Isolation of lectin from Musa acuminata for evaluation of antibacterial activity against Methicillin-Resistant Staphylococcus aureus and the interaction of banana proteins with certain Enterococcus species

Introduction

Methicillin-Resistant Staphylococcus aureus (MRSA) have become a major health concern globally with high number of cases being recorded in East Asia and United States (Dunachie et al. 2020). Since wound infections are major cause of morbidity and mortality worldwide especially in burn patients (Ladhani et al. 2021). Common pathogens isolated from burn wound infections are Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes and various coliforms (Afreen et al. 2021; Tasnim et al. 2020). Amongst them emphasis have been given to the biofilm formers due to evolution of antibiotic resistance as there is a strong association between chronic wound infection and biofilm formation (Hasannejad-Bibalan et al. 2021).

Medicinal plant extracts have been studied in detail for development of novel bioactive compounds (Ghuman et al. 2019). Lectins, a well-known class of carbohydrate binding protein possesses tremendous biological activity such as anti-tumor, immunomodulatory, insecticidal and most importantly antimicrobial (Singh et al. 2019). Lectins mediate the identification of micro-organisms through interaction with the carbohydrates present on microbial cell surfaces (Pereira et al. 2019). Banana Lectin (BanLec) from Musa acuminata and Musa paradisica species, is a subgroup of Jacalin related lectin family consisting two subunits of 15 KDa each (Meagher et al. 2005; Martinez et al. 2021). These subunits are unique in recognizing internal and terminal sugar linkages preferably glucose/mannose residues (Dragacevic et al. 2020). Molecularly engineered banana lectin has shown to exhibit antiviral potential by binding to surface glycans of HIV and Ebola viruses and it can also induce nitric oxide production by macrophages (Swanson et al. 2010; Covés-Datson et al. 2019). Recombinant banana lectin has been recognized as a potential immunomodulatory molecule for its potential to stimulate T- cell proliferation and the expression of cytokines (INF-γ, TNF-α and IL-2) (Wong et al. 2006). Banana lectin (BanLec) also inhibited proliferation of leukemia (L1210) cells and Hepatoma (HepG2) cells (Cheung et al. 2009). However, anti-biofilm activity of BanLec to the best of our knowledge is not evaluated up to date hence the synergistic effect of plant proteins with beneficial microorganisms will open new dimensions for the treatment of life threatening infections. Thermostability, extreme pH and trypsin stability of BanLec renders its research as an effective antimicrobial alternative (Dimitrijevic et al. 2010).

Furthermore, probiotics which are the beneficial live microorganisms have revitalized attention in medical field. The majority of probiotics are comprised of lactic acid bacteria (LAB), which are used to make a wide range of foods products but have also been demonstrated to have the capacity to prevent certain illnesses (Peng et al. 2022). Their potential therapeutic effects within gut have been demonstrated thoroughly yet studies related to skin health is under way. Moreover, with the increased use of probiotic LAB, there is growing concerns about safety, since incidences of adverse effects such as severe infections and antibiotic resistance gene transfer can occur (Crits-Christoph et al. 2022). Recently, it is shown that probiotics and antimicrobial substances have therapeutic potential in prevention of skin infections by combating drug-resistant bacteria (Onbas et al. 2019). Lactic Acid Bacteria (LAB) have long been used as probiotic due to its sizeable benefits. Currently, the use of Lactobacillus has been extended to ameliorate dermal health, studies have reported that Lactobacillus plantarum strain enhances the production of cytokines and chemokines (TNF-α, IFN-γ, IL-4, IL-6, TGF-β and MMPs) which resulted in fast wound recovery. Moreover, it showed promising anti-Staphylococcal activity by elaborating proteinaceous metabolites (Ong et al. 2019). Therefore, this study was conducted to explore the inhibitory activity of LAB strains isolated from various sources and proteins isolated from banana pulp also the synergy between them to combat MRSA growth and mainly on biofilm development because biofilms are known to cause major problems in food safety as they account for 80% of contamination during food processing, 65% of human illnesses, and 99% of the microbial population (Xu et al. 2019).

Materials And Methods

Isolation of Banana Proteins

Ripe banana fruits for this study were purchased from local market. After peeling, the pulp was sliced and soaked in 150 mM Acetic acid solution for overnight at 4°C using methods described by (Singh et al. 2004) with slight modification. After one week, solution was homogenized for 6 hours and centrifuged. Proteins in the supernatant were precipitated by using 65% (w/v) Ammonium sulphate after clarification of liquid through cheesecloth. Pellet obtained after centrifugation was resuspended in 10 mM Phosphate buffered saline (PBS) pH 7.4. Dialysis was performed with the same buffer and clear solution was stored at -20˚ until further use.

Purification of BanLec by Affinity Chromatography

Clear sample was loaded into Sephadex G-75 column equilibrated with PBS. The Fast protein liquid chromatography (Amersham Biosciences UV-900) system was set up as 1.0 ml /min flow rate by using UNICORN software. Bound proteins were eluted by using 0.4M Glaxose-D (GlaxoSmithKline) purchased from local market and 0.8M D-Glucose (Merck, Darmstadt, Germany) (Wearne et al. 2013). The concentration of proteins was determined by using NanoDrop 2000 (ThermoFisher Scientific) and molecular weight of bound as well as unbound proteins were confirmed by 15% SDS-PAGE. The gel was stained by using silver staining protocol (Hempelmann et al. 2017).

Isolation and identification of Lactic acid bacteria

The strains used in this study were isolated from different sources like milk, butter, cocoa beans, fermented cabbage and soya beans, purchased from local stores and those belonging to the genus Enterococcus were considered for further evaluation. These strains were isolated by standard plate technique where samples were serially diluted in sterile PBS and 0.1 ml of last two dilutions were plated on de Man Rogosa and Sharpe medium (MRS) medium for isolated colonies and were maintained under their optimum conditions (dos Santos Leandro et al. 2021). For the extraction of DNA each sample was inoculated in 2 ml of MRS broth and incubated overnight at 37 ºC. Extraction and purification of DNA was carried out by using GJC ® DNA purification kit method procedure as followed by manufacturer. Amplification of genomic DNA was performed by using Universal primers 27F' 5 ʹ AGAGTTTGATCMTGGCTCAG 3 ʹ and 1492R' 5ʹ TACGGYTACCTTGTTACGACTT 3ʹ by Macrogen (Seoul, Republic of Korea). Amplification was carried out in final volume of 50 µl using Taq PCR Master Mix 2X (BioBasic). Thermocycler (Thermoscientific) program was set at initial denaturation at 95ºC for 2 minutes followed by 35 cycles of denaturation at 95 ºC for 15 seconds, annealing at 63.3 ºC for 1 minute and extension at 72 ºC for 2 minutes and final hold at 72 ºC for 5 minutes. Amplified products were separated by electrophoresis in 2% (w/v) Agarose gel (Stevens et al. 2019). The samples were sequenced by Macrogen company (Korea).

Antibiofilm assay of Banana proteins

The banana proteins were evaluated for their inhibitory effect on growth and biofilm for which 100 µl of bound protein sample (1mg/ ml) and unbound protein (0.7mg/ml) were added in each of the 96- well microtitre plate (Nest Biotech, China) (Bai et al. 2019; Lopes et al. 2017). Then, 100 µl fresh culture of MRSA 0.5 McFarland standard was diluted to the ratio of 1:100 in BHI medium supplemented with 0.2% (w/v) glucose. The BHI medium alone served as negative control while 100 µl pathogen in 100 µl BHI served as positive control. Plates were sealed and incubated for 24 hours at 37ºC. After incubation, optical density (OD) was observed at 600 nm by using microplate reader (Diatek, China). The plates were washed thrice by distilled water and were kept under drying oven for an hour. Staining of plates was carried out by using 0.1% (w/v) crystal violet stain for 30 minutes at room temperature. After washing three times with distilled water plates were air dried, bound dye was resolubilized by using 30% (v/v) Glacial Acetic acid (Fisher Scientific). The OD of the bound dye was observed at 595nm wavelength, Percent inhibition (%) was calculated by using formula (Al Atya et al. 2016).

% Inhibition = 100 - [(OD_T – OD_B) / OD_C – OD_B) × 100]

OD_T = optical density of test

OD_B = optical density of blank (Negative control)

OD_C = optical density of control (Positive control)

Antibiofilm assay of Enterococcus strains

To evaluate antibiofilm activity the supernatants (CFS/NCFS) (100 µl) from each test strain including E. faecium (LCM001 and LCM002), E. lactis (LCM003) and E. durans (LCM004 and LCM005) were added in each of the 96- well microtitre plate (Bai et al. 2019; Lopes et al. 2017). Then, 100 µl fresh culture of MRSA 0.5 McFarland standard was diluted to the ratio of 1:100 in BHI medium supplemented with 0.2% glucose. Additionally, 100 µl of all probiotic samples (viable cells) in MRS broth were also tested for determination of biofilm forming probiotic strains followed by the procedure aforementioned.

Synergistic effect of Banana proteins with E. lactis

To evaluate the synergistic effect 50 µl of cell free supernatant from the selected strain (E. lactis) and 50 µl of banana proteins (bound and unbound) were added in wells and inhibition of MRSA biofilm was performed by using 96-well microtitre plate followed by the procedure aforementioned.

Statistical analysis

All the experiments were performed in triplicates and statistical analysis was conducted by using IBM SPSS statistics version 20. Software and significance of (p<0.05) was used as indicated by Dunnett's Multiple Comparison for OD of cells and biofilm. Additionally, the % inhibition of biofilm was statistically assessed by Tukey's Multiple Comparison Test using GraphPad Prism 9 software.

Results

Isolation of Banana Proteins

Proteins from banana pulp were isolated and were regarded as crude protein sample. Different aliquots of crude protein were made and was stored at 4ºC until further use. The clear solution was then loaded into Sephadex G-75 column for purification.

Purification of BanLec using Affinity Chromatography

Banana lectin (BanLec) was successfully isolated by using glaxose- D (GlaxoSmithKline), indicating affinity of BanLec with glucose and other components of Galaxose- D, hence, purified at a concentration of 0.3mg/ml by using affinity chromatography. The dextran nature of Sephadex G-75 material provide the binding site for carbohydrate binding protein, hence single peak of isolated protein was obtained. Additionally, unbound proteins were also collected and were concentrated up to 0.1mg/ml (Fig. 1).

SDS-PAGE (Sodium dodecyl polyacrylamide gel electrophoresis)

Molecular weight of bound and unbound proteins was determined by using 15% SDS-PAGE. BanLec appeared at 14.5 KDa (Fig. 2 lane ‘b’) while unbound proteins showed multiple bands of different molecular weights (Fig.2 lane ‘c and e’). A fraction of protein obtained as unbound samples showed a clear band around 21 KDa (Fig. 2 lane ‘d’).

Isolation and identification of Lactic acid bacteria

LAB strains were successfully isolated and were catalase negative, Gram positive cocci. The 16s rRNA sequence showed that they all belonged to genus Enterococcus. The amplified product after DNA extraction showed a clear band (Qiagen) (Fig. 3). The identified strains were assigned accession numbers by NCBI as, Enterococcus faecium LCM001(GenBank accession no. OM212651), Enterococcus faecium LCM002 (GenBank accession no. OM108304), Enterococcus lactis LCM003 (GenBank accession no. OM189468), Enterococcus durans LCM004 (GenBank accession no. OM131605) and Enterococcus durans LCM005 (GenBank accession no. OM212650) Table. 1.

Table 1. Isolated LAB and their characteristics.

Isolates

Strain

Source

Gram staining

Shape

Arrangement

Catalase test

E. faecium

E. lactis

E. durans

LCM001

LCM002

LCM003

LCM004

LCM005

Milk

Butter

Cocoa beans

Sour cabbage

Soya beans

Cocci

Scattered

Key: Key: + Gram Positive

Antibiofilm activity of Banana proteins

Antibiofilm activity of isolated banana lectin and unbound proteins from peak 1 (Fig. 1) were assessed. The unbound protein fraction showed significant reduction in growth of MRSA also the unbound protein showed approximately 80% inhibition of biofilm p- values < 0.05 while BanLec did not show any significant result on MRSA growth but it showed increased biofilm OD₅₉₅ (Fig.4a and b).

Antibiofilm activity of Enterococcus strains

The CFS of E. faecium (LCM002), E. lactis (LCM003) and E. durans (LCM004 and LCM005) showed inhibition of biofilm (p<0.05) and decrease in growth of pathogen while CFS of E. faecium LCM001 did not show any significant inhibition rather increase in biofilm as the cell OD₆₀₀ was decreased compared with the positive control (Fig. 5 a,d). The neutralized CFS from all the enterococcus strains showed increased attachment of biofilm and slight reduction in pathogenic growth (Fig. 4 b, d). When cells of Enterococcus strains were introduced along with MRSA the biofilm was completely inhibited (Fig 5 d) indicating the remaining cells OD₆₀₀ left were mostly Enterococcus strains (Fig. 5c). All the selected Enterococcus strains were unable to form biofilm when added alone in the assay (Supplementary Fig. 1).

Synergistic effect of Banana proteins with Enterococcus

E. lactis was selected for the synergistic effect with banana proteins. The CFS with unbound protein fractions showed 80% decrease in biofilm development with p value <0.05 while isolated lectin showed neither inhibition of growth nor the biofilm instead the combination increased the attached biofilm with the wells (Fig 6 a, b).

Discussion

Lectins are the proteins which have the ability to read sugar codes present on various surfaces, it is due to the versatility of lectins that allowed them to be used in a variety of diagnostic and therapeutic approaches (Naithani et al. 2021). Lectins are considered as carbohydrate binding protein which can play a key role in reduction of pathogen load due to its ability to bind sugars present on surface of microorganisms. Similarly, banana lectin having affinity for mannose/glucose residues was aimed to bind sugars expressed during biofilm formation and to evaluate its ability to inhibit the growth of MRSA. As the biofilm development in this study was mainly due to pathogenic strain MRSA, it has several virulence factors like sea gene that encodes Staphylococcal Enterotoxin A (SEA), Fibronectin binding proteins and Penicillin-binding protein 2a (PBP2a) all of which are key components in biofilm production (Guimarães et al. 2021). Other Staphylococcal components, such as extracellular DNA (eDNA), autolysins and proteases have also shown to influence biofilm development (Creutz et al. 2022).

Banana lectin was successfully isolated as bound protein (BanLec) and unbound protein (other than Lectin). One of the fraction from the unbound protein was of 21 KDa collected separately which was expected to be Thaumatin like protein (TLP). The TLP reported to showe antifungal activity against Fusarium oxysporum and Mycosphaerella arachidicola which stipulates that the unbound protein fraction could be utilized for isolation of antibacterial component (Ho et al. 2007). This unbound protein fraction showed a significant decrease in biofilm development although the BanLec showed no inhibition of biofilm which may be due to inadequate concentration to assess this protein against different parameters, hence this creates a demand for new ways to isolate it in bulk quantities and to further evaluate the outcome of having a novel antibiofilm agent by which we can cross the hurdle of antibiotic resistance. However, the inhibition of biofilm of Escherichia coli and Listeria monocytogenes by ConA lectin from Canavalia ensiformis has enlightened lectins as intriguing component as there exist a competitive inhibition of bacterial adhesion to particular surfaces mainly due to the affinity of lectins towards carbohydrates (Jin et al. 2019). The quest for compounds that might impact biofilm development has sparked the interest of scientists all around the world. Since antibiofilm action can aid to prevent the spread of microbial resistance for which lectins from different sources must be studied. The protein fraction (CasuL) from the leaves of Calliandra surinamensis was examined to have antibiofilm activity against E. coli, also the growth was not impeded by the lectin, indicating that the lectin may be used to develop a novel antibiofilm agent (Procópio et al. 2017).

Since the strains of LAB are known to inhibit biofilm formation of MRSA hence, the ability of LAB to reduce biofilm suggests a strategy for reducing infections (Nataraj and Mallappa 2021). Therefore, this research was conducted to determine the ability of CFS and NCFS of LAB to inhibit biofilm formation by MRSA. It is reported that LAB produces variety of biomolecules that showed antibacterial effect leading to deformation of cell, therefore autolysis caused by permeable channels could be a reason for antibacterial effect of bacteriocins (Li et al. 2021 and Messaoudi et al. 2013). However, the mechanism of bacteriocins on biofilm formation is incomprehensible which creates obstacles in development of new strategies to contend with biofilm formation by several pathogens.

This study explored the horizons of using Enterococci as potential therapy against MRSA. Enterococci are regarded as Lactic acid bacteria being widely present in many habitats, hostile biotopes, gastrointestinal tracts of humans (Cattoir 2022). These organisms are important due to their function in food degradation and fermentations as well as their usage as probiotics in humans and animals (Khanlari et al. 2021). Besides this they are able to cause serious infections for which they must be characterized before using in food fermentation processes (Mariam 2021). Some strains may exhibit both antibiotic resistance and virulence traits (Franz et al. 2011). The CFS of all the Enterococci showed inhibition of growth and reduction in biofilm except by LCM001 hence, the active component in CFS must further be isolated, identified and utilized for treatment purposes. The NCFS of probiotics showed no effect on growth but the increase in attached biofilm might be due to excessive amount of polysaccharides which facilitated the attachment of biofilm, this also indicates that some other component of supernatant might be effective against MRSA more specifically the organic acids. This is in accordance with a study conducted where pH dependent inhibition of Vibrio cholerae biofilm signifies organic acids as potent biofilm inhibitor (Kaur et al. 2018). In addition, the lactic acid produced by Pediococcus strain was thought to be a potential therapy for blocking bacterial communication of clinical isolate of Pseudomonas aeruginosa thus lowering its pathogenicity (Kiymaci et al. 2018). However, the results highlight the value of organic acids as antibacterial agents whereas a more thorough investigation of bacteriocins from Enterococci is required.

Co-culture of probiotics with MRSA showed competitive exclusion of MRSA growth and ultimately biofilm development. This suggests that in the presence of beneficial microorganisms even multidrug resistant organisms cannot prosper and hence could be possibly utilized in problems related to skin infections especially in burn patients. As reported, the oral administration of probiotic resulted in improvement and wound healing of post burn patients (El-Ghazely et al. 2013). The probiotics are effective in reducing the rate of infections cause by multi drug resistant pathogens isolated from burn patients (Ali and Aldujaili 2022). The administration of probiotic strains increased IgA level which act as an anti-inflammatory antibody released extensively in severe burn patients also defines the role of probiotics in treatment of burn patients (Saputro et al. 2019).

Enterococci have achieved recognition due to their safety and risk measures. They are tough bugs that can live for extended periods of time and their contribution to human health improvement has given rise to a new notion of holobiont (Kim et al. 2019). Since Enterococcus species often exhibit vancomycin resistance, the use of Enterococci as an alternate therapy becomes a question which require greater attention (Wada et al. 2021). Healthy people with VRE colonization rarely get infected unless immunocompromised (Büchler et al. 2022 and Johnstone et al. 2018). In short, Enterococcus strains must be devoid of virulence genes and considered acceptable for use in food industries or as an alternative medication therapy.

The synergistic effect of banana proteins with CFS of E. lactis showed a decrease in growth and biofilm development and to the greatest of our knowledge, this is the first study that utilizes this combination. Synergism is a popular domain in therapeutics, most of the lectins have recently been studied in the context of synergistic actions like concanavalin A protein isolated from Canavalia ensiformis seeds improves the performance of gentamicin against various multi drug resistant MDR strains and could be further evaluated for structural based functional analysis (Santos 2021). The study reported that lectin from Alpinia purpurata (Apul) showed synergistic effect with different drugs against resistant strains of S. aureus also it inhibited biofilm development of non-resistant isolates of S. aureus (Ferreira et al. 2018).

Overall this study exploits the application of banana proteins and LAB, also the synergistic impact and the risk of contracting an Enterococcus illness can also be reduced by combining it with plant proteins having medicinal properties. Moreover, it opens new ways to deal with resilient pathogens by development of an alternate therapy. Extensive studies regarding production and classification of anti-biofilm molecules from both banana proteins and LAB should be undertaken as well as detailed studies regarding mechanism behind biofilm inhibition and further characterization of Enterococcus as probiotics is required.

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Isolation of lectin from Musa acuminata for evaluation of antibacterial activity against Methicillin-Resistant Staphylococcus aureus and the interaction of banana proteins with certain Enterococcus species

Abstract

Introduction

Materials And Methods

Results

Discussion

Abbreviations

Declarations

References