was identified by Dr. John Britto, The Rapinat Herbarium, St. Joseph’s College,

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Tiruchchirappalli, Tamil Nadu, India (voucher number: ARK001). The root was separated from

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the plant and washed with tap water. The cleaned roots were shade dried and milled to a fine

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powder. The extraction was carried out using Soxhlet extraction method. In order to extract the

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components from the root of V. zizanioides, dichloromethane was used [18]. The extract was

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allowed for evaporation. The dried extract was weighed and dissolved in methanol to obtain final

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concentration of 100 mg/ml which was used for further assay. The yield of the extract was 2.9%.

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Bacterial strains and culture conditions

In the present study four clinical strains (GSA-44, GSA-45, GSA-410 and GSA-395) of

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MRSA were used along with the reference strain MRSA ATCC 33591. The clinical strains were

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identified at species level based on 16S rRNA gene sequencing and the GenBank accession

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numbers for GSA-44, GSA-45, GSA-410 and GSA-395 are JN315148, JN315149, JN390832

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and JN315150, respectively [5]. All the strains were maintained on tryptic soya agar (TSA)

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plates at 4°C and subcultured in tryptic soya broth (TSB) (Hi-Media, Mumbai, India). For assay

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purpose, 1% sucrose was used along with TSB for inducing the biofilm formation; methanol was

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used as negative control for all assays and the OD of the test pathogens were adjusted to 0.4 at

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OD600nm from overnight culture (1×108CFU/ml).

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2.3

Determination of minimal inhibitory concentration (MIC) The MIC of VREX was determined through microdilution method as described by the

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Clinical and Laboratory Standards Institute (CLSI) [19]. Briefly, 1% of test pathogens (0.4 OD at

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600nm) were added to Muller Hinton Broth (MHB) (Hi-Media, Mumbai, India) containing

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serially diluted VREX at concentrations ranging from 1024 to 64 µg/ml and incubated at 37°C

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for 24 h. The MIC was recorded as the lowest concentration of VREX that showed complete

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inhibition of bacterial growth as like medium control.

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Determination of minimal biofilm inhibitory concentration (MBIC) The MBIC was determined as the minimum concentration that shows maximum

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reduction in biofilm formation when compared to control. In order to determine MBIC, VREX at

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increasing concentration was tested against all the strains of MRSA on 24-well polystyrene

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microtitre plate (MTP) [20]. Briefly, VREX (100-400 µg/ml) was added in TSB containing 1%

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of the test organisms. The plates were then incubated at 37°C for 24 h. After incubation, the

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planktonic cells were removed and the biofilm was stained with 0.4% (w/v) crystal violet (CV)

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solution. The stained wells were eluted with 95% ethyl alcohol and quantified at OD570nm by UV-

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visible spectrophotometer (Hitachi U-2800, Tokyo, Japan). Simultaneously, VREX was analyzed

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for its effect on the growth of test pathogens at the tested concentration (100-400 µg/ml). After

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incubation of the test pathogens in the presence and the absence of VREX at 37°C for 24 h, the

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content of the wells was quantified using spectrophotometer at OD600nm.

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2.5

Microscopic analyses

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2.5.1

Confocal laser scanning microscopic (CLSM) analysis

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The test pathogens were incubated along with glass slides (1×1cm) in the 24 well MTP

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containing 1ml of TSB at 37°C for 24 h in the presence and the absence of MBIC of VREX. The

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slides were washed with phosphate buffered saline (PBS) pH 7.4 and stained with 0.1% acridine

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orange, which was imaged under CLSM (LSM 710, Carl Zeiss, Germany). The Z-Stack images

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(Zen 2009 software) were analyzed using COMSTAT software (kindly gifted by Dr. Claus

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Sternberg, DTU Systems Biology, Technical University of Denmark) to obtain biofilm

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biovolume, average thickness and surface to volume ratio of the biofilm formed in control and

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treated samples [21].

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For SEM analysis, the test pathogens were allowed to form biofilm on the glass slides in

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the presence and the absence of VREX as mentioned above. Sample preparation was performed

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as described by Gowrishankar et al. [22]. After fixing the biofilm on the glass slides by 2.5%

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glutaraldehyde solution for 2 h, the slides were washed with distilled water. The slides were then

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dehydrated using ethanol at increasing concentrations (20%, 40%, 60%, 80% and 100%) for 2

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min each. The fixed biofilm slides were sputter coated by gold and examined under SEM

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(VEGA 3 TESCAN, Czech Republic).

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In XTT reduction assay, metabolic activity of viable cells were assessed using XTT (2,3-

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Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) as substrate [22]. MRSA

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cells treated with and without VREX was washed twice with sterile PBS (pH 7.4) and

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resuspended in an equal volume of the same. The cell suspensions were further incubated at

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37°C for 8 h in dark, along with XTT (Sigma, St. Louis, MO, USA)/menadione (Hi-Media,

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Mumbai, India) solution in the ratio of 12.5:1 respectively. After incubation, calorimetric

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changes were measured at OD490nm using spectrophotometer.

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2.7

Quantification of EPS EPS quantification was carried out by following the method of Santhakumari et al. [23]

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with little modifications. Briefly, MTP containing 1ml of TSB along with test pathogens were

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incubated at 37°C for 24 h in the presence and the absence of VREX. After incubation, the wells

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were washed and the biofilm cells adhered on MTP was washed with 0.9% NaCl to which equal

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volume of 5% phenol was added. To the NaCl: phenol mixture, 5 volumes of concentrated

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H2SO4 was added and incubated in dark for 1 h. After centrifugation at 10,000 rpm for 10 min

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the absorbance was measured at OD490nm.

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2.8

Slime layer production

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To determine the slime production, Congo red agar (CRA) plate assay was done [24].

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The CRA plates (TSB-30 g/l, Sucrose-36 g/l and agar-20 g/l; Congo red dye-0.8 g/l autoclaved

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separately and added to the medium after it reaches the hand bearable heat) were prepared with

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and without VREX. All the test pathogens were streaked in the untreated and VREX treated

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plates and incubated at 37°C for 24 h to observe the changes.

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Hemolysin quantification

Fresh sheep blood was washed thrice with PBS (pH 7.4) and resuspended in the same to a

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final concentration of 2% (v/v). Equal volume of 2% blood and bacterial cell free culture

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supernatant (treated with and without VREX) were added together and incubated at 37°C for 2h.

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The tubes were then spun at maximum speed for 2 min. The hemolytic activity was determined

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by measuring the total amount of hemoglobin released in the supernatant at OD405nm. To obtain

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the total lysis, the erythrocytes were incubated with distilled water (positive control) and 9

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background lysis was determined by incubating the erythrocytes with PBS (negative control).

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The percentage lysis was determined by using the following formula [25].

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A405 of sample - A405 of negative control × 100 A405 of positive control - A405 of negative control

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FT-IR analysis

MRSA cells treated with and without VREX were washed thrice with sterile PBS (pH

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7.4). 100 mg of Potassium bromide (IR grade) with 1 mg of bacterial cells was used to prepare

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pellet. Prepared KBr pellet was analyzed by FT-IR spectroscopy (NicoletTM iS5, Thermo

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Scientific, U.S.A). A total of 64 scans were taken with 4 cm-1 resolution. The spectrum was

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scanned in the range of 4000–400 cm−1. All the IR spectra were plotted as absorbance and

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analyzed using OMNIC software [26].

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2.11

Antibiotic sensitivity assay

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As per the Clinical and Laboratory Standards Institute guidelines [19], the disc diffusion

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assay was done to determine the antibiotic sensitivity pattern of the test pathogens in the

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presence and the absence of VREX. All the strains at an optical density of 0.5 McFarland

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standard (1X108 CFU/ml) were swabbed over the surface of Muller-Hinton agar (MHA)

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(HiMedia, Mumbai) plates incorporated with and without VREX at MBIC. Amikacin (10 µg),

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clindamycin (10 µg), gentamycin (50 µg) and vancomycin (10 µg) (HiMedia, Mumbai) were the

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antibiotics used. The antibiotic discs were placed over the swabbed plates and kept under

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incubation for 24 h at 37°C. After incubation, the zone of inhibition was measured [27].

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Disintegration of mature biofilm

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To examine the effect of VREX on preformed biofilm, the preformed biofilm of all the

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test strains were incubated with VREX at MBIC for 24 h at 37 °C. After incubation, the biofilms

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on the glass slides were washed thrice with sterile PBS and stained with crystal violet solution as

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mentioned above. The stained slides were observed under light microscope (Nikon Eclipse Ti

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100, Japan) [28].

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Total RNA isolation and cDNA synthesis

Total RNA was isolated from the cells of MRSA ATCC 33591, treated with and without

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VREX, using TRIzol reagent. The isolated RNA was reverse transcribed to cDNA using High

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Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc., USA).

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Real time PCR (qPCR) analysis

The qPCR reactions were carried out as per manufacturer’s instructions using Power

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SYBR® Green PCR master mix (Applied Biosystems Inc., USA) and performed using 7500

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Sequence Detection System (Applied Biosystems Inc., USA). The data set was normalized with

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housekeeping (16S rRNA) gene of MRSA. In response to VREX, relative expression of the target

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genes were quantified using gene-specific primers as listed in table 1 with the help of standard

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formula described by Yuan et al. [29].

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Gas chromatography Mass spectroscopy (GC-MS) analysis The VREX was subjected to GC-MS analysis (SHIMADZU GC-MS-138 QP2010). Gas

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chromatograph column (Rxi®-5ms) (ID 0.25 mm thickness 0.25 139µm) was coupled to a mass

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detector (with 5% diphenyl and 95% 140 dimethylpolysoloxane) using standard GC-MS

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parameters. The oven temperature of the column was maintained at 60°C for 3 min, 250°C for 8

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min and 280°C for 2 min. The sample was introduced into the column by a splitless mode of

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injection. The interface and ion source temperature was set to 250°C and helium was used as

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carrier gas with a delay time of 2 min. The scan range was 40–600 m/z with the scan speed of

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1,250. The total GC-MS run time was 47 min. The major peaks were identified by comparison

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with the MS reference database of NIST software (National institute of standards and

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technology, Gaithersburg, USA).

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2.16

Statistics All the experiments were done in experimental triplicate for thrice, and the statistical

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analyses were performed using SPSS version 20.0. Values were expressed as mean ± standard

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deviation. A Dunnett–ANOVA and student-t tests were used to compare tests and controls.

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3.

Results

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3.1

Determination of minimal inhibitory concentration (MIC)

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To analyze the effect of VREX on the growth of the test pathogens, MIC was determined.

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Since VREX inhibited the growth of all tested strains at 1024 µg/ml, the MIC of VREX was

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fixed as 1024 µg/ml. To be an ideal antibiofilm agent, it should not possess any inhibitory effect

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on growth of the bacterium. Hence, the concentrations well below the sub-MIC (100-400 µg/ml)

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were assessed for the antibiofilm activity of VREX against MRSA and its clinical isolates.

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Determination of minimal biofilm inhibitory concentration (MBIC) To investigate whether VREX inhibits the biofilm formation of MRSA and its clinical

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isolates, biofilm forming potential of all the test strains was assessed in the absence and the

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presence of VREX at the concentration ranging from 100-400 µg/ml. As shown in Fig. 1A,

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biofilm formation was reduced by VREX in a concentration dependent manner without

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inhibiting the growth of the test pathogens at the tested concentrations (Fig. 1B). Since 300

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µg/ml of VREX effectively inhibited the maximum biofilm formation of all test strains (P≤0.05),

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300 µg/ml was fixed as MBIC.

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Microscopic visualization of biofilm formation

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Biofilm formation was assessed qualitatively using microscopic techniques. The results

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of CLSM analysis revealed a thick lawn of biofilm coverage in the control samples, wherein

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VREX treated strains showed a visible reduction in the thickness of biofilm (Fig. 2).

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Consequently, COMSTAT analysis also evidenced a tremendous reduction in parameters like

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biofilm biovolume and average thickness of all the strains upon treatment with VREX (Table 2).

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As anticipated, strains treated with VREX showed increase in surface to volume ratio compared

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to the respective control. SEM analysis was further done to explain the effect of VREX on

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MRSA

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exopolysaccharides production and biofilm formation in the VREX treated sample (Fig. 3).

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3.4

formation.

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XTT reduction assay

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XTT reduction assay was done for untreated and VREX treated cells at MBIC to evaluate

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its effects on cell viability. The spectrophotometric results of XTT reduction assay showed there

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was no significant difference in the cell viability of untreated control and VREX treated samples,

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which confirmed the non-lethal effect of VREX on the viability of MRSA and its clinical isolates

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(Fig. 4).

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Inhibition of EPS and slime layer production Since biofilm formation of S. aureus is directly correlated with the production of EPS and

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slime, an effort was taken to evaluate the effect of VREX on EPS and slime layer production.

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VREX exhibited a promising reduction in the EPS production of MRSA and its clinical isolates.

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At MBIC, VREX halts the EPS production to an extent of 68, 90, 83, 66 and 87% in MRSA

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ATCC 33591, GSA-44, GSA-45, GSA-410 and GSA-395 strains, respectively (Fig. 5). Further

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to ascertain the effect of VREX on slime production, CRA plate assay was done. In CRA plate

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assay, the level of black colour formation by test organism is directly proportional to the level of

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slime production. From fig. 6, it was clearly evident that MBIC of VREX suppresses the slime

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production in all the strains tested.

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3.6

Hemolysin quantification In the hemolysin quantification assay, test strains cultured along with VREX showed

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reduced production of hemolysin. Compared to the respective controls, VREX treatment led to

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significant reduction in hemolysin production at the concentration of 300 µg/ml (Fig. 7).

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FT-IR analysis

FT-IR analysis was carried out to monitor the changes in the cellular components of

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MRSA upon treatment with VREX. Variation in the FT-IR spectra of VREX treated samples

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validated the results of in vitro assays (represented by dashed rectangle). From Fig. 8 it was

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evidenced that the spectral difference observed in the region of cell membrane fatty acids (3050–

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2750 cm-1), region containing amide bonds of proteins and peptides (1700–1600 cm-1) and the

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region containing mixed profile of protein and fatty acid (1500–1000 cm−1).

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Antibiotic sensitivity assay

VREX was assessed for its activity along with antibiotics such as amikacin (10 µg),

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clindamycin (10 µg), gentamycin (50 µg) and vancomycin (10 µg) through disc diffusion assay.

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The increased zone of inhibition in disc diffusion assay (Table 3) clearly evidence that VREX

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enhances the sensitivity of MRSA and its clinical isolates towards the tested antibiotics.

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Effect of VREX on preformed biofilm

The impact of VREX on preformed biofilm was assessed through light microscopic

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analysis. In light microscopic analysis, no significant difference was observed as shown in Fig. 9

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when treated with VREX compared to that of respective control slides.

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Quantitative real-time PCR analysis 14

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To find out the effect of VREX on candidate genes, qPCR was performed. Expression of

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few genes involved in adhesins (fnbA, fnbB, cna & clfA) and genes responsible for biofilm

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maturation like icaD & icaA were studied. The expression levels of VREX treated cells were

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compared with control and normalized to one. The data of expression analysis displayed

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significant down regulation in the genes fnbA, fnbB and clfA. Genes icaD & icaA which encode

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for biofilm maturation and maintaining integrity, has no significant difference in expression

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compared to that of control (Fig.10).

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3.11

Characterization of VREX by GC-MS analysis

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In order to reveal the chemical constituent, VREX was subjected to GC-MS analysis. The

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results related to the GC-MS analysis are given in fig. S1. Major constituents of VREX were

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found to be the derivatives of sesquiterpenes as listed in table 4.

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Discussion

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The era of MDR been emerged together with the era of antibiotics owing to the acquired

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antibiotic resistance and vigorous biofilm forming ability of bacterial pathogens. Formation of

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biofilm is recognized as a key virulence in pathogenesis, which helps the inhabitants to survive

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in extreme conditions and fortify them against the invading antimicrobials and host immune

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responses. Given the prominence of biofilm in antibiotics resistance, the necessity of exploring a

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new alternate source that targets biofilm formation of bacterial pathogens is very much essential.

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Because of the breakthrough in the exploration of folk medicinal plants as therapeutics against a

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number of human diseases, attempts were made to investigate the efficiency of folk medicinal

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plants in suppressing the biofilm formation and its related virulence factors production in several

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bacterial and fungal pathogens [13, 30, 31]. The present study was carried out to explore the

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ability of VREX in suppressing the biofilm formation and biofilm-related phenotype production

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in MRSA. Based on the results obtained, it was evident that VREX significantly inhibited the

345

biofilm formation of MRSA ATCC 33591 and its clinical isolates (GSA-44, GSA-45, GSA-410

346

and GSA-395) in a concentration-dependent manner without inhibiting the bacterial growth (Fig.

347

1A & 1B). The minimal biofilm inhibitory concentration (MBIC) was found to be 300 µg/ml for

348

all the test pathogens and the same was used in all subsequent assays. The results of this study is

349

superior to the recent findings of Zhang et al. [32], wherein the polyphenolic extract of Rosa

350

rugosa at a concentration of 640 µg/ml inhibited the biofilm formation of E. coli K-12 and P.

351

aeruginosa PAO1 up to 67% and 73% respectively, with no effect on planktonic cells. In S.

352

aureus, the development of characteristic biofilm architecture is a crucial stage during

353

pathogenesis and it is also proven that conditioned surface induces the S. aureus adhesion and

354

subsequent microcolony formation [33]. In order to ascertain the changes exerted by VREX on

355

microcolonies formation and 3D biofilm architecture development, the confocal microscopy

356

followed by COMSTAT software analyses were performed. As anticipated, CLSM analysis

357

confirmed that VREX modulates the surface architecture of all the test strains (Fig. 2).

358

Furthermore, COMSTAT analysis evident the reduction in biofilm biovolume and the average

359

thickness in the VREX exposed samples compared to their respective controls (Table 2). In

360

addition, an increase in surface to volume ratio indicates reduced attachment of cells to the

361

surface upon VREX treatment. Therefore, it is envisaged that treatment of MRSA with VREX

362

resulted in the formation of weak biofilms possibly by reducing the surface adhesion and

363

subsequent microcolony formation. Towards gathering deep insights on the VREX induced

364

modifications on the surface topology and morphology of MRSA biofilms SEM analysis was

365

employed. The multilayered cell clusters bounded with exopolymeric matrix (a typical feature of

366

S. aureus biofilms) of MRSA was obvious in the untreated samples, whereas the slides treated

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with VREX portrayed the cells free of such matrix structures and segregated as single cells at the

368

surface covered (Fig. 3). In par with this result, Salini et al. [34] has reported the reduced biofilm

369

formation and maximum biofilm architecture disintegration in Serratia marcescens treated with

370

Hyptis suaveolens hexane extract. As far as the folk medicinal plants against staphylococcal

371

infections are concerned, the crude extract of Quercus cerris, Alnus japonica, Rubus ulmifolius,

372

Coriandrum sativum, Mentha piperita and Pimpinella anisum has been reported to limit the

373

staphylococcal biofilm formation [35, 36, 37, 38]. Despite the numerous reports on the use of

374

various folk plants against staphylococcal infection, the current study clearly explicated the

375

inhibitory potency of VREX towards the biofilm and virulence production of MRSA strains.

376

Owing to the notion that any bioactive molecule to be a promising antibiofilm agent it should not

377

affect the basic metabolic activity of the pathogen, hence the MBIC of VREX was subjected to

378

XTT reduction assay. Fig. 4 evident that there was only a meager difference between the

379

metabolically active cells of VREX treated and untreated samples, which suggests that VREX

380

target the biofilm formation rather than its cellular viability.

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EPS plays a pivotal part in tailoring the biofilm architecture and in maintaining structural

382

integrity in response to the host environment [33]. Destruction of EPS is expected to improve the

383

eradication of biofilm by preventing S. aureus cells to attain the irreversible phase during its

384

biofilm developmental cycle progression. In the present study, S. aureus strains treated with

385

VREX showed reduction in EPS production up to 65-89% among the test strains (Fig. 5). On line

386

to this finding, a study made by Packiavathy et al. [39] showed the reduced EPS production in

387

Vibrio spp. upon treatment with curcumin. In the recent past FT-IR technique has been

388

performed to analyze the difference in the chemical composition of prokaryotes and eukaryotes

389

[21, 40]. Likewise, in this study also FT-IR analysis was done to monitor the changes in the

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cellular components in MRSA upon treatement with VREX. Reduction in the spectral region

391

(3050–2750 cm-1, 1700–1600 cm-1 and 1500–1000 cm−1) indicates that VREX could possibly

392

interfere with fatty acid and protein components present in the biofilms and those reductions

393

were turned out because of the reduction in EPS synthesis upon VREX treatment under in vitro

394

conditions (Fig. 8). Elmasri et al. [41] reported the presence of sesquiterpenes in the plant

395

Teucrium polium is responsible for inhibition of biofilm formation in S. aureus through

396

modulating the membrane fatty acid. Similar to these findings, GC-MS analysis of VREX

397

revealed the presence of plant specific sesquiterpenes such as cyclosativen, α –gurjunene, γ-

398

himachalene, β-vatirenene, trichoacorenol, valerenic acid, valerenyl acetate, nootkatone,

399

longifolene, α-vetivone, α-eudesmol and α-copaene which might contribute towards the

400

antibiofilm activity through membrane fatty acid modulation [18, 42] (Table 4). Despite these

401

findings, recent literature evidenced the use of fatty acid biosynthesis pathway to inhibit biofilm

402

formation of other bacterial pathogens [43]. In concordance with these findings, essential oil

403

from commercial and wild Patchouli was also found to act on biofilm formation by modulating

404

the fatty acid membrane of S. pyogenes and which was evidenced through the reduction in the

405

fatty acid region of FT-IR spectrum [24].

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Thus, if any bioactive compound disintegrates the biofilm architecture by means of EPS

407

inhibition, it is possible to reduce the biofilm mediated MDR by enhancing the antibiotics

408

penetration. To substantiate the fact, Rogers et al. [44] in 2010 stated that the compounds

409

possessing ability of reducing biofilm and its associated virulence factors production may likely

410

to act synergistically with conventional antibiotics towards surpassing the resistance prevalence

411

among bacterial pathogens. In the current study, as VREX inhibits biofilm formation as well as

412

its associated candidate virulence factors like EPS and slime production of S. aureus, VREX was

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intended to assess its synergistic activity along with antibiotics through disc diffusion assay. The

414

increased zone of inhibition in disc diffusion assay (Table 3) clearly evidences the antibiofilm

415

potential of VREX in enhancing the susceptibility of MRSA and its clinical isolates to the tested

416

antibiotics. In an earlier report by Musthafa et al. [45], an increased susceptibility to antibiotics

417

in S. marcescens was observed upon treatment with SS4 extract, which goes in hand with the

418

results obtained in this study.

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Slime synthesis is also one of the important biofilm related virulence traits recognized in

420

most of the coagulase negative staphylococci (CNS). Slime producing CNS are considered to

421

bear increased ability to colonize host tissue than the non-slime producing variants and have

422

better fortification from opsonisation and phagocytosis [46, 47]. Classical black colour colony

423

appearance is the characteristic feature of slime producing S. aureus in CRA plate [23], which

424

was used as a qualitative method to assess the impact of VREX on slime production of MRSA.

425

In comparison to the control CRA plates of test pathogens, the level of black colouration was

426

considerably reduced in VREX treated samples, particularly the single colonies at the end of the

427

fourth quadrant streak (Fig. 6). Consistent with this result, Gowrishankar et al. [19] observed a

428

significant decline in slime production in the S. aureus and its clinical strains treated with CAB

429

extract. Similar to the role of slime and EPS on biofilm formation, alpha-hemolysin (a 33-kDa

430

pore forming protein encoded by hla gene) which is also responsible for biofilm formation in S.

431

aureus and erythrocyte lysis [48]. The result indicated that VREX was found to be effective in

432

inhibiting the hemolysin production in all the test strains up to the range of 72-80% upon

433

treatment (Fig. 7). In accordance with this finding, hemolytic activity of S. aureus ATCC 6538

434

was strongly inhibited by the flavonoid quercetin which was identified from the extract of A.

435

japonica [37].

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Once formed biofilms are hard to eradicate. To be a potent antibiofilm compound, it

437

should disintegrate the preformed biofilms. In this context to evaluate the efficiency of VREX on

438

the preformed biofilm, the mature biofilm disruption assay was performed. From fig. 9, it is

439

more evident that VREX targets the initial attachment of cells and not the established biofilms.

440

Furthermore, in order to ascertain the data of in vitro assays and to understand the antibiofilm

441

mechanism of VREX at the molecular level, expression analysis was performed. Since VREX

442

inhibit only the initial attachment of biofilm cells, qPCR was performed using adhesins genes

443

and fewer biofilm related genes of MRSA. Adhesins genes of MSCRAMM family initiate host

444

surface colonization by means of anchoring cell wall peptidoglycan [49]. Besides multiple

445

factors responsible for the attachment and accumulation, PIA or polymeric N-acetyl-glucosamine

446

(PNAG) encoded by ica operon is a well understood mechanism in staphylococcal biofilm

447

formation [50]. As anticipated, expression analysis data of VREX treated MRSA showed that

448

genes involved in the initial attachment of biofilm formation (fnbA, fnbB and clfA) were down

449

regulated (Fig. 10). However, it was also observed that upon VREX treatment the genes icaA and

450

icaD, which encodes for the biofilm maturation and maintaining integrity has no significant

451

changes in the expression, which may be the reason that VREX has no effect on preformed

452

biofilm. Apart from biofilm and adhesins genes, MBIC of VREX was also assessed for its

453

potential on other virulence gene (sea) expressions as well. staphylococcal enterotoxin A (sea) is

454

a pyrogenic exotoxin responsible for the cause of food poisoning and autoimmune diseases.

455

Down regulation of sea gene and inhibition of hemolysin production validates that VREX not

456

only inhibit the biofilm and biofilm related virulence factors, it also targets the production of

457

other toxin genes produced by the MRSA.

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In conclusion, the present work delineates that VREX could inhibit the biofilm formation

459

of MRSA and its clinical isolates in a concentration dependent manner. Significant reduction in

460

the biofilm and biofilm-related virulence factors production viz. slime, EPS, α-hemolysin and

461

reduction in the cellular component studied through FT-IR spectroscopy validates the efficacy of

462

VREX against MRSA biofilm formation. Furthermore, the results of qPCR analysis and mature

463

biofilm disruption assay unveiled the plausible antibiofilm mechanism of VREX.

464

Characterization of VREX through GC-MS analysis revealed the presence of sesquiterpenes and

465

other important compounds which may be accountable for the antibiofilm activity. Hence, the

466

results of the present study divulged the therapeutic value of V. zizanioides root and could prove

467

to be a promising anti-virulence agent in treating staphylococcal biofilm mediated infections.

468

5. Acknowledgement

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The authors sincerely acknowledge the computational and bioinformatics facility

470

provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by DBT,

471

GOI; File No. BT/BI/25/015/2012, BIF). Financial assistance rendered to AR. Kannappan by

472

University Grants Commission in the form of UGC-BSR Fellowship is thankfully acknowledged

473

[Grant No. F.25-1/2014-15(BSR)/7-326/2011(BSR)]. This work is partly supported by a grant

474

from the DBT sponsored research project [BT/PR4815/AAQ/3/587/2012] awarded to Dr. A.

475

Veera Ravi.

476

6. Conflict of Interest

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Authors have no conflict of interest to declare.

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Figure legends

607

Fig. 1. Antibiofilm activity of VREX (A) and its effect on the growth (B) of MRSA and its

608

clinical isolates at the concentrations ranging from 100 – 400 µg/ml. Mean values of triplicate

609

independent experiments and SD are shown. ANNOVA was used to compare the treated and

610

untreated controls. * indicates significance at P≤0.05.

611

Fig. 2. CLSM analysis of biofilm formed by MRSA and clinical isolates in the presence and

612

absence of MBIC of VREX.

613

Fig. 3. SEM micrograph of MRSA ATCC 33591 biofilm formed on glass slides in the presence

614

and absence of VREX at MBIC.

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S. Arslan, F. Ozkardes, Slime production and antibiotic susceptibility in staphylococci

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615

Fig. 4. Effect of VREX at MBIC on the viability of MRSA and clinical isolates. Image

616

representing the spectroscopic reading of metabolically active cells treated with and without

617

VREX. Mean values of triplicate independent experiments and SD are shown. Student-t Test was

618

used to compare the control and treated. * indicates significant at

619

significant at P≤ 0.0005 and *** indicates significant at P≤ 0.025 of treated compared to control.

620

Fig. 5. Effect of VREX at MBIC on EPS production. Mean values of triplicate independent

621

experiments and SD are shown. Student-t Test was used to compare the control and treated. *

622

indicates significant at P≤ 0.005, ** indicates significant at P≤ 0.025 and *** indicates

623

significant at P≤ 0.0005 of treated compared to control.

624

Fig. 6. The level of slime production by MRSA and its clinical isolates in the presence and

625

absence of VREX at MBIC in CRA plates.

626

Fig. 7. Effect of VREX on hemolysin production of MRSA and its clinical isolates. Mean values

627

of triplicate independent experiments and SDs are shown. Student-t Test was used to compare

628

the control and treated. * indicates significant at P≤ 0.005, ** indicates significant at P≤ 0.0005

629

of treated compared to control.

630

Fig. 8. FT-IR spectra of MRSA and clinical strains treated with and without the VREX. Dashed

631

rectangles represent regions having spectral alterations in the treated samples when compared to

632

the untreated control.

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Fig. 9. Light microscopic images showing non-inhibitory activity of VREX at MBIC on

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preformed biofilms of MRSA and clinical isolates.

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Fig. 10. Gene expression analysis represents transcription level of MRSA ATCC 33591 after the

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treatment with MBIC of VREX. Mean values of triplicate independent experiments and SDs are

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shown. Student-t Test was used to compare the control and treated. * indicates significant at

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P≤ 0.01, ** indicates

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P≤ 0.0005, ** indicates significant at P≤ 0.005 and *** indicates significant at P≤ 0.025 **

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indicates significant at P≤ 0.001 of treated compared to control.

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Fig. S1. GC-MS chromatogram of VREX.

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ACCEPTED MANUSCRIPT Table 1 List of primer sequences used for Real time PCR analysis

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icaA icaD sea clfA cna fnbA fnbB 16S rRNA

Nucleotide sequence of primers (5'-3') Forward primer Reverse primer ACACTTGCTGGCGCAGTCAA TCTGGAACCAACATCCAACA ATGGTCAAGCCCAGACAGAG AGTATTTTCAATGTTTAAAGCA ATGGTGCTTATTATGGTTATC CGTTTCCAAAGGTACTGTATT ATTGGCGTGGCTTCAGTGCT CGTTTCTTCCGTAGTTGCATTTG AAAGCGTTGCCTAGTGGAGA AGTGCCTTCCCAAACCTTTT ATCAGCAGATGTAGCGGAAG TTTAGTACCGCTCGTTGTCC AAGAAGCACCGAAAACTGTG TCTCTGCAACTGCTGTAACG ACTCCTACGGGAGGCAGCAG ATTACCGCGGCTGCTGG

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Surface to bio-volume ratio (µm2/µm3) Control Treated 0.0308 0.0468 0.0305 0.0413 0.0344 0.0587 0.0262 0.0477 0.0307 0.0400

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Avg. Thickness (µm) Control Treated 36.46 22.55 36.83 25.99 31.97 17.58 44.26 22.1 36.52 26.94

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MRSA GSA - 44 GSA – 45 GSA – 410 GSA - 395

Biofilm Biomass (µm3/µm2) Control Treated 38.39 23.74 38.79 27.36 33.65 18.51 46.59 23.26 38.44 28.36

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Zone of inhibition (mm) Clindamycin (10µg) Gentamycin (50µg) control Treated control Treated R R 18.67±0.58 22.67±0.58 24.33±1.15 32.66±0.58 6.33±0.58 13.33±1.15 25.33±0.58 30.66±0.58 7.67±1.15 14.00±1.00 22.00±1.00 26.66±1.15 12.67±1.53 15.67±1.53 24.33±0.58 27.00±1.00 18.33±1.53 21.67±1.58

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Amikacin (10µg) control Treated 14.67±0.57 20.67±0.58 11.33±0.57 14.00±0.58 7.00±1.00 15.33±1.15 12.66±0.58 17.66±0.57 15.00±1.00 20.66±1.15

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MRSA GSA – 44 GSA – 45 GSA –410 GSA – 395

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Table 3 Antibiotic sensitivity pattern of MRSA and its clinical isolates in the presence and the absence of VREX. Data represents the mean values of triplicate independent experiments and SD. R - Resistant

Vancomycin (10µg) control Treated 13.33±0.58 17.33±0.58 13.67±0.58 18.33±0.58 13.00±0.00 17.67±1.53 12.33±1.53 18.33±0.58 11.33±0.58 26.33±1.53

ACCEPTED MANUSCRIPT Table 4: List of compounds identified from VREX through GC-MS analysis Peak Area (%) 1.26 1.77 2.02 1.50 4.83 2.14 3.15 3.11 11.43 2.12 2.16 4.93 4.82 10.63 4.01 2.02 0.95 2.56 1.06 1.17

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Name of the major compounds identified (common name) Methyl steviol α-Eudesmol* Cyclosativen* Trichoacorenol* 2-Propenoic acid, tetradecyl ester 6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydro-naphthalen-2-ol α-Gurgujene* Methyl zizanoate gamma.-Himachalene* 2,3,5,5,8A-Pentamethyl-6,7,8,8a-tetrahydro-5h-chromen-8-ol Valerenic acid* Valerenyl acetate* Germacrone 12-Oxatetracyclo[4.3.1.1(2,5).1(4,10)]dodecane, 11-isopropylideneNootkatone* 1H-2-Indenone,2,4,5,6,7,7a-hexahydro-3-(1-methylethyl)-7a-methyl 1,4-Dimethyl-2-cyclohexylbenzene Longifolene* Campasterol 9.beta.-Acetoxy-3,5.alpha.,8-trimethyltricyclo[6.3.1.0(1,5)]dodec-3-ene

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RT-Retention time, * - Sesquiterpenes

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RT 18.874 21.101 21.182 21.404 21.801 22.052 22.594 22.706 23.022 23.197 23.408 23.821 24.318 24.506 24.754 25.906 28.045 45.678 46.033 46.258

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Highlights

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Biofilm formation is the major cause of bacterial pathogenesis and its drug resistance. V. zizanioides root extract (VREX) deflates the biofilm formation of MRSA and its clinical strains. At MBIC, VREX reduces the synthesis of virulence factors which was evidenced by FTIR analysis. GC-MS analysis revealed the presence of sesquiterpenes as major constituents in the VREX. Down regulation of adhesion genes portrays the action mechanism of VREX.