Bacterial strains
All clinically isolated Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Pseudomonas aeruginosa, Acinetobacter baumannii from various specimens were screened for their Drug Resistance status according to CLSI guidelines – CLSI M100-S22, 201215. Multi drug resistant isolates were further tested for biofilm production and categorized into 3 groups – strong, moderate and weak biofilm producers16. Referral ATCC Bacterial strains of the similar isolates that have been previously characterized in Microbiology laboratory of SGT Medical College, Hospital and Research Institute, Gurugram were simultaneously tested in triplicates for antibacterial activity and single testing for biofilm inhibition assay.
Collection and certification of medicinal plants
Syzygium aromaticum – UHF herbarium no. 13632, Camellia sinensis – 13633, Allium sativum – 13590 and Coriandrum sativum – 13634 were obtained and certified from Department of Forestry, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh.
Plant Extract Preparation
The methanolic extracts of the above mentioned plants were prepared. Flower buds (Syzygium aromaticum), dried leaves (Camellia sinensis, Coriandrum sativum) and bulb part (Allium sativum) of plants were crushed to powder and soaked into 50ml of methanol. Further, it was continuously boiled for 3 minutes for 3 times, with a gap of 2 minutes interval between each boiling time. The extract or supernatant was collected, subjected to centrifugation for 5minutes at 3600g until clear supernatant was obtained. The supernatant was then filtered using 0.2 um filter (Micropore filters), and stored at 4⁰C until further use17.
Antimicrobial activity by using Agar well diffusion method
Sterile petri dish plates containing 20 ml Muller Hinton agar were prepared. Fresh culture suspensions (0.5 McFarland unit) of isolated pathogenic bacteria were swabbed on the respective plates. Sterile gel puncher was used to make wells over the agar plates in which plant extracts were added at various concentrations (50, 25, 12.5 & 6.25 mg/mL). These plates were further incubated for 24 hours at 37°C. After incubation,the diameter of inhibitory zones around each disc were measured in mm and recorded17,18.
Determination of Minimum Bactericidal Concentration (MBC)
MBC is defined as the concentration producing a 99.9% reduction in colony forming units (CFU) number in the initial inoculum. Serial two-fold dilutions of the plant extracts were made at concentration of 50, 25, 12.5 and 6.25 mg/mL to which 100uL of microorganism suspension at a final density of 105cells/ml were added. The tubes were incubated at 37°C for 24 h. The tubes after 24 h of incubation were sub-cultured on Mueller Hinton agar and the bacterial growth was observed on the very next day. MBC was determined as the lowest concentration of plant extract that failed to yield any bacterial growth in the subcultures19.
Determination of Biofilm Formation by bacterial isolate using modified crystal violet assay
Sterile 96-well tissue culture plates were used to which 50 µl of Mueller–Hinton broth per well was added. Fresh bacterial suspensions (1.0 McFarland) were made and 50µl were added to the wells and incubated for 48 hours at 37°C. To check for the biofilm formation, contents from the wells were removed by washing with 200µl normal saline after which 200 µl of 0.1% crystal violet stain was added and incubated again for 20 minutes. Then, each well was thoroughly washed with deionized water and later the wells were added with 200 µl of 96% ethanol. Optical density (OD) of the adherent bacteria was calculated using ELISA reader at 630 nm. Formation of biofilm was calculated using the formula.
OD of bacteria= [(OD growth control – OD sample) / OD growth control] × 100.
Strains were classified as follows20:
OD ≤ ODc= No biofilm producer
ODc< OD ≤ 2 × ODc= Weak biofilm producer
2 × ODc< OD ≤ 4 × ODc= Moderate biofilm producer
4 × ODc< OD= Strong biofilm producer.
ODc: Optical density of growth control
Determination of Anti Biofilm Activity of plant extracts using modified crystal violet assay
Sterile 96-well tissue culture plates were used to which 50 µl of Mueller–Hinton broth was added to each well. Two-fold serial dilutions of plant extract were made in the tissue culture plates. Final concentrations to be tested were 50, 25, 12.5 and 6.25 mg/mL. Fresh bacterial suspensions (1.0 McFarland turbidity standard matched) were made and 50 µl was added to the wells containing plant extract at different concentrations. Bacteria without plant extract was used as growth control. After 24 hrs of incubation modified crystal violet assay was performed as described above. The percentage of biofilm inhibition was calculated by using the following formula:
[(OD growth control – OD sample) / OD growth control] × 100.
The biofilm inhibition concentration (BIC50) was defined as the lowest concentration of extracts that showed 50% inhibition on the biofilm formation20.
