INTRODUCTION:

Biofilms are intricate bacterial community encapsulated within a polymeric matrix and possess strong adherent properties to an abiotic or biotic surface. It remains as one of the most challenging area of modern medicinal research as there is an increase in biofilm colonization on medical implants leading to chronic infections.

The treatment strategies established so far are not successful due to their inherent property of antibiotic resistance. A hydrated polyanionic complex of exopolysaccharide (EPS) forms the major volume of biofilms and protects the bacteria from adverse environmental conditions, aid in pathogenesis, antibiotic resistance and immune response¹. Another important characteristic featureis the microenvironment that makes bacteria to adapt and become more resistant to antimicrobial agents by inactivation of antimicrobial targets². The various modes of resistance strategies adopted by biofilms makes the pathogen more difficult to treat when compared to their planktonic counterparts³.

According to National Institute of Health (NIH), about 65% of bacterial infections are due to biofilm formation which can lead to 80% of chronic infections. In general, the microorganisms which reside within biofilms are difficult to eradicate and becomes more vulnerable in case of Gram negative bacteria namely Pseudomonas aeruginosa. P. aeruginosa is a virulent human pathogen responsible for wide range of hospital acquired infections, a major cause of deaths in cystic fibrosis patients, and are resistant to multiple antibiotics^4,5. It forms a highly structured biofilm in patients with chronic infections and effectively colonizes various medical materials⁶. The mortality rate due to its infections are increasing because of its ability to form strong biofilms and its resistance towards traditional antibiotics. In Gram negative bacteria like P. aeruginosa, a chemical signaling process known as quorum sensing (QS) upholds a crucial role in regulation of biofilm formation. It is an intercellular cell-to-cell communication system that regulates gene expressions involved in various biological functions⁷.

QS sense a critical number of bacteria and regulates various transcriptional genes⁸. QS signaling molecules referred to as autoinducers (AIs) have the ability to recognize and induce or suppress QS controlled gene expressions by binding to their target transcriptional receptors. P. aeruginosa QS system possess two acyl homoserine lactone Lux group of systems namely lasI/lasR and rhlI/rhlR, that regulates the genes accountable for production of virulence factors and a third non-LuxI/LuxR QS system referred to as the Pseudomonas quinolone signal (PQS). In lasI/lasR system, lasI produces its signaling molecule3-oxo-dodecanoylhomoserine lactone (3-oxo-C12HSL) which binds to its cognate receptor LasR to activate the gene expression associated with its corresponding virulence factors namely elastases, proteases, and exotoxin A. In the other system, rhlI synthesize its signaling molecule butanoyl homoserine lactone (C4HSL) which is regulated by LasR-3-oxo-C12HSL complex. C4HSL binds to RhlR and activate genes necessary for pyocyanin, elastases, siderophores and rhamnolipids production⁹. Thus, targeting these QS networks might serve as an alternative strategy to decrease the pathogenesis of P. aeruginosa.

P. aeruginosa synthesizes three exopolysaccharides namely Pel, Psl and alginate which contributes to biofilm formation in P. aeruginosa as matrix components. In addition, the extracellular DNA (eDNA) and rhamnolipids are involved in micro colony formation¹⁰. Medicinal plants are believed to be the indigenous cure and serve as an effective alternative form of health care medicine in traditional medical systems over centuries that aims to modern drug discovery^11,12. Over the years, the active constituents of plants demonstrated better healing and medicinal properties and established the finding that drugs from plants are safer than conventional medicines^13,14. In this study, Amomum subulatum (Cardamom), Holarrhena pubescens (Kurchi bark), Laurusnobilis (Bay leaf) and Punica granatum (Pomegranate) were selected based on their medicinal value and antimicrobial properties. A. subulatum is one of the Indian spices and an antimicrobial agent used in Ayurveda in the treatment of nausea, itching and tuberculosis¹⁵. H. pubescens is a medicinal plant with antimicrobial and anti-inflammatory agent and a rich source of steroid alkaloids specially used in the treatment of jaundice¹⁶. L. nobilis is one of the ancient herb with strong antifungal activity, anticancer activity and has found to be more effective in inhibition of biofilms. P. granatum is a shrub whose peels are used in treatment of various skin infections and serve as excellent source of compounds with anticancer, antimicrobial, and antibiofilm activities¹⁷.

MATERIALS AND METHODS:

Bacterial strain and preparation of plant extracts:

Bacterial strain namely Pseudomonas aeruginosa PAO1 MTCC 2453 was used as the model organism for the study. Prior to each assay, a single colony of bacteria from the nutrient agar plate was allowed to grow in Luria Bertani (LB) medium for 16-18 hrs at 37°C and the density of broth was adjusted to 0.5 optical density with LB broth. Plant materials namely Amomum subulatum (Cardamom), Holarrhena pubescens (Kurchi bark), Laurus nobilis (Bay leaf), and Punica granatum (Pomegranate) were purchased from a local market in Coimbatore city. The purchased plant materials were rinsed under running tap water followed by sterile distilled water. The samples were shade dried for one week and ground to a fine powder. 10g of powdered material was extracted in methanol (100ml) over a period of 24 hrs. The filtered extracts were concentrated to dryness using a rotatory evaporator at 50℃ to yield crude extracts and stored at 4°C until further use.

Minimum Inhibitory Concentration:

The minimum inhibitory concentration (MIC) of A. subulatum, H. pubescens, L. nobilis, and P. granatum extracts against P. aeruginosa PAO1 was identified by microbroth dilution method as per the guidelines of Clinical and Laboratory Standards Institute (CLSI). Two fold serially diluted extracts in LB medium with their final concentrations ranging from 0.25 to 32 mg/ml for A. subulatum and H. pubescens whereas 1.56 to 200 mg/ml for L. nobilis and P. granatum were used. The plates were incubated at 37°C for 18-24 hrs. The lowest concentration of the extract which completely inhibited the bacterial growth was determined as the MIC of the extract. Azithromycin at its sublethal concentration was used as the positive control¹⁸.

Antibiofilm activity: Crystal violet staining assay:

The inhibitory effect of extracts on formation of biofilm in P. aeruginosa PAO1 was evaluated using the method of Lee et al.¹⁹. In brief, overnight culture of P. aeruginosa PAO1 was diluted (1:100) and treated with different concentrations of plant extracts for 24 hrs at 37°C. Bacterial growth was measured and the free floating planktonic cells were removed. The plates were washed with phosphate buffer saline (PBS, pH 7.4) and 0.1% crystal violet solution was added and incubated at room temperature for 20 mins. The wells were again washed with PBS to remove the excess stain. To the dried plate, 95% ethanol was added and allowed to solubilize for 5-10mins. Finally, the ethanol re-suspended cells were measured by measuring the absorbance at 570nm.

Exopolysaccharide production inhibition: Congo red binding assay:

The production of exopolysaccharide (EPS) in P. aeruginosa PAO1 was assessed using the procedure reported by Goswami et al.²⁰. Overnight culture of P. aeruginosa PAO1 (diluted 1:100) was allowed to grow in LB broth with presence and absence of plant extracts for 24 hrs at 37°C. The planktonic cells were removed,and the adhered cells were washed with PBS. 1% Congo red was added and incubated for 30 mins in dark. Excess dye was removed, and the bound dye was solubilized by adding DMSO. Finally, the solubilized dye was measured by reading the absorbance at 490nm.

Virulence factors assay:

To determine the effect of the plant extracts on production of virulence factors namely pyocyanin, rhamnolipids, and alginate, P. aeruginosa PA01 was cultured in LB broth with and without plant extracts, followed by incubation at 37°C for 24 hrs. The cultures were centrifuged for 15 mins at 4°C. The filter sterilized (0.22μm) supernatants were utilized for the bioassays. Assay for pyocyanin was carried out by the protocol described by Essar et al.²¹ where 1ml of cell-free supernatant was added to chloroform and mixed vigorously. The pyocyanin pigment that is extracted in the chloroform layer was fetched into another tube and 0.2M hydrochloric acid was added to obtain a deep red colour solution. The absorbance of the solution was measured at 520nm. Production of rhamnolipid was assayed using the cetyltrimethylammonium bromide (CTAB) agar plate method described by Pinzon et al.²². Briefly, culture filtrates were inoculated on M9-glutamate minimal medium agar plates containing 0.2g of CTAB and 5mg methylene blue and incubated for 24 hrs at 37°C. Post 24hrs, the plates were kept for 48 hours at room temperatureand followed by refrigerator for two days. A dark blue halo zone indicates rhamnolipids production. Alginate production was identified using the procedure discussed by Owlia et al.²³. 600µl of boric acid/ H₂SO₄ solution (4:1) was added to 70µl of filter-sterilized supernatant. 0.2% carbazole solution was added, vortexed and incubated for 30mins at 55°C. After incubation, the absorbance was read at 530nm.

Motility assays:

Swimming and swarming motility assay:

Swimming and swarming motility assay was assessed according to the protocol mentioned by Packiavathy et al.²⁴. For swimming motility, diluted overnight culture of P. aeruginosa PAO1 was point inoculated at the center of the swimming agar medium that contained 1% tryptone, 0.5% NaCl and 0.3% agar. For swarming motility, the cultures were inoculated at the center of the swarming agar medium which contained 1% peptone, 0.5% NaCl, 0.5% agar and 0.5% filter sterilized D-glucose. The plates were kept for 24hrs at 37°C to observe the motilities.

RESULTS:

Yield percentage of plant extracts:

The yield percentage for methanol extracts of H. pubescens, L. nobilis, and P. granatum were observed to be 10%, 9%, and 8%, respectively (Table 1). The percentage of yield for A. subulatum methanol extract was found to be 4% which is the least compared to other plant extracts.

Table 1: Yield percentage of plant extracts

Plant extract	Yield (%)
Amomum subulatum (Cardamom)	4
Holarrhenapubescens (Kurchi bark)	10
Laurus nobilis (Bay leaf)	9
Punica granatum (Pomegranate)	8

Determination of minimum inhibitory concentration of plant extracts:

The MICs of A. subulatum, H. pubescens, L. nobilis, and P. granatum against P. aeruginosa PAO1 assessed by microbroth dilution were determined to be 4mg/ml, 0.5 mg/ml, 6.25mg/ml, and 25mg/ml, respectively (Table 2a and 2b). The MIC of the plant extracts was defined as the lowest concentration that completely inhibited the bacterial growth²⁵. The sub-MICs (1/2 MIC, 1/4 MIC) of the extracts were used for further studies since the growth of P. aeruginosa was not inhibited at their sub-MICs. Azithromycin, a standard antibiotic with MIC of 0.05 mg/ml was employed as the positive control.

Table 2a: MIC of methanolic extracts of A. subulatum and H. pubescens on P.aeruginosa

Extract (mg/ml)	32	16	8	4	2	1	0.5	0.25
A.subulatum	-	-	-	-	+	+	+	+
H. pubescens	-	-	-	-	-	-	-	+

Table 2b: MIC of methanolic extracts of L. nobilis and P. granatum on P. aeruginosa

Extract (mg/ml)	200	100	50	25	12.5	6.25	3.12	1.56
L. nobilis	-	-	-	-	-	-	+	+
P.granatum	-	-	-	-	+	+	+	+

Effect of extracts on biofilm formation in P. aeruginosa:

The inhibitory action of plant extracts on P. aeruginosa PAO1 biofilms was examined by crystal violet staining assay (Figure 1a). The results inferred that increase in concentration of the extracts increased the rate of biofilm inhibition. The percentage of biofilm inhibitory activity at 1/2 MICs of A. subulatum, H. pubescens, L. nobilis, and P. granatum were determined to be 45.2%, 39.9%, 67%, and 56.7%, respectively. The highest percentage of biofilm inhibition was exhibited by L. nobilis (Figure 1a).

Effect of extracts on EPS production in P. aeruginosa PAO1:

The effect of the extracts on EPS production in P. aeruginosa PAO1 was ascertained by Congo red binding assay. The amount of aggregated EPS was drastically decreased on treatment with all the four plant extracts (Figure 1b). Among them, greater inhibitory effect (79.48% ) was shown by P. granatum at a concentration of 12.5mg/ml whereas lower effect was exhibited by H. pubescens (7.74%) at its sub-MIC of 0.12mg/ml.

Figure 1: Effect of methanolic plant extracts on a) biofilm formation b) EPS production inP. aeruginosa PAO1

Effect of extracts on QS-associated production of virulence factors in P. aeruginosa PAO1:

Pyocyanin is a virulence factor which plays a significant role in invasion of bacteria into the host cells²⁶. It was observed that all the extracts decreased the production of pyocyanin in a dose dependent way (Figure 2a). Among them, A. subulatum exhibited the highest percentage of inhibition (53.54%) on pyocyanin production at its sub-MICof 2 mg/ml and the lowest inhibition percentage of 31.48% was shown by L. nobilis at its sub-MIC of 1.56 mg/ml. Rhamnolipids are bio-surfactants that play a major role in biofilm architecture and swarming motility. The blue halo zone that appears due to the precipitation of CTAB in presence of methylene blue indicates the production of rhamnolipids²⁷. Reduction in rhamnolipid production when treated with all the four extracts were evidenced (Figure 2b) by the presence of small diameter of halo zone around P. aeruginosa PAO1 growth. The efficiency of the plant extracts to inhibit alginate production in P. aeruginosa PAO1 was determined as it is an important component of the extracellular matrix material²⁸. It was seen that, with an increase in concentration of the extracts, the production of alginate was decreased. Among the four extracts, A. subulatum at its sub-MICof 2 mg/ml reduced alginate production to 56.53%. On the other hand, H. pubescens exhibited the least effect on inhibiting alginate production (27.45%) at its sub-MIC of 0.12 mg/ml (Figure 2c).

Figure 2: Effect of methanolic plant extracts on production of a) pyocyanin b) rhamnolipid c) alginatein P. aeruginosa PAO1

Effect of extracts on motilities in P. aeruginosa PAO1:

Swimming and swarming motilities play a crucial role in QS regulated biofilm formation in P. aeruginosa²⁹.The highest inhibitory action on swimming and swarming motility was observed for A. subulatum and P. granatum at its sub-MICs (Figure 3&4). The obtained results were comparable to that of the positive control (Azithromycin).

DISCUSSION:

P. aeruginosa, one of the major causative organism is responsible for biofilm formation and owes to major types of chronic infections in nature. The architecture of P. aeruginosa biofilms consist of various components and are highly complex in nature. Rather than its structural complexity, the expression of biofilm related genes and the presence of persister cells makes these pathogens more resistant to conventional medicines. Targeting QS that regulates the expression of disease-associated features could be an alternative treatment strategy against multidrug resistant organisms as it may attenuate pathogenesis without affecting the bacterial growth³⁰. Plants have remained as a great source of novel drug compounds since they have vast benefits to human health. Numerous studies have been so far undertaken based on plant derived products for their antibiofilm and antivirulence properties³¹. Hence in the present study, four unique medicinal plants namely A. subulatum, H, pubescens, L. nobilis, P. granatum were examined for their effectiveness as inhibitors of biofilm formation and QS regulated virulence factors production against P. aeruginosa PAO1. Additionally, the motility behaviour of P. aeruginosa PAO1 on treatment with plant extracts were also determined.

The MIC analysis revealed that none of the plant extracts showed its direct antibacterial effect on P. aeruginosa PAO1 grow that early stationary phase and there by could reduce the QS-controlled virulence factors levels without retarding the growth. Initially, the selected medicinal plants were screened for their antibiofilm effect to inhibit formation of biofilm in P. aeruginosa PAO1 by crystal violet assay. The results revealed that the methanolic plant extracts effectively inhibited the formation of biofilm in a concentration dependent manner. Coherent to our findings, Ouyang et al.¹⁸ has stated that quercetin impaired biofilm formation by inhibiting the adherence of P. aeruginosa PAO1 to microtitre plate surfaces at its sub-MIC concentration of 16μg/ml. EPS which is regulated by QS mechanism plays a predominant role as a protection barrier to retard the invasion of antimicrobial agents into the cells²⁹. There was a remarkable decrease in the production of EPS in P. aeruginosa PAO1 when treated with the selected plant extracts. Among them, P. granatum demonstrated the highest inhibitory effect on EPS production.

Pyocyanin is a toxic secondary metabolite which regulates P. aeruginosa PAO1 infections by its effects on DNA damage, reactive oxygen species generation, and destabilization of redox sensitive lysosomes²⁶. The production of pyocyanin was greatly affected by A. subulatum extract in a concentration dependent manner (Figure 2a). Jain et al.³² has also witnessed certain medicinal plants namely Ipomea pesticides, Citrullus colocynthis, and Digera muricata for their potential to inhibit the production of pyocyanin. Musthafa et al.³³ stated that aqueous extracts of edible fruits and plants namely Musa paradisiaca, Ocimum sanctum, Ananas cmosus, and Manilkara zapota inhibited production of violacein pigmentin C. violaceum, pyocyanin pigment, and biofilm formation capacity of P. aeruginosa PAO1 and can serve as quorum sensing inhibitors (QSIs). The Rhl controlled rhamnolipids and alginate productionin P. aeruginosa PAO1 are essential for adherence to host cells and to protect from environmental conditions within the hosts, respectively^27,28. The methanolic extracts of the selected plants decreased the production of rhamnolipids and alginate at their sub-MICs effectively in a concentration dependent manner (Figure 2b and 2c). These results agreed with the report by Rasamarivaka et al.³⁴who stated dose dependent inhibitory effect of quercetin on production of rhamnolipids and alginate. Kim et al.³⁵ has stated that production of rhamnolipid was drastically suppressed to about 36-60% on treatment with 0.1-100μM concentrations of 6-Gingerol. Among the four plant extracts, A. subulatum showed the highest inhibitory activity on pyocyanin, rhamnolipid, and alginate production at its sub-MIC.

The flagellar motility behaviour of P. aeruginosa is necessary for initiation and maturation of biofilms²⁹. All of the screened plants impaired the swimming and swarming motilities of P. aeruginosa with shorter diameters on agar plates (Figure 3 and 4). These findings highlighted the potency of the screened plant extracts as a source of anti-QS compounds. Al-Hussaini and Mahasneh³⁶, have also evidenced that extracts from different plants namely Laurus nobilis, Combretum albiflorum, and Sonchus oleraceus to possessed anti-QS activity. Thus, the study suggested that all the four screened plants namely A. subulatum, H. pubescens, L. nobilis, and P. granatum demonstrated antibiofilm and antiquorum sensing activity against P. aeruginosa PAO1. These findings may serve as a preliminary screening to extract new active compounds and can be counted as new leads that can be used as a combination therapy along with antibiotics to manage antibiotic resistant P. aeruginosa infections in near future.

LIST OF ABBREVIATIONS:

MIC - Minimum Inhibitory Concentration

EPS - Exopolysaccharide

NIH - National Institute of Health

QS - Quorum Sensing

AIs - Autoinducers

PQS - Pseudomonas quinolone signal

3-oxo-C12HSL - 3-oxo-dodecanoylhomoserine lactone

C4HSL-Butanoyl homoserine lactone

eDNA - Extracellular DNA

LB medium - Luria Bertani medium

CLSI - Clinical and Laboratory Standards Institute

PBS - Phosphate buffer saline

DMSO - Dimethyl sulfoxide

CTAB - Cetyltrimethylammonium bromide

QSIs - Quorum sensing inhibitors

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENTS:

The authors wholeheartedly express their gratitude to the Management of Avinashilingam Institute for Home Science and Higher Education for Women for providing necessary facilities to carry out this research work.

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Received on 10.02.2022 Modified on 07.12.2022

Research J. Pharm. and Tech 2023; 16(11):5218-5224.

DOI: 10.52711/0974-360X.2023.00846