Antibacterial and antibiofilm potential of leaves extracts of Mirabilis jalapa L . and Ajuga bracteosa wall . against Pseudomonas aeruginosa

In the Present study the antibacterial and antibiofilm potential of Mirabilis jalapa and Ajuga bracteosa crude methanolic extracts of leaves against P1, P2 and P3 clinical strains of Pseudomonas aeruginosa was explored. Antibacterial activity was investigated by agar well diffusion method and the antibiofilm potential was determined by Anti-Pellicle assay and exopolysaccharide inhibition (EI) by Congo red (CR) assay. In antibacterial assay, M. jalapa extract showed good antibacterial activity against P. aeruginosa with maximum 13.5 mm inhibition zone (ZI) against P2 strain and minimum 12.8 mm ZI against P3 strain of P. aeruginosa as compared to A. bracteosa which showed a maximum 13.4 mm ZI and a minimum of 12.7 mm against both P2 and P3 strains of P. aeruginosa, respectively. Both M. Jalapa and A. bracteosa extracts showed moderate (+++) and weak (++++) effect on pellicle inhibition against the tested strains of P. aeruginosa. In CR assay, a moderate antibiofilm effect was recorded on EI for both plants. Based on the results it was concluded that methanol extracts of M. jalapa and A. bracteosa possessed good antibacterial activity and moderate antibiofilm potential against P. aeruginosa.


Introduction
Many of the bacterial species have survived in unfavourable conditions by attaching each other and often cells adhere to surfaces to form highly organized matrix sheathed structure known as biofilm [1, 2].The structure of biofilms shields the microbes from the effect of antimicrobial agents and immune cells, as it stops their penetration.It has been reported that biofilms are involved in about 60% of human infections [3].The bacterial species that form the biofilm structure are physiologically different from their planktonic counterparts.Bacteria survive in biofilm form exhibited increased resistance to antibiotics [2, 4].Therefore, the emergence of resistance to antibiotics resulted in the enhance need of natural antibacterial drugs active against microorganisms involved in biofilm formation and spared of infections [5].Pseudomonas aeruginosa (P.aeruginosa) has emerged as a key opportunistic pathogen involved in the production of various virulence factors responsible for the infections especially in peoples with compromised immunity, burn wounds and cystic fibrosis [6, 7].P. aeruginosa have this remarkable ability to live as biofilms and results in chronic infections and also showed increased resistance to antimicrobials agents

Plant extraction
The aerial parts of M. jalapa and A. bracteosa were collected from the area of Shaheed abad, Lower Dir, Khyber Pakhtunkhwa, Pakistan as shown in Figure 1.The plant specimens collected were then identified by Dr. Uzma Khan, Associate professor, Department of Botany, Hazara University Mansehra.The voucher specimens of both collected plants were placed in Hazara University Herbarium.The shade-dried leaves of plants (1kg) were crushed into powder and the methanol crude extract of both plants were prepared by keeping it in methanol solvent for 3 days on vigorous shaking.The extracts present in methanol were then filtered and methanol was evaporated via rotary evaporation process and the semi-solid extracts of both plants were stored at 4°C.The extraction process was repeated 3-4 times in order to get maximum plants extraction.The crude methanol extracts of plants were then used for further processing.

Exopolysaccharide inhibition assay
The Exopolysaccharide inhibition assay was performed for crude extracts of both M. jalapa and A. bracteosa against three tested strains of P. aeruginosa.In this assay Congo red agar plates were prepared by adding 40 µg/ml Congo red dye, 10 g/l tryptone and 20 µg/ml coomassie brilliant blue.Then (2.5 μl) of inoculum of each strain in nutrient broth with crude methanolic extract (15 mg/ml) of both plants and with no extract (control) was transfer into the CR agar plates and were kept for 4 days at room temperature [20].In the absence of plant extracts, P3 strain of P. aeruginosa formed a dark red color bacterial colonies as compared to P1 and P2 strains, which showed that this strain have a higher tendency to form biofilm due to increase production of polysaccharides.When extracts were used, a color change in the bacterial colonies was recorded and was consider as polysaccharides inhibition and antibiofilm effect.

Results and discussion
Numerous studies have been reported that numbers of plant extracts were used to screen out their antimicrobial potential In antibiofilm assay, the effect of M. jalapa and A. bracteosa methanolic extracts was observed on inhibition of pellicle layer at different concentrations as shown in (Table 1 and 2).A moderate disruption (+++) of pellicle layer was recorded for both M. jalapa and A. bracteosa against all 3 P. aeruginosa strains tested at 15-12.5 mg/ml concentrations.Results revealed that with increase in concentration from 12.5 to 15 mg/ml there is no increase in antipellicle effect signifying 12.5 mg/ml to be the lowest effective dose for all the tested strains.It has been documented that when low concentration of antibiotics or other antimicrobial drugs is able to prevent initial adherence of bacteria to surfaces, the steps of mature biofilm formation would also be effected [16].A weak antipellicle activity (++++) was observed for both plants against P1, P2 and P3 strains biofilm formation from concentration 10 mg/ml to 7.5 mg/ml, respectively.During antipellicle activity, the extracts inhibition of bacterial cell attachment confirmed that prevention of bacterial attachment to surface is easier than mature biofilm [17].In the current findings, a moderate to weak antibiofilm activity in pellicle inhibition assay was observed for M. jalapa and A. bracteosa at different concentrations.There is no data exist on the pellicle inhibition for M. jalapa and A. bracteosa.The effect of plant extracts on pellicle inhibition has been previously reported [28].The antibiofilm potential of M. jalapa and A. bracteosa on exopolysaccharide production in Congo red assay was recorded at 15 mg/ml and a moderate antibiofilm effect was observed.The changes in color of the bacterial colonies due to extracts was recorded and compared to controls used against the tested strains as shown in Figure 4 (A, B and C).In CR assay, without plant extract, maximum polysaccharide production was observed in P3 strain forming dark red color colonies as compared to P1 and P2 strain (Figure 4 Figure 1.(A) M. jalapa (B) A. bracteosa A).It was observed that the extracts clearly suppressed exopolysaccharide production, form light red color bacterial colonies and as a result inhibited biofilm formation.There is no data present on biofilm inhibition by CR assay for A. bracteosa and M. jalapa.However, the antibiofilm effects of plants extracts by CR assay have been previously reported [17].In antibacterial and antibiofilm assays, an increase in resistance was found to extracts in biofilm mode [29].It is well known that bacteria in biofilm form showed more resistant to antibacterial agents [30].

Figure 2 .Figure 3 .
Figure 2. Inhibition zone (mm) against different strains of P. aeruginosa by the methanolic extract of M. jalapa after 24h incubation.Data represent as mean ±standard error.(n=3)

[21, 22].
[23,25]antages of biofilm formation are numerous for bacteria.Bacteria that involved in the formation of biofilms are protected from host, antibiotics and thus exhibited increased drug resistance[23,25].
From the results it is concluded that the methanolic extracts of M. jalapa and A. bracteosa leaves exhibited antibacterial and antibiofilm potential against clinical strains of P. aeruginosa.Further study is needed to isolate antibacterial compounds in order to use it for control strategies against P. aeruginosa biofilm formation in bacterial infections.