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Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus
Accepted Manuscript Title: Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus Authors: Sai Nithya, T.R. Nimal, Gaurav Baranwal, Maneesha K. Suresh, Anuj C.P., V. Anil Kumar, C. Gopi Mohan, R. Jayakumar, Raja Biswas PII: DOI: Reference:
S0141-8130(17)34750-5 https://doi.org/10.1016/j.ijbiomac.2018.01.083 BIOMAC 8915
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
30-11-2017 5-1-2018 13-1-2018
Please cite this article as: Sai Nithya, T.R.Nimal, Gaurav Baranwal, Maneesha K.Suresh, Anuj C.P., V.Anil Kumar, C.Gopi Mohan, R.Jayakumar, Raja Biswas, Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2018.01.083 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.
Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus Sai Nithya1, T R Nimal1, Gaurav Baranwal1, Maneesha K Suresh1, Anju C P1, V Anil
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Kumar2, C Gopi Mohan1, R Jayakumar1*, Raja Biswas1*
Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical
Department of Microbiology, Amrita Institute of Medical Sciences and Research Centre,
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Sciences and Research Centre, Amrita Vishwa Vidyapeetham,Kochi-682041, India
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Amrita Vishwa Vidyapeetham, Kochi-682041, India
__________________________ Authors contributed equally.
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*Corresponding authors: R. Jayakumar ([email protected]) Raja Biswas ([email protected]) Tel: +91-484-2801234 (ext. 8762); Fax: +91-484-2802020. 1
Abstract: Lysostaphin (LST) is a bacteriocin that cleaves within the pentaglycine cross bridge of Staphylococcus aureus peptidoglycan. Previous studies have reported the high efficiency of LST even against multi drug resistant S. aureus including methicillin resistant S. aureus (MRSA). In this study, we have developed a new chitosan based hydrogel formulation of LST to exploit its anti-staphylococcal activity. The atomic
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interactions of LST with chitosan were studied by molecular docking studies. The
rheology and the antibacterial properties of the developed LSTC gel were evaluated. The
developed LST containing chitosan hydrogel (LSTC gel) was flexible, flows smoothly and remains stable at physiological temperature. The in vitro studies by agar well
diffusion and ex vivo studies in porcine skin model exhibited a reduction in S. aureus
survival by ~3 Log10CFU/mL in the presence of LSTC gel. The cytocompatibility of the
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gel was tested in vitro using macrophage RAW 264.7 cell line and in vivo in Drosophila
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melanogaster. A gradual disruption of S. aureus biofilms with the increase of LST concentrations in the LSTC gel was observed which was confirmed by SEM analysis. We
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conclude that LSTC gel could be highly effectual and advantageous over antibiotics in
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treating staphylococcal-topical and biofilm infections.
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1. Introduction
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Keywords: Staphylococcus aureus skin infection; lysostaphin; chitosan gel.
About 20–60 per cent of human populations are carriers of Staphylococcus aureus (S.
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aureus) [1]. This Gram positive cocci colonizes our skin and mucus membranes; and acts as a causative agent for variety of skin infections e.g., folliculitis, furuncles and
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carbuncles, abscess, sycosis, impetigo, ecthyma, cellulites, scabies, mastitis, diabetic ulcers, staphylococcal scalded skin syndrome, scarlet fever and urinary tract infections
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[2,3].
In nosocomial setting transmission of S. aureus occurs often between patients;
and between patients and care takers at higher rates. Staphylococcal biofilm once formed on an implanted medical device or on catheters, are extremely difficult to disrupt and treat using antibiotics [2,4,5]. Currently mupirocin, bacitracin, triclosan and mersacidin are popularly used for topical treatment to prevent S. aureus skin colonization and infections, however 2
resistance against these anti-microbial agents are often reported in the literature [6–9]. Therefore, there is a need to develop an anti-staphylococcal agent to successfully eradicate S. aureus infections. Lysostaphin (LST), a 26 kDa zinc metalloprotease that rapidly lyse the cell wall of S.aureus [10], is reported in the literature as an excellent anti-staphylococcal agent for
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the treatment of topical infections in experimental animals [11,12]. Lysostaphin cleaves
between the third and fourth Glycine residues of the pentaglycine cross bridge of S.
aureus peptidoglycan [13]; and has no effect on bacteria of other genera [14]. Lysostaphin was previously tested in several animal models to prevent nasal colonization,
for the treatment of burn wound infections, against aortic valve endocarditis and sepsis [15–18]. These results indicate LST was highly effective anti-microbial agent.
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Experiments in neonatal mice model demonstrated lysostaphin is even better than
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vancomycin in treating methicillin resistant S. aureus (MRSA) sepsis [19]. Chitosan is a natural, biodegradable and non-toxic polymer, obtained
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commercially by the deacetylation of chitin, which is a naturally occurring
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polysaccharide [20]. It is insoluble at neutral and alkaline pH. Some of the important applications of chitosan as an excipient in pharmaceutical products include solid dosage
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forms, controlled release dosage forms, gels, microspheres, microcapsules and wound
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healing products [21,22]. Chitin and chitosan derivatives have become a major focus of interest in biomedical and biopharmaceutical research applications especially as promising biomaterials in tissue engineering, in wound healing, in drug delivery and in
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gene delivery [23–25].
In this study, the lysostaphin-chitosan (LSTC) gel was prepared; and it’s
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rheological and antibacterial properties were studied. The efficacy of LSTC gel to disrupt biofilms formed by S. aureus was tested. The antimicrobial activity of LSTC gel, was
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also tested in an ex vivo porcine skin model. Our in vitro and ex vivo studies performed with LSTC gel have shown that this staphylococcin can be used to prevent or treat staphylococcal infections. 2. Materials and methods 2.1 Materials
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The polymer chitosan (MW: 100-150 kDa; degree of deacetylation: 85%) was obtained from Koyo Chemical Industry Ltd, Japan. Dulbecco’s Modified Eagle’s Medium (DMEM) and Alamar Blue were obtained from Invitrogen, India. Fetal Bovine Serum (FBS), Luria Bertani (LB) Broth and Agar-agar were purchased from Himedia, India. All chemicals were used without further alteration and purification.
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2.2 Computational Studies
2.2.1 Preparation of Lysostaphin and Chitosan gel Structures
The three-dimensional (3D) X-ray crystal structure of LST from Staphylococcus simulans at 3.5 Å resolution was retrieved from protein databank (PDB) with PDB id: 4LXC [10,26]. The 3D structure was then prepared by assigning bond orders and the missing hydrogen atoms were added using PrepWiz module of Schrӧdinger (Protein Preparation
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Wizard, Schrödinger, LLC, New York, NY, 2016). Further, restrained energy
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minimization of the structure was performed using OPLS-2005 force field. Chemical structure of chitosan consisting of five monomer units was modelled
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using ChemBio Ultra 11.0 software. The number of chitosan monomer units was
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restricted to five to reduce the structural complexity as well as the computational cost. The modelled chitosan structure was geometry optimized to attain a stable atomic
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ChemBio Ultra software.
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arrangement using MMFF94 (Merk Molecular Force Field) method implemented in
2.2.2 Molecular Docking of LST with Chitosan gel Molecular docking calculation was performed to understand the LST-chitosan atomic
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level interactions. Prior to docking, the input structure in .pdbqt format was prepared using AutoDock 4.0 [27]. The active site of LST was reported by Sabala et al [10] and the
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docking grid box was specified such that it covers the entire active site region of the protein with X, Y and Z dimensions of 18 x 18 x 18 Å respectively. Autodock Vina [28]
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software was used further to perform LST-chitosan molecular docking calculations. Vina is an open source docking program based on the sophisticated gradient optimization method for local optimization procedure. Its scoring function was computed as: ΔGbind = ΔGvdW + ΔGelec + ΔGH-bond + ΔGhydrophobic + ΔGtors Where ΔGbind is the total binding energy, ΔGvdW is the binding energy owing to Vander Waal’s forces, ΔGelec is the binding energy due to electrostatic interactions, ΔGH-bond term 4
accounts for the hydrogen bonding interactions, ΔGhydrophobic is the binding energy due to hydrophobic interactions and finally ΔGtors is the energy due to the rotational bonds and atoms that are involved in torsions. Furthermore, the LST-chitosan complexes obtained after molecular docking were inspected for various 3D interactions using PyMOL (The PyMOL Molecular Graphics
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System, Version 1.8 Schrödinger, LLC) molecular visualization tool. 2.3 Bacterial strain
S. aureus strain SA113 (American Type Culture Collection 35556) and other methicillin
resistant S. aureus (MRSA) clinical strains were routinely grown in Tryptic soy broth (TSB) medium at 37°C with shaking at 160 rpm. For biofilm experiments, S. aureus were grown in filter sterilized TSB media containing 0.5% glucose under static
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conditions for 48 h [26, 27].
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2.4 Preparation of LSTC gel
2 g chitosan powder was dissolved in a 100 mL solution of 1% (v/v) acetic acid and left
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for stirring overnight. 1% (w/v) sodium hydroxide (NaOH) was added in drop-wise
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manner in order to neutralize the obtained viscous chitosan solution to a pH of 7.4. The gel was then harvested by centrifugation at 3,000 rpm for 10 min (Beckman Coulter
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Avanti J-26XP centrifuge using JA-17 rotor). The gel was further washed thrice with
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Phosphate-buffered saline (PBS) to remove traces of acetic acid and stored at 4°C. Different concentrations of LST were added to the chitosan gel and mixed well to prepare LSTC gel containing 0%, 0.0125%, 0.025% and 0.05% lysostaphin [29].
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2.5 Rheological studies of the lysostaphin-chitosan gel 2.5.1 Viscoelastic study
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Rheological studies were performed in order to assess the strength, flow and stability of the LSTC gel using Malvern Kinexus pro rheometer with a stainless steel parallel plate
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with a diameter of 20 mm [30]. A constant gap of 0.5 mm was maintained between the upper and lower plates at a temperature of 25ºC. Chitosan gel without LST served as control. First, the amplitude sweep was performed to find the Linear Viscoelastic Region (LVER) of the gel. The storage modulus (G’), loss modulus (G’’) and phase angle (δ) at LVER were measured at different strain percentage. The amplitude sweep was started from a strain percentage of 10-1 and the end point was determined automatically by the 5
rheometer. The gel strength and solid/liquid dominating behavior of LSTC gel was determined by studying the frequency sweep between 101 to 10-1 Hz in the LVER region of the gel. 2.5.2 Temperature stability tests The complex modulus and complex viscosity of the gel was measured from 25 to 45ºC at
gel from room temperature to human body temperature. 2.5.3 Analysis of yield stress
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constant frequency and shear [30]. This study was performed to test the stability of the
Yield stress analysis was performed by plotting stress against rheological shear strain.
The shear strain was applied from 10−1%. The higher the yield stress, more stress is required to initiate the flow of sample.
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2.5.4 Power law model fit
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The Power law can be used to mathematically express the power law region of the flow
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curve as follows: σ = kỳn
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Eq. (1)
law index.
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2.5.5 Injectability
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where ‘σ’ is the shear stress, ‘k’ is consistency index, ‘ỳ’ is shear rate and ‘n’ is Power
The injectability of the gel was observed by loading them into a 1 mL syringe without
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needle and subjecting them to manual shear. The flow of the gel from the syringe was observed visually [30].
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2.5.6 Inversion test
The flow of gel under the influence of gravity was observed using inversion test [31]. The inversion test confirms the property of the gel to remain in the infection site without
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falling. LSTC gel was taken in a flat bottomed cylindrical bottle and placed on a flat surface without any disturbance. A control was also used. The flow of the gel was observed visually at 0th, 1st, 2nd, 3rd, 4th and 5th day time points. 2.6 In vitro lysostaphin release study To quantify the LST released from chitosan gel, 100 µl of double distilled water was added to100 mg of LST loaded chitosan gel and the gel suspension was incubated at 37ºC 6
with ambient shaking. At frequent time intervals the gel suspension was centrifuged at 5000 rpm for 5 min (EBA 21 Hettich Centrifuge). 10 µl supernatant was collected from the sample at 0, 3, 6, 12, 24, 48 and 72 h and the double distilled water was replaced each time. The supernatant collected at different time intervals was analysed by Bradford assay. Protein standards were prepared using bovine serum albumin (BSA). Percentage of
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lysostaphin release was calculated using the following formula [32]:
Release (%) = (Amount of lysostaphin released at definite time)/ (Total amount of lysostaphin entrapped within the gel) X 100.
Eq. (2)
2.7 Hemolysis Assay
The hemolysis assay was performed to measure the extent of hemolysis caused by LSTC
gel when it comes into contact with human blood. 200 µL of diluted human whole blood
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(1:10 in PBS) was taken in several 1.5 mL vials and increasing amount of 50 mg CL gel
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(containing 0%, 0.0125%, 0.025% and 0.05% LST) were added into the diluted blood. The vials were incubated in 37ºC incubator for 48 h. 0.1% (v/v) Triton-X 100 treated
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blood samples served as positive control. Following incubation, the samples were
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centrifuged at 5000 rpm for 5 min (Minispin centrifuge) and the absorbance of supernatant was measured at 610 nm using microtiter plate reader [33,34]. The percent of
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hemolysis was calculated using the formula:
CONTROL)
X 100.
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Hemolysis (%) = (O.DTEST – O.DNEGATIVE CONTROL)/( O.DPOSITIVE CONTROL - O.DNEGATIVE Eq. (3)
2.8 pH and temperature dependent activity assay
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Overnight culture of S. aureus was pelleted down and the cell density was adjusted to 0.5. The cells were resuspended in 50 mM sodium acetate buffer (pH 4), PBS (pH7.0) and 50
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mM Tris-HCl buffer (pH 10). 10 µg/mL of LST was added and O.D. was measured at 610 nm for different time intervals. The bacterial culture where no gel was added served
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as the control. To study the temperature dependent activity of LST, LSTC gel was incubated at temperatures ranging from 20°C to 100°C for 10 min and added to the S. aureus culture. The O.D. of the S. aureus culture was measured at 610 nm to understand the lytic activity of LST. 2.9 Cell Culture
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Macrophage RAW 264.7 (ATCC® TIB-71™) cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO2 atmosphere. 2.9.1 Cell viability Studies The effect of LSTCL gel on the viability of macrophage RAW 264.7 cell lines were
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tested using Alamar blue (resazurin) assay. Alamar blue (resazurin) can be used as a direct indicator to quantitatively measure cytotoxicity. Alamar blue reagent is weakly fluorescent blue indicator dye. Resazurin reagent can acts as an intermediate electron acceptor in the electron transport chain without interfering with the normal electrons
transfer process. Within viable cell resazurin gets reduced to resorufin which is highly fluorescent and pink in color. The RAW 264.7 macrophage cells were seeded in 96 well
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plates at a density of 10,000 cells/well in DMEM medium supplemented with 10% FBS.
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50 mg LSTC gel (containing 0%, 0.0125%, 0.025% and 0.05% lysostaphin) were added into the wells. The untreated and 0.1% (v/v) Triton X-100 treated RAW 264.7
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macrophage cells were used as positive and negative control, respectively. Following
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incubation the culture medium was replaced with 10% (v/v) Alamar blue in DMEM and further incubated for 4 h. The absorbance of the solution from each well was measured at
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570 nm and 600 nm using microtiter plate reader [33]. The percent cell viability was
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measured using the following formula: Cell viability (%) = (O.D
TEST
at 570 nm - O.D
TEST
570 nm - O.D POSITIVE CONTROL at 600 nm) X 100.
at 600 nm)/( O.D POSITIVE CONTROL at Eq. (4)
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2.10 In vitro antibacterial activity 2.10.1 Agar Well Diffusion
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The agar well diffusion assay was used to determine the susceptibility of S. aureus to LSTC gel. S. aureus cultures adjusted to 0.5 McFarland standards and were spread plated
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on LB agar plate. Wells of 8 mm diameter were made using biopsy punch. 100 mg of gel containing varying concentrations of LST were added into the wells and incubated at 37ºC for 24 h. The inhibition of bacterial growth around the wells was observed [35–37]. 2.10.2 Growth curve 1% of SA113 overnight cultures were added to fresh LB medium and the growth of S. aureus was measured at defined time interval for 48 h. 100 mg of LSTC gel containing 8
varying concentrations of LST was added to the 5 mL of cultures after 6 h of growth and the O.Ds were determined using a spectrophotometer (BioTek) at 600 nm [35]. 2.10.3 Lysis assay in simulated nasal fluid The activity of lysostaphin was tested in a buffer that simulates the ionic composition of nasal fluid and sweat. The simulated nasal fluid (SNF) contains 0.87% NaCl, 0.088%
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CaCl2. 2H20, 0.31% KCl and 0.636% bovine serum albumin (BSA) [38]. Stationary phase cultures of S. aureus were pelleted down, washed twice with SNF and resuspended
in the same buffer. The cell density was adjusted to 0.5 at 600 nm. 100 mg of LSTC gel containing varying concentrations of LST was added to 5 mL of SNF buffer containing S. aureus and O.Ds were measured at hourly intervals for 6 h. S. aureus cells resuspended in SNF without the addition of the LST was served as control.
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2.10.4 Microscopic studies
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Overnight culture of S. aureus was pelleted down by centrifugation at 5000 rpm for 10 min and resuspended in 2 mL of PBS. 10 µg/mL of lysostaphin in 100 mg chitosan gel
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microscope at different time intervals.
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was added to the culture. An aliquot was taken and viewed under the phase contrast
2.11 Ex vivo antibacterial activity
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Pig ear skin was obtained from a local slaughter house and ex vivo testing of S. aureus
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survival in pig skin was performed. The skins were thoroughly washed with sterile saline and hair was removed. Excess fat deposits were removed using a sterile scalpel and were cut into sections of 2 cm × 2 cm. The skin sections were disinfected by soaking in 70%
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ethanol for 5 min. These disinfected sections were then placed on sterile petri-plates and allowed to dry. 100 µl of microbial suspension (~ 5 X 109 cells) was added to each
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section. The sections were incubated at 37°C for 30 min. After 30 min, 100 mg of LSTC gel containing 0, 0.0125, 0.025 and 0.05% lysostaphin was added onto the sections. The
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samples were incubated further for a period of 6 h at 37°C. Following incubation, the sections were taken in 15 mL falcon tubes containing 1 mL PBS and vortexed for 5 min vigorously to release the microbes adhered onto the skin [39]. To determine the number of viable bacteria present on the skin the samples were serially diluted and plated on mannitol salt agar plates. The plates were incubated at 37°C overnight for colony enumeration. 9
2.12 Stability assay An aliquot of LSTC gel was taken in 3 vials and stored at different temperatures (4, 25 and 37˚C). For every 5 days upto 30 days, well diffusion assay was performed with the gel stored at different temperatures. The zone of inhibition around the wells was measured [35].
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2.13 Biofilm assay
Catheter tubes which were cut into small pieces of approximately 2 cm, were cleaned by
dipping in 70% ethanol and placed into 1.5 mL centrifuge tubes containing 750 µL of LB medium with 0.5% glucose (LBG). The biofilms were allowed to form on catheter tubes
for 48 h at 37˚C. After incubation, the catheter tubes were washed once with PBS and
placed in a new tube with fresh LBG media and varying concentrations of lysostaphin (0,
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5, 10, 20 and 80 µg/mL) containing chitosan gel. The catheter tubes were then dipped
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into these vials and left for 24 h incubation. Catheter tube dipped in the microbial culture without any lysostaphin or mupirocin was used as the control. Post incubation, the
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catheter tubes were dehydrated, sputter coated and imaged using SEM (Scanning electron
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microscope). To further assess the effect of LSTC gel on S. aureus biofilm, the above mentioned procedure was repeated. The catheter tubes were washed after the LSTC gel
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treatment and rolled on mannitol salt agar base plates as described before. The plates
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were incubated for a period of 24 h and the growth of the bacteria was observed [35]. 2.14 In vivo toxicity studies in Drosophila model Wild-type D. melanogaster (Flybase Stock 2: FBst0000002) were cultured on classical
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banana agar medium at room temperature (26ºC) with 60% humidity. 2 to 5 day old female flies were used for the toxicity assays. 10 female flies were fed with banana media
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that contained 0.05% LSTC gel (Chitosan gel containing maximum % of LST). The flies were allowed to feed for 14 days and the survivability was observed. Another set of flies
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were fed with classical banana media (without the LSTC gel) which served as a control [35]. To confirm the authenticity of the results these experiments were repeated thrice.
3. Results and discussion 3.1. Molecular docking simulation studies Initial computational studies were carried out to rule out possible interactions between LST and chitosan. LST-chitosan interaction study was performed using crystal structure 10
of LST and computationally modelled five units of chitosan. Molecular docking calculations are being used in recent studies to understand the protein-polymer and polymer-drug interactions [40]. We performed molecular docking of chitosan polymeric gel of five units at the active site of LST. LST has a Zn2+ ion at the active site tunnel region [10] as shown in Figure 1 which resulted in positive potential at this regions.
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Experimentally, an instant release of LST from chitosan gel was observed and the mechanism of binding was computed using the molecular docking technique.
Our molecular docking studies resulted in positive binding energy 10.1 kcal/mol
between chitosan and lysostaphin, suggesting its weak (repulsive) binding affinity.
Docking studies showed hydrogen bonding of chitosan gel with the amino acid residues ASN 372, GLY 309, GLY 310, HIS 362 and THR 357 at the active site of lysostaphin
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(Fig. 1B-D). However, the positive binding energy indicates weak binding affinity
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between protein-gel complexes. This might be the reason why chitosan, a positively charged polymer shows very weak interaction with lysostaphin. In support with the
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experimental lysostaphin release studies, the instant release of enzyme from the chitosan
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gel could be due to the weak binding of lysostaphin and chitosan.
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Fig. 1. (A) Structure of S. aureus peptidoglycan and lysostaphin cleavage site. (B-D)
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Docked complexes of Lysostaphin – chitosan (5 units) (B), chitosan interacting residues at the active site of lysostaphin (C), molecular surface representations of lysostaphin with
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chitosan showing Zn2+ ion and tunnel region (D). Interactions viewed using PyMOL software. The cartoon represents the protein, the sticks represent the Chitosan (5 unit),
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blue sphere shows the zinc ion (Zn2+) at the active site of lysostaphin and the yellow dotted line shows the hydrogen bonding interactions. 3.2. Rheological studies
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3.2.1. Viscoelastic analysis First, the amplitude sweep was performed which records the linear viscoelastic region (LVER) of the gel. LVER is the region in which any material can elastically strain and return to its original state when the strain is removed. To understand the gel behavior
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over a period of time, a frequency sweep was carried out. The G` of the gel was obtained by subjecting it to a frequency range of 101 to 10-1 Hz (Fig. 2A). G` of control and lysostaphin-chitosan gel was found to be 14.58 ± 0.27 and 12.68 ± 0.38 respectively
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(Table 1). The viscosity of the gel was analyzed using the flow curve. The viscosity (η) of the gel was measured at different shear rates (Y) from 10 -1 to 102 s-1. The flow curve
indicated that the viscosity reduced as shear rate was increased from 0.001 to 100 s-1 (Fig. 2D). For any material to have a gel-like nature, it must have a dominant solid nature with a liquid component. The phase angle (δ) of the gel was below 10°, which indicated the
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solid dominating nature of the gels (Fig. 2C). For pure solids, δ=0° and for liquids δ= 90°.
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Phase angle of gel showed between 0-10° shows that it is of a solid nature with a minor
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liquid component.
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Table 1. Rheological properties of control, chitosan gel and test, lysostaphin-chitosan gel.
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Storage Modulus(K Material Name Pa)
Power Law Index (n)
Correlati on Coefficie nt
Yield Stres s (Pa)
Comple x Shear Strain (%)
Shear Viscoci ty (Pa s)
14.586+0.27 7
0.0385 4
0.9733
5970
74.6442
1.9
Chitosa_lysosta phin
12.686+0.38 6
0.0335 6
0.9888
6256
69.274
1.343
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Chitosan
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Fig. 2. Rheological analysis. (A) Frequency sweep analysis of control and chitosan containing lysostaphin representing storage modulus (G’), loss modulus (G”) and phase
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angle (δ). (B) Temperature stability analysis. (C) Power law model fit. (D) Viscosity vs
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shear rate. (E) Yield stress analysis. (F) Injectability test. (G) Inversion test. (H) LST release assay from LST-chiotsan gel.
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3.2.2. Temperature stability analysis The gel appeared to be considerably stable within the given temperature range (Fig. 2B).
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This indicated that lysostaphin-chiotsan gel would be stable under physiological temperatures without much variation in their strength. 3.2.3. Yield stress analysis
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Yield stress analysis was performed by plotting stress against rheological shear strain. The shear strain was applied from 10−1%. Yield stress analysis showed that there was a slight increase in the complex shear strain percentage of the gel containing lysostaphin when compared to the control (Fig. 2E). This indicated that the lysostaphin-chitosan gel was flexible in nature and can flow smoothly upon shearing. 3.2.4. Power law fit analysis 14
To further understand the rheological properties of the gel as pseudoplastic, Newtonian or dilatant, data were modeled using the power law Eq. (1). Both the control and lysostaphin-chitosan gel showed good fitting with correlation coefficient of 0.97 and 0.98 respectively (Fig. 2C). The n values for both the test and control were below 1, which confirmed they were pseudoplastic, i.e. shear thinning in nature.
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3.2.5. Injectability
Injectability test was performed by applying manual shear using a 1 mL syringe without needle and observing the evenness of the flow of the gel. The gel showed good injectability and continuous flow (Fig. 2F). 3.2.6. Inversion test
From the inversion test it was noted that the gel did not flow under the influence of
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gravity even after 5 days (Fig. 2G), which may be due to the adhesive nature of the
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chitosan gel. This property indicated that the gel will not flow unless an external shear is applied, thus making it remain on the infection site without falling.
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3.3 Release assay
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The supernatant collected by carrying out release assay was estimated using Bradford assay. The results show that the chitosan gel containing lysostaphin gave a sustained
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release of the enzyme into the supernatant over a period of time (Fig. 2H) and almost 90-
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95% of the enzymes get released in 24 h. 3.4 LSTC gel inhibited the growth of S. aureus We first tested the in-vitro antimicrobial activity of LSTC gel against S. aureus. Agar
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plate well diffusion assay results demonstrated that LSTC gel inhibited the growth of S. aureus. The growth inhibition zone diameter of S. aureus was increased with increasing
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concentrations of LST within the LSTC gel (Fig. 3A). Addition of 40 mg of 0, 0.0125, 0.025 and 0.05% LSTC gel per well resulted in the production of growth inhibitory zone
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diameters of 1.2 cm, 1.5 cm and 1.8 cm, respectively. No growth inhibition of S. aureus was observed when chitosan gel alone (without LST) was used for the agar plate well diffusion assay. Mupirocin (Mup) ointment was used as positive control (Fig. 3A). Similar results were obtained when the growth of S. aureus was tested in broth culture. When cultures of S. aureus were treated using LSTC gel, growth of S. aureus was inhibited irrespective of it’s growth state. LG gel rapidly induces the lysis of S. aureus 15
culture (Fig.
3B). As expected when tested against
other pathogens,
such
as Pseudomonas aeruginosa LSTC gel did not show any anti-microbial activity (data not
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shown).
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Fig. 3. (A) Well diffusion assay carried out with different concentrations of LST in 100
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mg chitosan gel. (B) Graph curve of S. aureus. Increasing concentrations of LST was added to S. aureus culture after 6 h of its growth. The growth of S. aureus was monitored
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further for 48 h. (C) LST induced lysis of S. aureus. The S. aureus cells were suspended
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in simulated nasal fluid (SNF) at a cell density of 0.5 at 600 nm. 100 mg of LSTC gel containing varying concentrations of LST was added to SNF buffer containing S. aureus and O.Ds were measured at hourly intervals for 6 h. (D) Microscopic images
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demonstrating the lytic activity of LSTC gel against S. aureus at different time intervals. (E) LSTC gels were incubated at different temperatures for 10 min and then added to S.
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aureus to test their lytic activity. (F) pH dependent activity of LST from LSTC gel. Maximum LST activity was observed at pH 7.0.
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In order to confirm the lytic activity of LSTC gel against S. aureus in the nasal secretion we used simulated nasal fluid. Rapid lysis of S. aureus cells were observed following addition of the 100 mg of LSTC gel within the simulated nasal fluid containing S. aureus of OD 600 nm. Complete lysis of S. aureus cells were observed within 1 h when 0.05% LSTC gel was added to the S. aureus culture (Fig. 3C, D). 3.5 Temperature and pH dependent enzymatic activity of LSTC gel 16
In order to understand the stability of LSTC gel we prepared the gel and incubated at different temperatures before the lysis assays. Incubation of LSTC gel till 90ºC for 10 min caused only slight alteration of it’s enzymatic activity, however incubation above 90ºC caused rapid loss of it’s enzymatic activity (Fig. 3E). As the pH of all human secretions such as sweat, nasal fluid, urine and tears varies between 4.5 -7.0, we tested
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the enzymatic activity of LSTC gel at pH 2.0 and 10.0. The lytic activity of lysostaphin
from the LSTC gel was maximum at pH 7.0. Around 90% of S. aureus cells were lysed in 2 h in presence of LSTC gel. However, activity of the LST from the LSTC gel was reduced by 40% at pH 4.5. Results indicate LSTC gel showed maximum lytic activity at neutral pH (Fig. 3F).
3.6 In vitro cytocompatibility and in vivo biocompatibility of LSTC gel
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The blood compatibility is an important parameter of biological safety for any therapeutic
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formulations. Therefore, hemolysis assays were carried out using LSTC gels containing different concentrations of lysostaphin (Fig. 4A). No significant erythrocyte lysis was
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were added into human blood.
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observed with LSTC gels containing different concentrations of lysostaphin (Fig. 4B)
The cytotoxic effect of the LSTC gel was evaluated in RAW 264.7 cell lines and
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using alamar blue assay. The cell viability plot showed that more than 97% cells were
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viable after 24 and 48 h of incubation even in presence of varying concentrations of lysostaphin containing LSTC gel (Fig. 4C). The non-toxic nature of chitosan gel alone was well documented by us in our several previous studies. No alteration of cell
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morphology of RAW 264.7 cell lines was observed under the microscope in presence of LSTC gel. The alamar assay confirmed that LSTC gels are non-toxic and are cyto-
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compatible.
Further the toxicity of the LSTC gel was tested using fly feeding assays. D.
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melanogaster flies were allowed to be fed with LSTC gel containing different concentrations of lysostaphin for 1 week. The experiment was carried out to check whether the gel was toxic to the flies. At the end of the 14 day observation period, it was found that lysostaphin-chitosan gel was non toxic to the flies and all flies survived the experiment (Fig. 4D).
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Fig. 4. (A) Varying concentrations of lysostaphin in chitosan gel were added to diluted
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blood, the tubes were incubated for a period of 48 h and the hemolytic activity was
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observed. (B) Bar diagram showing the percentage hemolysis of different concentrations of lysostaphin in chitosan gel. (C) Bar diagram representing the cytotoxicity assay carried
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out in cell lines. (D) The survival plot showing the number of flies that survived the toxicity assay. At the end of the 14 day observation period, it was found that lysostaphin-
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chitosan gel was not toxic to the flies and that all the flies survived the experiment. 3.7 Disruption of S. aureus biofilm by LSTC gel
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The ability of LSTC gels to disrupt S. aureus biofilms were tested using silicon catheter tubes. S. aureus was allowed to adhere with the catheter tubes for 48 h and then treated
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LSTC gels containing different concentrations of LST. We found gradual disruption of S. aureus biofilms with the increase of LST concentrations. Biofilm formation was
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decreased by 2.5, 3.0 and 5.5 fold in presence of 0%, 0.0125%, 0.025% and 0.05% LST containing LSTC gel (Fig. 5A-C). Scanning electron microscopic images confirmed that S. aureus biofilms were disrupted after treatment with LSTC gels and maximum biofilm disruption was observed when the biofilm was treated with 0.5 % LST containing LSTC gel (Fig. 5D).
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Fig. 5. (A) Catheter tubes were taken in centrifuge tubes containing S. aureus culture,
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incubated for 48 h, washed, transferred to media containing varying concentrations of
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LST in LSTC gel and incubated for 24 h. (B) The rolling assays were performed to
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demonstrate reduced S. aureus attachment with the silicon catheters when treated with LSTC gel. (C) Graph showing the percentage survival of S. aureus cells on the catheter
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tubes after treatment with LSTC gel. (D) SEM images of catheter tubes after treatment with LSTC gels containing different percentages of LST.
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3.8 Ex vivo activity of LSTC gel against S. aureus The S. aureus infected skin sections were treated with LSTC gel (Fig. 6A). The amount
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of live bacteria from the skin surface were extracted and enumerated. Dose dependent decrease in S. aureus survival was observed when infected porcine skin was treated with
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LSTC gel containing increasing concentrations of LST. Bacterial survival was decreased
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by 1.0, 2.3 and 4.2 Log10CFU/mL after treatment with LSTC gels (Fig. 6B).
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Fig. 6. (A) Porcine ear skin was cut into sections; S. aureus culture was added; incubated
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for 30 min; and then treated with the LST gel containing 0, 0.0125, 0.025 and 0.05% LST for 6 h. Viable bacterial colonies were counted. (B) Bar diagram showing LST gel
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containing 0.05% LST showed decreased S. aureus survival by 4.2 log10CFU/mL
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compared to the control. Unpaired student’s t-test was used to calculate statistical significance. P values ≤ 0.05 were considered statistically significant and were indicated
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with asterisks: *p ≤ 0.05. 4. Conclusion
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Lysostaphin is a possible alternative to antibiotics for eradication of S. aureus topical and
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biofilm infection. Here we formulated LSTC gel and characterized further in terms of it’s rheological properties and anti-staphylococcal activity. Rheological tests were conducted
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in order to prove that the gel is flexible, can flow smoothly and remains stable at physiological temperature as well as tested how rapidly the gel was capable of lysing the
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bacterial cells. The gel was added to an overnight culture of S. aureus and it was observed under microscope at various time points. Within 15 min of addition, the LSTC gel caused lysis of more than 95% of S. aureus cells in the culture. In silico molecular
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docking calculation of LST with chitosan structure further supports the experimental release of LST from the chitosan gel. Agar well diffusion assay showed that with increasing concentration of LST added to chitosan gel, the zones of inhibition increased, thus preventing the growth of S. aureus. The non-toxic nature of the LSTC gel was demonstrated using hemolysis and cytotoxicity assays. Further studies carried out with the model organism, D. melanogaster proved that gel was non-toxic. 20
The gel also had the advantage of disrupting S. aureus biofilm. The specific activity of lysostaphin for staphylococci has the added benefit of not disrupting normal flora, thus minimizing the occurrence of antibiotic resistance in other opportunistic pathogens. In summary, we have demonstrated that LSTC gel may be a superior alternative to mupirocin ointment for the eradication of S. aureus topical infection and
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will be a valuable tool for both prevention of community acquired and nosocomial S. aureus infections. Acknowledgement
We thank the Center for Nanoscience and Molecular Medicine, Kochi, Amrita
University, India for infrastructural support. G.B is an ICMR SRF fellow (80/924/2015ECD-1). MKS is supported by senior research fellowship (09/963 (0037) 2Kl7- EMR-I)
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from Council of Scientific & Industrial Research (CSIR), India.
[1]
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S0141-8130(17)34750-5 https://doi.org/10.1016/j.ijbiomac.2018.01.083 BIOMAC 8915
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
30-11-2017 5-1-2018 13-1-2018
Please cite this article as: Sai Nithya, T.R.Nimal, Gaurav Baranwal, Maneesha K.Suresh, Anuj C.P., V.Anil Kumar, C.Gopi Mohan, R.Jayakumar, Raja Biswas, Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2018.01.083 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.
Preparation, characterization and efficacy of lysostaphin-chitosan gel against Staphylococcus aureus Sai Nithya1, T R Nimal1, Gaurav Baranwal1, Maneesha K Suresh1, Anju C P1, V Anil
1
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Kumar2, C Gopi Mohan1, R Jayakumar1*, Raja Biswas1*
Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical
Department of Microbiology, Amrita Institute of Medical Sciences and Research Centre,
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Sciences and Research Centre, Amrita Vishwa Vidyapeetham,Kochi-682041, India
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Amrita Vishwa Vidyapeetham, Kochi-682041, India
__________________________ Authors contributed equally.
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#
*Corresponding authors: R. Jayakumar ([email protected]) Raja Biswas ([email protected]) Tel: +91-484-2801234 (ext. 8762); Fax: +91-484-2802020. 1
Abstract: Lysostaphin (LST) is a bacteriocin that cleaves within the pentaglycine cross bridge of Staphylococcus aureus peptidoglycan. Previous studies have reported the high efficiency of LST even against multi drug resistant S. aureus including methicillin resistant S. aureus (MRSA). In this study, we have developed a new chitosan based hydrogel formulation of LST to exploit its anti-staphylococcal activity. The atomic
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interactions of LST with chitosan were studied by molecular docking studies. The
rheology and the antibacterial properties of the developed LSTC gel were evaluated. The
developed LST containing chitosan hydrogel (LSTC gel) was flexible, flows smoothly and remains stable at physiological temperature. The in vitro studies by agar well
diffusion and ex vivo studies in porcine skin model exhibited a reduction in S. aureus
survival by ~3 Log10CFU/mL in the presence of LSTC gel. The cytocompatibility of the
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gel was tested in vitro using macrophage RAW 264.7 cell line and in vivo in Drosophila
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melanogaster. A gradual disruption of S. aureus biofilms with the increase of LST concentrations in the LSTC gel was observed which was confirmed by SEM analysis. We
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conclude that LSTC gel could be highly effectual and advantageous over antibiotics in
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treating staphylococcal-topical and biofilm infections.
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1. Introduction
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Keywords: Staphylococcus aureus skin infection; lysostaphin; chitosan gel.
About 20–60 per cent of human populations are carriers of Staphylococcus aureus (S.
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aureus) [1]. This Gram positive cocci colonizes our skin and mucus membranes; and acts as a causative agent for variety of skin infections e.g., folliculitis, furuncles and
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carbuncles, abscess, sycosis, impetigo, ecthyma, cellulites, scabies, mastitis, diabetic ulcers, staphylococcal scalded skin syndrome, scarlet fever and urinary tract infections
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[2,3].
In nosocomial setting transmission of S. aureus occurs often between patients;
and between patients and care takers at higher rates. Staphylococcal biofilm once formed on an implanted medical device or on catheters, are extremely difficult to disrupt and treat using antibiotics [2,4,5]. Currently mupirocin, bacitracin, triclosan and mersacidin are popularly used for topical treatment to prevent S. aureus skin colonization and infections, however 2
resistance against these anti-microbial agents are often reported in the literature [6–9]. Therefore, there is a need to develop an anti-staphylococcal agent to successfully eradicate S. aureus infections. Lysostaphin (LST), a 26 kDa zinc metalloprotease that rapidly lyse the cell wall of S.aureus [10], is reported in the literature as an excellent anti-staphylococcal agent for
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the treatment of topical infections in experimental animals [11,12]. Lysostaphin cleaves
between the third and fourth Glycine residues of the pentaglycine cross bridge of S.
aureus peptidoglycan [13]; and has no effect on bacteria of other genera [14]. Lysostaphin was previously tested in several animal models to prevent nasal colonization,
for the treatment of burn wound infections, against aortic valve endocarditis and sepsis [15–18]. These results indicate LST was highly effective anti-microbial agent.
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Experiments in neonatal mice model demonstrated lysostaphin is even better than
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vancomycin in treating methicillin resistant S. aureus (MRSA) sepsis [19]. Chitosan is a natural, biodegradable and non-toxic polymer, obtained
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commercially by the deacetylation of chitin, which is a naturally occurring
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polysaccharide [20]. It is insoluble at neutral and alkaline pH. Some of the important applications of chitosan as an excipient in pharmaceutical products include solid dosage
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forms, controlled release dosage forms, gels, microspheres, microcapsules and wound
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healing products [21,22]. Chitin and chitosan derivatives have become a major focus of interest in biomedical and biopharmaceutical research applications especially as promising biomaterials in tissue engineering, in wound healing, in drug delivery and in
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gene delivery [23–25].
In this study, the lysostaphin-chitosan (LSTC) gel was prepared; and it’s
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rheological and antibacterial properties were studied. The efficacy of LSTC gel to disrupt biofilms formed by S. aureus was tested. The antimicrobial activity of LSTC gel, was
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also tested in an ex vivo porcine skin model. Our in vitro and ex vivo studies performed with LSTC gel have shown that this staphylococcin can be used to prevent or treat staphylococcal infections. 2. Materials and methods 2.1 Materials
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The polymer chitosan (MW: 100-150 kDa; degree of deacetylation: 85%) was obtained from Koyo Chemical Industry Ltd, Japan. Dulbecco’s Modified Eagle’s Medium (DMEM) and Alamar Blue were obtained from Invitrogen, India. Fetal Bovine Serum (FBS), Luria Bertani (LB) Broth and Agar-agar were purchased from Himedia, India. All chemicals were used without further alteration and purification.
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2.2 Computational Studies
2.2.1 Preparation of Lysostaphin and Chitosan gel Structures
The three-dimensional (3D) X-ray crystal structure of LST from Staphylococcus simulans at 3.5 Å resolution was retrieved from protein databank (PDB) with PDB id: 4LXC [10,26]. The 3D structure was then prepared by assigning bond orders and the missing hydrogen atoms were added using PrepWiz module of Schrӧdinger (Protein Preparation
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Wizard, Schrödinger, LLC, New York, NY, 2016). Further, restrained energy
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minimization of the structure was performed using OPLS-2005 force field. Chemical structure of chitosan consisting of five monomer units was modelled
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using ChemBio Ultra 11.0 software. The number of chitosan monomer units was
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restricted to five to reduce the structural complexity as well as the computational cost. The modelled chitosan structure was geometry optimized to attain a stable atomic
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ChemBio Ultra software.
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arrangement using MMFF94 (Merk Molecular Force Field) method implemented in
2.2.2 Molecular Docking of LST with Chitosan gel Molecular docking calculation was performed to understand the LST-chitosan atomic
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level interactions. Prior to docking, the input structure in .pdbqt format was prepared using AutoDock 4.0 [27]. The active site of LST was reported by Sabala et al [10] and the
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docking grid box was specified such that it covers the entire active site region of the protein with X, Y and Z dimensions of 18 x 18 x 18 Å respectively. Autodock Vina [28]
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software was used further to perform LST-chitosan molecular docking calculations. Vina is an open source docking program based on the sophisticated gradient optimization method for local optimization procedure. Its scoring function was computed as: ΔGbind = ΔGvdW + ΔGelec + ΔGH-bond + ΔGhydrophobic + ΔGtors Where ΔGbind is the total binding energy, ΔGvdW is the binding energy owing to Vander Waal’s forces, ΔGelec is the binding energy due to electrostatic interactions, ΔGH-bond term 4
accounts for the hydrogen bonding interactions, ΔGhydrophobic is the binding energy due to hydrophobic interactions and finally ΔGtors is the energy due to the rotational bonds and atoms that are involved in torsions. Furthermore, the LST-chitosan complexes obtained after molecular docking were inspected for various 3D interactions using PyMOL (The PyMOL Molecular Graphics
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System, Version 1.8 Schrödinger, LLC) molecular visualization tool. 2.3 Bacterial strain
S. aureus strain SA113 (American Type Culture Collection 35556) and other methicillin
resistant S. aureus (MRSA) clinical strains were routinely grown in Tryptic soy broth (TSB) medium at 37°C with shaking at 160 rpm. For biofilm experiments, S. aureus were grown in filter sterilized TSB media containing 0.5% glucose under static
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conditions for 48 h [26, 27].
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2.4 Preparation of LSTC gel
2 g chitosan powder was dissolved in a 100 mL solution of 1% (v/v) acetic acid and left
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for stirring overnight. 1% (w/v) sodium hydroxide (NaOH) was added in drop-wise
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manner in order to neutralize the obtained viscous chitosan solution to a pH of 7.4. The gel was then harvested by centrifugation at 3,000 rpm for 10 min (Beckman Coulter
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Avanti J-26XP centrifuge using JA-17 rotor). The gel was further washed thrice with
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Phosphate-buffered saline (PBS) to remove traces of acetic acid and stored at 4°C. Different concentrations of LST were added to the chitosan gel and mixed well to prepare LSTC gel containing 0%, 0.0125%, 0.025% and 0.05% lysostaphin [29].
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2.5 Rheological studies of the lysostaphin-chitosan gel 2.5.1 Viscoelastic study
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Rheological studies were performed in order to assess the strength, flow and stability of the LSTC gel using Malvern Kinexus pro rheometer with a stainless steel parallel plate
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with a diameter of 20 mm [30]. A constant gap of 0.5 mm was maintained between the upper and lower plates at a temperature of 25ºC. Chitosan gel without LST served as control. First, the amplitude sweep was performed to find the Linear Viscoelastic Region (LVER) of the gel. The storage modulus (G’), loss modulus (G’’) and phase angle (δ) at LVER were measured at different strain percentage. The amplitude sweep was started from a strain percentage of 10-1 and the end point was determined automatically by the 5
rheometer. The gel strength and solid/liquid dominating behavior of LSTC gel was determined by studying the frequency sweep between 101 to 10-1 Hz in the LVER region of the gel. 2.5.2 Temperature stability tests The complex modulus and complex viscosity of the gel was measured from 25 to 45ºC at
gel from room temperature to human body temperature. 2.5.3 Analysis of yield stress
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constant frequency and shear [30]. This study was performed to test the stability of the
Yield stress analysis was performed by plotting stress against rheological shear strain.
The shear strain was applied from 10−1%. The higher the yield stress, more stress is required to initiate the flow of sample.
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2.5.4 Power law model fit
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The Power law can be used to mathematically express the power law region of the flow
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curve as follows: σ = kỳn
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Eq. (1)
law index.
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2.5.5 Injectability
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where ‘σ’ is the shear stress, ‘k’ is consistency index, ‘ỳ’ is shear rate and ‘n’ is Power
The injectability of the gel was observed by loading them into a 1 mL syringe without
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needle and subjecting them to manual shear. The flow of the gel from the syringe was observed visually [30].
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2.5.6 Inversion test
The flow of gel under the influence of gravity was observed using inversion test [31]. The inversion test confirms the property of the gel to remain in the infection site without
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falling. LSTC gel was taken in a flat bottomed cylindrical bottle and placed on a flat surface without any disturbance. A control was also used. The flow of the gel was observed visually at 0th, 1st, 2nd, 3rd, 4th and 5th day time points. 2.6 In vitro lysostaphin release study To quantify the LST released from chitosan gel, 100 µl of double distilled water was added to100 mg of LST loaded chitosan gel and the gel suspension was incubated at 37ºC 6
with ambient shaking. At frequent time intervals the gel suspension was centrifuged at 5000 rpm for 5 min (EBA 21 Hettich Centrifuge). 10 µl supernatant was collected from the sample at 0, 3, 6, 12, 24, 48 and 72 h and the double distilled water was replaced each time. The supernatant collected at different time intervals was analysed by Bradford assay. Protein standards were prepared using bovine serum albumin (BSA). Percentage of
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lysostaphin release was calculated using the following formula [32]:
Release (%) = (Amount of lysostaphin released at definite time)/ (Total amount of lysostaphin entrapped within the gel) X 100.
Eq. (2)
2.7 Hemolysis Assay
The hemolysis assay was performed to measure the extent of hemolysis caused by LSTC
gel when it comes into contact with human blood. 200 µL of diluted human whole blood
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(1:10 in PBS) was taken in several 1.5 mL vials and increasing amount of 50 mg CL gel
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(containing 0%, 0.0125%, 0.025% and 0.05% LST) were added into the diluted blood. The vials were incubated in 37ºC incubator for 48 h. 0.1% (v/v) Triton-X 100 treated
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blood samples served as positive control. Following incubation, the samples were
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centrifuged at 5000 rpm for 5 min (Minispin centrifuge) and the absorbance of supernatant was measured at 610 nm using microtiter plate reader [33,34]. The percent of
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hemolysis was calculated using the formula:
CONTROL)
X 100.
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Hemolysis (%) = (O.DTEST – O.DNEGATIVE CONTROL)/( O.DPOSITIVE CONTROL - O.DNEGATIVE Eq. (3)
2.8 pH and temperature dependent activity assay
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Overnight culture of S. aureus was pelleted down and the cell density was adjusted to 0.5. The cells were resuspended in 50 mM sodium acetate buffer (pH 4), PBS (pH7.0) and 50
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mM Tris-HCl buffer (pH 10). 10 µg/mL of LST was added and O.D. was measured at 610 nm for different time intervals. The bacterial culture where no gel was added served
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as the control. To study the temperature dependent activity of LST, LSTC gel was incubated at temperatures ranging from 20°C to 100°C for 10 min and added to the S. aureus culture. The O.D. of the S. aureus culture was measured at 610 nm to understand the lytic activity of LST. 2.9 Cell Culture
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Macrophage RAW 264.7 (ATCC® TIB-71™) cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO2 atmosphere. 2.9.1 Cell viability Studies The effect of LSTCL gel on the viability of macrophage RAW 264.7 cell lines were
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tested using Alamar blue (resazurin) assay. Alamar blue (resazurin) can be used as a direct indicator to quantitatively measure cytotoxicity. Alamar blue reagent is weakly fluorescent blue indicator dye. Resazurin reagent can acts as an intermediate electron acceptor in the electron transport chain without interfering with the normal electrons
transfer process. Within viable cell resazurin gets reduced to resorufin which is highly fluorescent and pink in color. The RAW 264.7 macrophage cells were seeded in 96 well
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plates at a density of 10,000 cells/well in DMEM medium supplemented with 10% FBS.
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50 mg LSTC gel (containing 0%, 0.0125%, 0.025% and 0.05% lysostaphin) were added into the wells. The untreated and 0.1% (v/v) Triton X-100 treated RAW 264.7
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macrophage cells were used as positive and negative control, respectively. Following
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incubation the culture medium was replaced with 10% (v/v) Alamar blue in DMEM and further incubated for 4 h. The absorbance of the solution from each well was measured at
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570 nm and 600 nm using microtiter plate reader [33]. The percent cell viability was
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measured using the following formula: Cell viability (%) = (O.D
TEST
at 570 nm - O.D
TEST
570 nm - O.D POSITIVE CONTROL at 600 nm) X 100.
at 600 nm)/( O.D POSITIVE CONTROL at Eq. (4)
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2.10 In vitro antibacterial activity 2.10.1 Agar Well Diffusion
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The agar well diffusion assay was used to determine the susceptibility of S. aureus to LSTC gel. S. aureus cultures adjusted to 0.5 McFarland standards and were spread plated
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on LB agar plate. Wells of 8 mm diameter were made using biopsy punch. 100 mg of gel containing varying concentrations of LST were added into the wells and incubated at 37ºC for 24 h. The inhibition of bacterial growth around the wells was observed [35–37]. 2.10.2 Growth curve 1% of SA113 overnight cultures were added to fresh LB medium and the growth of S. aureus was measured at defined time interval for 48 h. 100 mg of LSTC gel containing 8
varying concentrations of LST was added to the 5 mL of cultures after 6 h of growth and the O.Ds were determined using a spectrophotometer (BioTek) at 600 nm [35]. 2.10.3 Lysis assay in simulated nasal fluid The activity of lysostaphin was tested in a buffer that simulates the ionic composition of nasal fluid and sweat. The simulated nasal fluid (SNF) contains 0.87% NaCl, 0.088%
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CaCl2. 2H20, 0.31% KCl and 0.636% bovine serum albumin (BSA) [38]. Stationary phase cultures of S. aureus were pelleted down, washed twice with SNF and resuspended
in the same buffer. The cell density was adjusted to 0.5 at 600 nm. 100 mg of LSTC gel containing varying concentrations of LST was added to 5 mL of SNF buffer containing S. aureus and O.Ds were measured at hourly intervals for 6 h. S. aureus cells resuspended in SNF without the addition of the LST was served as control.
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2.10.4 Microscopic studies
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Overnight culture of S. aureus was pelleted down by centrifugation at 5000 rpm for 10 min and resuspended in 2 mL of PBS. 10 µg/mL of lysostaphin in 100 mg chitosan gel
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microscope at different time intervals.
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was added to the culture. An aliquot was taken and viewed under the phase contrast
2.11 Ex vivo antibacterial activity
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Pig ear skin was obtained from a local slaughter house and ex vivo testing of S. aureus
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survival in pig skin was performed. The skins were thoroughly washed with sterile saline and hair was removed. Excess fat deposits were removed using a sterile scalpel and were cut into sections of 2 cm × 2 cm. The skin sections were disinfected by soaking in 70%
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ethanol for 5 min. These disinfected sections were then placed on sterile petri-plates and allowed to dry. 100 µl of microbial suspension (~ 5 X 109 cells) was added to each
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section. The sections were incubated at 37°C for 30 min. After 30 min, 100 mg of LSTC gel containing 0, 0.0125, 0.025 and 0.05% lysostaphin was added onto the sections. The
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samples were incubated further for a period of 6 h at 37°C. Following incubation, the sections were taken in 15 mL falcon tubes containing 1 mL PBS and vortexed for 5 min vigorously to release the microbes adhered onto the skin [39]. To determine the number of viable bacteria present on the skin the samples were serially diluted and plated on mannitol salt agar plates. The plates were incubated at 37°C overnight for colony enumeration. 9
2.12 Stability assay An aliquot of LSTC gel was taken in 3 vials and stored at different temperatures (4, 25 and 37˚C). For every 5 days upto 30 days, well diffusion assay was performed with the gel stored at different temperatures. The zone of inhibition around the wells was measured [35].
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2.13 Biofilm assay
Catheter tubes which were cut into small pieces of approximately 2 cm, were cleaned by
dipping in 70% ethanol and placed into 1.5 mL centrifuge tubes containing 750 µL of LB medium with 0.5% glucose (LBG). The biofilms were allowed to form on catheter tubes
for 48 h at 37˚C. After incubation, the catheter tubes were washed once with PBS and
placed in a new tube with fresh LBG media and varying concentrations of lysostaphin (0,
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5, 10, 20 and 80 µg/mL) containing chitosan gel. The catheter tubes were then dipped
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into these vials and left for 24 h incubation. Catheter tube dipped in the microbial culture without any lysostaphin or mupirocin was used as the control. Post incubation, the
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catheter tubes were dehydrated, sputter coated and imaged using SEM (Scanning electron
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microscope). To further assess the effect of LSTC gel on S. aureus biofilm, the above mentioned procedure was repeated. The catheter tubes were washed after the LSTC gel
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treatment and rolled on mannitol salt agar base plates as described before. The plates
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were incubated for a period of 24 h and the growth of the bacteria was observed [35]. 2.14 In vivo toxicity studies in Drosophila model Wild-type D. melanogaster (Flybase Stock 2: FBst0000002) were cultured on classical
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banana agar medium at room temperature (26ºC) with 60% humidity. 2 to 5 day old female flies were used for the toxicity assays. 10 female flies were fed with banana media
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that contained 0.05% LSTC gel (Chitosan gel containing maximum % of LST). The flies were allowed to feed for 14 days and the survivability was observed. Another set of flies
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were fed with classical banana media (without the LSTC gel) which served as a control [35]. To confirm the authenticity of the results these experiments were repeated thrice.
3. Results and discussion 3.1. Molecular docking simulation studies Initial computational studies were carried out to rule out possible interactions between LST and chitosan. LST-chitosan interaction study was performed using crystal structure 10
of LST and computationally modelled five units of chitosan. Molecular docking calculations are being used in recent studies to understand the protein-polymer and polymer-drug interactions [40]. We performed molecular docking of chitosan polymeric gel of five units at the active site of LST. LST has a Zn2+ ion at the active site tunnel region [10] as shown in Figure 1 which resulted in positive potential at this regions.
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Experimentally, an instant release of LST from chitosan gel was observed and the mechanism of binding was computed using the molecular docking technique.
Our molecular docking studies resulted in positive binding energy 10.1 kcal/mol
between chitosan and lysostaphin, suggesting its weak (repulsive) binding affinity.
Docking studies showed hydrogen bonding of chitosan gel with the amino acid residues ASN 372, GLY 309, GLY 310, HIS 362 and THR 357 at the active site of lysostaphin
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(Fig. 1B-D). However, the positive binding energy indicates weak binding affinity
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between protein-gel complexes. This might be the reason why chitosan, a positively charged polymer shows very weak interaction with lysostaphin. In support with the
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experimental lysostaphin release studies, the instant release of enzyme from the chitosan
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gel could be due to the weak binding of lysostaphin and chitosan.
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Fig. 1. (A) Structure of S. aureus peptidoglycan and lysostaphin cleavage site. (B-D)
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Docked complexes of Lysostaphin – chitosan (5 units) (B), chitosan interacting residues at the active site of lysostaphin (C), molecular surface representations of lysostaphin with
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chitosan showing Zn2+ ion and tunnel region (D). Interactions viewed using PyMOL software. The cartoon represents the protein, the sticks represent the Chitosan (5 unit),
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blue sphere shows the zinc ion (Zn2+) at the active site of lysostaphin and the yellow dotted line shows the hydrogen bonding interactions. 3.2. Rheological studies
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3.2.1. Viscoelastic analysis First, the amplitude sweep was performed which records the linear viscoelastic region (LVER) of the gel. LVER is the region in which any material can elastically strain and return to its original state when the strain is removed. To understand the gel behavior
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over a period of time, a frequency sweep was carried out. The G` of the gel was obtained by subjecting it to a frequency range of 101 to 10-1 Hz (Fig. 2A). G` of control and lysostaphin-chitosan gel was found to be 14.58 ± 0.27 and 12.68 ± 0.38 respectively
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(Table 1). The viscosity of the gel was analyzed using the flow curve. The viscosity (η) of the gel was measured at different shear rates (Y) from 10 -1 to 102 s-1. The flow curve
indicated that the viscosity reduced as shear rate was increased from 0.001 to 100 s-1 (Fig. 2D). For any material to have a gel-like nature, it must have a dominant solid nature with a liquid component. The phase angle (δ) of the gel was below 10°, which indicated the
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solid dominating nature of the gels (Fig. 2C). For pure solids, δ=0° and for liquids δ= 90°.
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Phase angle of gel showed between 0-10° shows that it is of a solid nature with a minor
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liquid component.
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Table 1. Rheological properties of control, chitosan gel and test, lysostaphin-chitosan gel.
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Storage Modulus(K Material Name Pa)
Power Law Index (n)
Correlati on Coefficie nt
Yield Stres s (Pa)
Comple x Shear Strain (%)
Shear Viscoci ty (Pa s)
14.586+0.27 7
0.0385 4
0.9733
5970
74.6442
1.9
Chitosa_lysosta phin
12.686+0.38 6
0.0335 6
0.9888
6256
69.274
1.343
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Chitosan
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Fig. 2. Rheological analysis. (A) Frequency sweep analysis of control and chitosan containing lysostaphin representing storage modulus (G’), loss modulus (G”) and phase
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angle (δ). (B) Temperature stability analysis. (C) Power law model fit. (D) Viscosity vs
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shear rate. (E) Yield stress analysis. (F) Injectability test. (G) Inversion test. (H) LST release assay from LST-chiotsan gel.
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3.2.2. Temperature stability analysis The gel appeared to be considerably stable within the given temperature range (Fig. 2B).
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This indicated that lysostaphin-chiotsan gel would be stable under physiological temperatures without much variation in their strength. 3.2.3. Yield stress analysis
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Yield stress analysis was performed by plotting stress against rheological shear strain. The shear strain was applied from 10−1%. Yield stress analysis showed that there was a slight increase in the complex shear strain percentage of the gel containing lysostaphin when compared to the control (Fig. 2E). This indicated that the lysostaphin-chitosan gel was flexible in nature and can flow smoothly upon shearing. 3.2.4. Power law fit analysis 14
To further understand the rheological properties of the gel as pseudoplastic, Newtonian or dilatant, data were modeled using the power law Eq. (1). Both the control and lysostaphin-chitosan gel showed good fitting with correlation coefficient of 0.97 and 0.98 respectively (Fig. 2C). The n values for both the test and control were below 1, which confirmed they were pseudoplastic, i.e. shear thinning in nature.
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3.2.5. Injectability
Injectability test was performed by applying manual shear using a 1 mL syringe without needle and observing the evenness of the flow of the gel. The gel showed good injectability and continuous flow (Fig. 2F). 3.2.6. Inversion test
From the inversion test it was noted that the gel did not flow under the influence of
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gravity even after 5 days (Fig. 2G), which may be due to the adhesive nature of the
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chitosan gel. This property indicated that the gel will not flow unless an external shear is applied, thus making it remain on the infection site without falling.
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3.3 Release assay
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The supernatant collected by carrying out release assay was estimated using Bradford assay. The results show that the chitosan gel containing lysostaphin gave a sustained
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release of the enzyme into the supernatant over a period of time (Fig. 2H) and almost 90-
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95% of the enzymes get released in 24 h. 3.4 LSTC gel inhibited the growth of S. aureus We first tested the in-vitro antimicrobial activity of LSTC gel against S. aureus. Agar
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plate well diffusion assay results demonstrated that LSTC gel inhibited the growth of S. aureus. The growth inhibition zone diameter of S. aureus was increased with increasing
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concentrations of LST within the LSTC gel (Fig. 3A). Addition of 40 mg of 0, 0.0125, 0.025 and 0.05% LSTC gel per well resulted in the production of growth inhibitory zone
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diameters of 1.2 cm, 1.5 cm and 1.8 cm, respectively. No growth inhibition of S. aureus was observed when chitosan gel alone (without LST) was used for the agar plate well diffusion assay. Mupirocin (Mup) ointment was used as positive control (Fig. 3A). Similar results were obtained when the growth of S. aureus was tested in broth culture. When cultures of S. aureus were treated using LSTC gel, growth of S. aureus was inhibited irrespective of it’s growth state. LG gel rapidly induces the lysis of S. aureus 15
culture (Fig.
3B). As expected when tested against
other pathogens,
such
as Pseudomonas aeruginosa LSTC gel did not show any anti-microbial activity (data not
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shown).
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Fig. 3. (A) Well diffusion assay carried out with different concentrations of LST in 100
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mg chitosan gel. (B) Graph curve of S. aureus. Increasing concentrations of LST was added to S. aureus culture after 6 h of its growth. The growth of S. aureus was monitored
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further for 48 h. (C) LST induced lysis of S. aureus. The S. aureus cells were suspended
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in simulated nasal fluid (SNF) at a cell density of 0.5 at 600 nm. 100 mg of LSTC gel containing varying concentrations of LST was added to SNF buffer containing S. aureus and O.Ds were measured at hourly intervals for 6 h. (D) Microscopic images
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demonstrating the lytic activity of LSTC gel against S. aureus at different time intervals. (E) LSTC gels were incubated at different temperatures for 10 min and then added to S.
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aureus to test their lytic activity. (F) pH dependent activity of LST from LSTC gel. Maximum LST activity was observed at pH 7.0.
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In order to confirm the lytic activity of LSTC gel against S. aureus in the nasal secretion we used simulated nasal fluid. Rapid lysis of S. aureus cells were observed following addition of the 100 mg of LSTC gel within the simulated nasal fluid containing S. aureus of OD 600 nm. Complete lysis of S. aureus cells were observed within 1 h when 0.05% LSTC gel was added to the S. aureus culture (Fig. 3C, D). 3.5 Temperature and pH dependent enzymatic activity of LSTC gel 16
In order to understand the stability of LSTC gel we prepared the gel and incubated at different temperatures before the lysis assays. Incubation of LSTC gel till 90ºC for 10 min caused only slight alteration of it’s enzymatic activity, however incubation above 90ºC caused rapid loss of it’s enzymatic activity (Fig. 3E). As the pH of all human secretions such as sweat, nasal fluid, urine and tears varies between 4.5 -7.0, we tested
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the enzymatic activity of LSTC gel at pH 2.0 and 10.0. The lytic activity of lysostaphin
from the LSTC gel was maximum at pH 7.0. Around 90% of S. aureus cells were lysed in 2 h in presence of LSTC gel. However, activity of the LST from the LSTC gel was reduced by 40% at pH 4.5. Results indicate LSTC gel showed maximum lytic activity at neutral pH (Fig. 3F).
3.6 In vitro cytocompatibility and in vivo biocompatibility of LSTC gel
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The blood compatibility is an important parameter of biological safety for any therapeutic
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formulations. Therefore, hemolysis assays were carried out using LSTC gels containing different concentrations of lysostaphin (Fig. 4A). No significant erythrocyte lysis was
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were added into human blood.
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observed with LSTC gels containing different concentrations of lysostaphin (Fig. 4B)
The cytotoxic effect of the LSTC gel was evaluated in RAW 264.7 cell lines and
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using alamar blue assay. The cell viability plot showed that more than 97% cells were
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viable after 24 and 48 h of incubation even in presence of varying concentrations of lysostaphin containing LSTC gel (Fig. 4C). The non-toxic nature of chitosan gel alone was well documented by us in our several previous studies. No alteration of cell
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morphology of RAW 264.7 cell lines was observed under the microscope in presence of LSTC gel. The alamar assay confirmed that LSTC gels are non-toxic and are cyto-
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compatible.
Further the toxicity of the LSTC gel was tested using fly feeding assays. D.
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melanogaster flies were allowed to be fed with LSTC gel containing different concentrations of lysostaphin for 1 week. The experiment was carried out to check whether the gel was toxic to the flies. At the end of the 14 day observation period, it was found that lysostaphin-chitosan gel was non toxic to the flies and all flies survived the experiment (Fig. 4D).
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Fig. 4. (A) Varying concentrations of lysostaphin in chitosan gel were added to diluted
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blood, the tubes were incubated for a period of 48 h and the hemolytic activity was
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observed. (B) Bar diagram showing the percentage hemolysis of different concentrations of lysostaphin in chitosan gel. (C) Bar diagram representing the cytotoxicity assay carried
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out in cell lines. (D) The survival plot showing the number of flies that survived the toxicity assay. At the end of the 14 day observation period, it was found that lysostaphin-
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chitosan gel was not toxic to the flies and that all the flies survived the experiment. 3.7 Disruption of S. aureus biofilm by LSTC gel
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The ability of LSTC gels to disrupt S. aureus biofilms were tested using silicon catheter tubes. S. aureus was allowed to adhere with the catheter tubes for 48 h and then treated
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LSTC gels containing different concentrations of LST. We found gradual disruption of S. aureus biofilms with the increase of LST concentrations. Biofilm formation was
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decreased by 2.5, 3.0 and 5.5 fold in presence of 0%, 0.0125%, 0.025% and 0.05% LST containing LSTC gel (Fig. 5A-C). Scanning electron microscopic images confirmed that S. aureus biofilms were disrupted after treatment with LSTC gels and maximum biofilm disruption was observed when the biofilm was treated with 0.5 % LST containing LSTC gel (Fig. 5D).
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Fig. 5. (A) Catheter tubes were taken in centrifuge tubes containing S. aureus culture,
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incubated for 48 h, washed, transferred to media containing varying concentrations of
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LST in LSTC gel and incubated for 24 h. (B) The rolling assays were performed to
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demonstrate reduced S. aureus attachment with the silicon catheters when treated with LSTC gel. (C) Graph showing the percentage survival of S. aureus cells on the catheter
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tubes after treatment with LSTC gel. (D) SEM images of catheter tubes after treatment with LSTC gels containing different percentages of LST.
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3.8 Ex vivo activity of LSTC gel against S. aureus The S. aureus infected skin sections were treated with LSTC gel (Fig. 6A). The amount
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of live bacteria from the skin surface were extracted and enumerated. Dose dependent decrease in S. aureus survival was observed when infected porcine skin was treated with
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LSTC gel containing increasing concentrations of LST. Bacterial survival was decreased
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by 1.0, 2.3 and 4.2 Log10CFU/mL after treatment with LSTC gels (Fig. 6B).
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Fig. 6. (A) Porcine ear skin was cut into sections; S. aureus culture was added; incubated
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for 30 min; and then treated with the LST gel containing 0, 0.0125, 0.025 and 0.05% LST for 6 h. Viable bacterial colonies were counted. (B) Bar diagram showing LST gel
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containing 0.05% LST showed decreased S. aureus survival by 4.2 log10CFU/mL
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compared to the control. Unpaired student’s t-test was used to calculate statistical significance. P values ≤ 0.05 were considered statistically significant and were indicated
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with asterisks: *p ≤ 0.05. 4. Conclusion
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Lysostaphin is a possible alternative to antibiotics for eradication of S. aureus topical and
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biofilm infection. Here we formulated LSTC gel and characterized further in terms of it’s rheological properties and anti-staphylococcal activity. Rheological tests were conducted
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in order to prove that the gel is flexible, can flow smoothly and remains stable at physiological temperature as well as tested how rapidly the gel was capable of lysing the
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bacterial cells. The gel was added to an overnight culture of S. aureus and it was observed under microscope at various time points. Within 15 min of addition, the LSTC gel caused lysis of more than 95% of S. aureus cells in the culture. In silico molecular
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docking calculation of LST with chitosan structure further supports the experimental release of LST from the chitosan gel. Agar well diffusion assay showed that with increasing concentration of LST added to chitosan gel, the zones of inhibition increased, thus preventing the growth of S. aureus. The non-toxic nature of the LSTC gel was demonstrated using hemolysis and cytotoxicity assays. Further studies carried out with the model organism, D. melanogaster proved that gel was non-toxic. 20
The gel also had the advantage of disrupting S. aureus biofilm. The specific activity of lysostaphin for staphylococci has the added benefit of not disrupting normal flora, thus minimizing the occurrence of antibiotic resistance in other opportunistic pathogens. In summary, we have demonstrated that LSTC gel may be a superior alternative to mupirocin ointment for the eradication of S. aureus topical infection and
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will be a valuable tool for both prevention of community acquired and nosocomial S. aureus infections. Acknowledgement
We thank the Center for Nanoscience and Molecular Medicine, Kochi, Amrita
University, India for infrastructural support. G.B is an ICMR SRF fellow (80/924/2015ECD-1). MKS is supported by senior research fellowship (09/963 (0037) 2Kl7- EMR-I)
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from Council of Scientific & Industrial Research (CSIR), India.
[1]
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