Lavandula dentata leaves as potential natural antibiofilm agents against Pseudomonas aeruginosa

Preparation of plant extract

Liquorice (roots and rhizomes of Glycyrrhiza glabra, Fabaceae) was obtained from Agricultural Horticulture, Faculty of Agriculture, El-Azhar University, Cairo, Egypt. Carrot (roots of Daucus carota subsp. sativus, Apiaceae), Red Cabbage (leaves of Brassica oleracea var. capitata f. rubra, Brassicaceae) and beetroot (roots of Beta vulgaris, Amaranthaceae) were purchased from the Local Market in Cairo, Egypt. Turmeric was purchased from Local Plant Store. Neem (leaves of Azadirachta indica, Meliaceae) was purchased from El Mansoreya Road and placed in the Medicinal Plants station at the Faculty of Pharmacy, Ain Shams University, Cairo, Egypt. French Lavender (leaves of L. dentata, Lamiaceae) was obtained from Agricultural Horticulture, Faculty of Agriculture, El-Azhar University, Cairo, Egypt. Voucher specimens (PHG-P-GG-453, PHG-P-DC-454, PHG-P-BO-455, PHG-P-BV-456, PHG-P-CL-458, PHG-P-AI-457 and PHG-P-LD-459 respectively) were kept at the Pharmacognosy Department Herbarium, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt. The plant names were checked with http://www.theplantlist.org in May 2024.

The collected plant parts were air-dried in the shade and cut into very small pieces/grated/sliced/crushed using hands followed by mortar and pestle/crushed. (150 g) of each plant were macerated at room temperature in (600 mL) of distilled analytical grade methanol (El Nasr Pharmaceuticals Chemicals Company (ADWIC), Egypt, PioChem for laboratory chemicals, Egypt and Lab Chem, USA) till complete exhaustion, and then the extracts were evaporated using rotavapor (BUCHI R-300, Switzerland) at 45°C using pump followed by concentrating through placing them in the hood (Flores Valles, Spain). Each plant extract was prepared in dimethyl sulfoxide (DMSO)-d6 (Cambridge Isotope Laboratories, Inc. Company, USA) ^45,46 in a concentration of 10 mg/mL_DMSO in Eppendorf’s tubes and kept in the refrigerator until further use.

Preparation of the reference drug

ACC (Mash Premiere company, Egypt) was prepared in a concentration of 10 mg/mL_DMSO in Eppendorf’s tubes and kept in the refrigerator. ACC is used as a reference compound; as it is considered a non-antibiotic drug with good antibacterial properties against P. aeruginosa^7,47,48,,49 and it is a biofilms-disrupter that showed the ability to interfere with biofilm formation and is the well-known preventer of the biofilm attachment⁴⁷.

Preparation of the bacterial strain and its growth

P. aeruginosa can infect both the skin and the urinary tract, but the way it causes these infections may differ, however its virulence factor in the two types of infection is the same which is the biofilm formation which is one of the most important virulence determinants⁵⁰, which helps it adhere to the skin or wounds and resist antibiotic treatment. Similarly to skin infections, P. aeruginosa can adhere to the urinary tract lining in case of urinary tract infection and form biofilms. A multiple drug-resistant P. aeruginosa clinical isolate (tested against P. aeruginosa ATCC^® 27853 reference strain⁵¹) was recovered from the urine of patients suffering from urinary tract infection by the Department of Microbiology & Immunology, Faculty of Pharmacy, Ain Shams University and stored frozen in glycerol at -80 °C. It exhibits the highest resistance rates to Ciprofloxacin (100%), Levofloxacin (100%), Metropenem (94.7%), Ceftazidime (94.7%), Imipenem (89.5%), Gentamicin (89.5%) and Cefepime (78.9%) and lower rates of resistance to Amikacin (47.4%) and Doripenem (42.1%)⁵¹.

Different media are suitable for P. aeruginosa growth, such as nutrient broth, tryptone soya broth, Luria-Bertani broth, Luria-Bertani broth supplemented with 0.1% glucose and others⁵². Two media were preliminary tested which were nutrient broth and Luria-Bertani broth supplemented with 0.1% glucose. Glucose was found to efficiently promote P. aeruginosa biofilm formation by upregulating the expression of the extracellular polysaccharide-related gene pslA⁵³. In case of nutrient broth medium, it was observed that the optical density (OD) of the bacterial sub-culture at 630 nm is below the cut-off value OD₆₃₀. The cut-off OD is defined as three standard deviations above the mean OD of the negative control. In contrast, the OD₆₃₀ in case of Luria-Bertani Broth medium supplemented with 0.1% glucose is above the cut-off OD₆₃₀, which means that Luria-Bertani Broth supplemented with 0.1% glucose may be better used as shown in Fig. 9.

The effect of the medium on P. aeruginosa biofilm formation (NB: Nutrient Broth Medium, LB: Luria-Bertani broth medium supplemented with 0.1% glucose, 24: 24 h incubation period, 48: 48 h incubation period).

Aseptically, (1 loopful) was cultured in a test tube containing (5 mL) of autoclaved (HICLAVE HVA-110 – HIRAYAMA Manufacturing Corporation, Japan) nutrient broth ‘E’ medium (LabM, United Kingdom), incubated at 37 °C in incubator shaker (New Brunswick Scientific C25KC Classic Series – EDISON, NJ, USA) for 24 h, then (250 µL) from this culture were sub-cultured in a flask containing (25 mL) Luria-Bertani broth medium (TM MEDIA, Delhi, India) supplemented with 0.1% glucose (ADWIC), incubated at 37 °C in incubator shaker for 18 h.

Evaluation of antimicrobial activity of methanol extract of the seven plants against P. aeruginosa

Since antibiofilm activity is usually assessed sub-MIC values⁵⁴ in order to state that the observed antibiofilm potential is not due to the ability of the extract to kill bacteria before biofilm formation, the antibacterial activity of the methanol extract of the seven plants against P. aeruginosa needs to be evaluated. The microplate method provided a potentially useful technique for determining the MICs of large numbers of natural product samples, requiring small amounts of substances. This method is not expensive and presents reproducible results⁵⁵.

Aseptically, double-strength Luria-Bertani broth medium supplemented with 0.1% glucose (100 µL) was added into 96-well flat-bottomed sterile polystyrene microtiter plate wells. (100 µL) of prepared plant extract/reference were vortexed (XH-II, ARI Medical Technology Co., Ltd, China), then added to the wells, mixed well, followed by two-fold serial dilution five times, then (100 µL) were discarded; to keep fixed final volume (100 µL) in all wells. Bacterial suspensions (10 µL of adjusted approximately equal to 10⁸ CFU/mL) were then added to these wells. Uninoculated wells are considered negative control. Microplates were incubated statically at 37 °C for 24 h. The MIC was recorded as the highest dilution showing no visible growth.

Microtiter plate assay to evaluate the antibiofilm activity of methanol extract of the seven plants

P. aeruginosa was specifically chosen as it is a well-known biofilm producer and is considered the most common biofilm model organism, in addition to its relative ease of establishing biofilms formed by it in vitro. The ability of the plant extracts under investigation to reduce the biofilm formation by P. aeruginosa was assessed using microtiter plate assay⁵⁶. Aseptically, (100 µL) of double-strength Luria-Bertani broth medium supplemented with 0.1% glucose was added into 96-well flat-bottomed sterile polystyrene microtiter plate wells. (100 µL) of prepared plant extracts were vortexed, then added to the wells, mixed well, followed by two-fold serial dilution five times. Bacterial suspensions (10 µL of adjusted approximately equal to 10⁸ CFU/mL) were then added to these wells. The control used was composed of (10 µL) of bacterial sub-culture on (100 µL) Luria-Bertani broth medium supplemented with 0.1% glucose. The positive control used was composed of (100 µL) of prepared concentration of ACC added to (100 µL) of double-strength Luria-Bertani broth medium supplemented with 0.1% glucose, mixed well, followed by two-fold serial dilution five times. To exclude the DMSO effect⁵⁷, DMSO was added to other media-contained wells (DMSO: Media: 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32), then (10 µL) of bacterial suspensions were added to these wells; in order to achieve the same concentrations of DMSO used in preparing plant extracts for the assay. Microplates were incubated statically at 37 °C for 24 h and for 48 h.

Planktonic cells in wells of microplates were discharged by washing three times with distilled water and shaking out water. Wells were then left to dry. Biofilms formed on the walls of microplate wells, from sessile isolates, were stained with (100 µL) of filtered crystal violet (0.1% w/v) for 10 min. Crystal violet, a common dye that has been used to quantitatively assess biofilms, binds to proteins and DNA of viable cells, and thus attached cells are stained with this dye and allows visualization of the adherent biomass⁵⁸. Crystal violet-stained wells of microplates were washed three times with distilled water, then wells were dried and blotted on a stack of paper towels to rid the plate of all excess cells and crystal violet. Acetic acid (ADWIC) (100 µL of 30%) was added to each well of the microtiter plates to solubilize the crystal violet. Microtiter plates were incubated at room temperature for 15 min, then (100 µL) of the solubilized crystal violet was transferred to new flat-bottomed polystyrene microtiter plates. The microplates were then measured spectrophotometrically using a plate reader (ELx808 – BioTek Instruments, Inc., USA) at 630 nm. This study was done in triplicates. Readings were processed using Gen5 Reader Control software and calculations were performed using Microsoft Excel 2016 software, Microsoft, Washington, DC, USA.

The mean % reduction of biofilm formation was determined for each plant extract at a certain concentration against P. aeruginosa after both 24 h and 48 h incubation period, in triplicates, using the equation below:

OD (control): OD of control wells (bacterial sub-culture on Luria-Bertani broth medium supplemented with 0.1% glucose); OD (DMSO): OD of DMSO wells at a specific concentration (bacterial sub-culture on DMSO at a specific concentration and Luria-Bertani broth medium supplemented with 0.1% glucose); OD (plant extract): OD of plant extract/reference wells at specific concentration (bacterial sub-culture on plant extract/reference at specific concentration and Luria-Bertani broth medium supplemented with 0.1% glucose).

Statistical analysis of the results of % reduction of biofilm formation of methanol extract of the seven plants

A Two-way ANOVA was performed to examine the effects of plant extract/reference and concentration (5, 2.5, 1.25, 0.625, 0.3125, 0.15625 mg/mL) on % reduction of biofilm formation using a total sample size (the product of the number of replicating the study, the number of plant extract/reference and the number of different used concentrations) of 144 (3 × 8 × 6) in case of both 24 h and 48 h incubation periods. These analyses aimed to determine whether or not there were significant main effects of two factors: extract type (factor 1) and concentration (factor 2), as well as any interactions between these factors.

Chemometric analysis of the % reduction of biofilm formation of methanol extract of the seven plants

Chemometric analysis was performed via PCA⁵⁹ using Unscrambler^® 9.7, CAMO SA, Oslo, Norway software. Cross-validation method was utilized, and the number of PCs was adjusted to 4. PCA score plot was constructed using the % reduction of biofilm formation using the six concentrations used for the seven plant extracts under investigation after 24 h and 48 h incubation periods.

Chemical investigation of methanol extract of L. dentata leaves using LC-MS analysis

The hyphenated high performance liquid chromatography-mass spectrometry (HPLC-MS) technique is an important method used for identifying complex mixtures especially the phenolics in plant extract, by comparing the mass spectrum obtained with literature (tentative identification)^13,60. LC-MS analysis was performed on methanol extract of L. dentata leaves which showed the highest bioactivity. LC-MS analysis was performed using HPLC (Nexera LC-30AD) equipped with an autosampler (SIL-30AC), temperature-controlled column oven (CTO-20AC), and coupled to triple quadrupole mass spectrometer (Nexera with LCMS-8045, Shimadzu Corporation, Kyoto, Japan) that was equipped with reversed-phase (RP)-C18 UPLC column (shimpack 2 mm × 150 mm) possessing 2.7 µm particle size. The following gradient elution, using HPLC-grade acetonitrile (ACN) and water (Sigma Aldrich Company), was used (ACN, 0. 1% HCOOH in H₂O) 0–2 min (10% ACN); 2–5 min (30% ACN-80% ACN), 5–15 min (50% ACN), 15–25 min (70% ACN), 25–28 min (80% ACN), 28–30 min (80% ACN) and 30–33 min (10% ACN), with 0.2 mL/min flow rate. Positive and negative modes were operated during LC-MS with electrospray ionization. LC-MS data were collected and processed by Lab Solutions software.

Molecular docking analysis of the identified compounds of methanol extract of L. dentata leaves

Since P. aeruginosa is the most studied microorganism with regard to QS⁵⁰ which is a communication mechanism used by bacteria to regulate gene expression in response to population density, further research was needed to fully elucidate the interactions between L. dentata-derived compounds and QS systems in P. aeruginosa. Molecular docking analysis was done against LasR, the QS regulatory protein, of P. aeruginosa. Molecular docking analysis was done against LasR using BIOVIA Discovery Studio 2016 Client. The most common approximation for docking is to hold the protein in a rigid conformation and dock a series of ligand conformations into the active site. Fast docking based on binding site features (“hotspots”) was done using LibDock which is an algorithm for docking small molecules into an active receptor site. Initially, a hotspot map is calculated for the receptor active site which contains polar and apolar groups. This hotspot map is subsequently used to rigidly align the ligand conformations to form favorable interactions. After a final energy-minimization step (allowing the ligand poses to be flexible), the top-scoring ligand poses are saved.

Protein structure was downloaded from https://www.rcsb.org/structure/2UV0. Water was removed from the downloaded protein, then the protein was cleaned to add hydrogens, check bonds and bond orders, and correct them, if necessary, standardize atom order in amino acids, and modify terminal residues. The force field is then applied. Fixed atom constraints were created from atoms rather than hydrogens. The prepared protein was then defined as a receptor and the binding site sphere was defined. Ligands (identified compounds from methanol extract of L. dentata leaves along with ACC) were subjected to adding hydrogens (if absent), optimizing their geometries using a fast, Dreiding-like forcefield, then were applied to force field using CHARMm which has a wide coverage for general organic molecules.

Ligands were then prepared to fix bad valencies, generate 3D coordinates, and remove duplicate structures. LibDock docking program performs the following steps using a set of pre-generated ligand conformations and a receptor with a specified binding site: removing hydrogen atoms, ranking ligand conformations and pruning by solvent-accessible solvent area, finding hotspots using a grid placed into the binding site and using polar and apolar probes. The number of hotspots was pruned by clustering to 200. Docking ligands pose was done by aligning to the hotspots. This was performed by using triplets (i.e., three ligand atoms are aligned to three receptor hotspots). Poses that result in protein clashes are removed. A final Broyden-Fletcher-Goldfarb-Shanno pose optimization stage is performed using a simple pair-wise score (similar to Piecewise Linear Potential). The top-scoring ligand poses are retained, then hydrogen atoms are added.