Plants have the potential to serve as a solution to address the shortage of new antibacterials and the increasing problem of antibiotic resistance. Plants possess a variety of efficient defense mechanisms, including the synthesis of secondary metabolites, to counteract pests and pathogens before they can inflict significant harm. Plants and other species have engaged in co-evolution for over 350 million years [1], and have devised tactics to bypass the protective mechanisms of one another. Plant secondary metabolites are crucial for plant adaptation to their environment and serve as a surveillance system. They are secondary products of non-essential metabolic pathways that are accountable for the distinct smells, flavours, and pigments of plant tissues. Plant secondary metabolites have a crucial role in enabling plants to withstand abiotic challenges such as UV radiation and facilitate communication with other species such as herbivores, diseases, neighboring plants, pollinators, and fruit dispersers. Consequently, these metabolites are essential for the overall growth and development of plants [2]. As Nigella sativa L. which used in Arab and Islamic countries as an alternative medicine for treatment the hypertension, diabetes, bronchial pulmonary disorders, gastrointestinal disorders, various infections, inflammations and allergies cases [3]. Ziziphus spina-christi belonged to the family Rhamnaceae show antibacterial as well as antifungal activity [4]. These plants typically include substances that fall within one of three major chemical classes known for their biological activity: terpenoids, phenolics, and alkaloids. Terpenoids are a much diversified subclass of secondary metabolites, consisting of over 50,000 identified chemicals [5].

The Microorganisms Used in Study

The isolated bacterial Staphylococcus aureus, Enterococcus feacalis, lactobacillus spp, Proteus mirabilis, Citrobacter freundii and Pseudomonas aeruginosa obtained from the microbiology laboratory in Al Hassein teaching hospital.

Prepare the Aqueous Extract of Plants

All of the aqueous extracts were prepared depending on previous studies for Ziziphus rugosa Lam aqueous extract prepared by [6]. Finally Nigella sativa seeds extract achieved depending on [7], commonly all extracts was dried, the concentrations was prepared.

Determination the MIC of plants extract

100 µl of bacterial suspensions (Adjusted 0.5 McFarland standard) and 50 µl of MIC the tested agent (plant extracts) added to Eppendorf tubes, incubated for 24 hrs. Then the tubes were examined for any turbidity], the tubes that lack turbidity were identified as the minimum inhibitory concentration these step repeated under anaerobic condition [8].

Effect MIC of Extracts on Preformed Biofilm

100 μl of bacterial suspension added into wells (96-well microtiter plate), followed by incubation at 37°C for 24 hr, the formed biofilm was washed at least three time with PBS to remove the non-adherent cells. 200 μl of MIC plant extracts added. Well was left empty as a negative control, in other side same steps were repeated but without MIC of plants as positive control these step repeated under anaerobic condition [9].

Statistical Analysis

The results were expressed by difference between means were analyzed statistically according to LSD, the differences were considered significant when P≤0.05 by using the software SPSS statistic 23.

The MIC of the extracts showed variations between isolates under aerobic and anaerobic condition in both extract (Figure 1).

Figure 1: The MIC of the plant extracts.

Anti Biofim Activity of Nigella Sativa

The results showed that the Nigella sativa extract revealed anti biofilm activity against the studied isolates also the results found there was no significant difference of N. sativa antibiolfim in anaerobic and aerobic except in P. aeruginosa, the antibiofilm was lower in aerobic condition (0.212+0.00) than anaerobic (0.280+0.026) as well as in the biofilm in P, mirabilis in aerobic condition (0.207+0.064) was lower than anaerobic (0.234+0.044). However the biofilm formation in E.feacalis in an aerobic (0.208+0.005c) was lower than anaerobic (0.253+0.016b) as shown in table (1).

Table 1: The Anti-biofilm effect of Nigella sativa under aerobic and anaerobic condition.

Anti Biofim Activity of Ziziphus Spina-Christi

The results showed that the Ziziphus spina-christi extract revealed a good anti biofilm activity against the studied isolates also the results found there was no significant difference of Ziziphus spina-christi antibiolfim in anaerobic and aerobic except in P. aeruginosa, the biofilm formation in aerobic which (0.222+0.00) lower than anaerobic (0.290+0.026) as in table (2).

Table 2: The Anti biofilm effect of Ziziphus spina-christi under aerobic and anaerobic condition.

The findings in (Table1) were agreed with [10], who found the antimicrobial activity of black seeds against gram positive and negative bacteria as S.aures, E coli, P. aeruginosa and salmonella spp, also the results agreed with [11]. That Nigella sativa seed extracts showed antibacterial and antibiofilm against ten human pathogenic bacteria including four biofilm producing bacterial strains: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, other study [12], showed that antibiofilm activity of the biosynthesized AgNPs from N. sativa seed extract was 88.42% for E. faecalis, 84.92% for E. coli, 81.86% for K. pneumonia, and 82.84% for S. aureus, respectively at 12.5 µg/mL, while with the same concentration, the biofilm formation was reduced by 49.9% in the case of P. aeruginosa. The results of the study agreed with [13], on Algerian Nigella sativa L. seeds confirmed the effectiveness of the essential oil and methanol extract in inhibiting the growth of various microbial strains found in the oral cavities of periodontal patients. These strains include S. aureus, S. epidermidis, S. pneumoniae, E. faecalis, K. pneumoniae, Proteus sp., A. baumannii, and A. calcoaceticus. The concentrations used in the study were 16,500 mg/ml and 8,250 mg/ml for the essential oil, and 125,000 mg/ml and 62,500 mg/ml for the methanol extract. The antibacterial activity of Nigella sativa was ascribed to its polyphenol and flavonoid contents, as reported by Kiari et al. in 2018. Additionally, the antibiofilm activity of Nigella sativa was related to the presence of thymoquinone and melanin [14].

The results in (Table 2) were consistent with the findings of [15], regarding the effectiveness of N. sativa, Z. spina-christi, Origanum majorana, Allium sativum and Rosmarinus officinal is medicinal plants against highly resist bacteria. These strains included K. pneumoniae (Gr-ve), Escherichia coli (Gr-ve), P.aeruginosa (Gr-ve), S.aureus (Gr+ve), and Methicillin-resistant Staphylococcus aureus (Gr+ve) obtained from clinical specimens. Demonstrated the antimicrobial activity of the most effective extract against biofilm formation. Z. spina-christi whole extract exhibited a potent antibacterial activity against all the multi-drug resistant (MDR) strains that were tested. In addition, its polyphenol component exhibited a more potent impact. While the polyphenol fraction exhibited strong antibacterial properties, its ability to prevent biofilm formation was comparatively weaker than that of the entire extract. The results indicated that the whole extract of Z. spina-christi had a potent antibiofilm action against biofilms of S. aureus, P. aeruginosa, and MRSA strains. Other research have corroborated the antibiofilm activity of the plant extract being examined, demonstrating its ability to inhibit the production of biofilms in certain pathogens, such as P. aeruginosa [16] and S. aureus [17].

Additionally, the present findings demonstrated that the alkaloids component of the Z. spina-christi extract had a moderate antibacterial action. This may be attributed to the fact that it has the capacity to interchelate with the DNA of Gram-positive and Gram-negative bacteria, as well as to interfere with the process of cell division [18]. Variety of secondary metabolites that found in the plants have been proven to possess antimicrobial activity such as phenols, flavonoid, alkaloids, quinones, tannins and terpenoids [19]. In response to plant pathogen attack, these secondary metabolites are synthesized by plants. The plant antimicrobial activity mechanism is weakly understood, there are some reports on the probable mechanism of action of phytochemicals, for example flavonoids have been mentioned to inhibit DNA synthesis [20], another class of phytochemicals is quinone which known to provide a source of stable free radicals , also bind irreversibly to proteins and inhibit bacterial growth [21].

Furthermore, the findings recorded a different between biofilm formation in P. aeruginosa, E.feacalis, P.mirabilis and between anaerobic and aerobic condition with extract this attributed to ROS produce in higher in aerobic condition beside to induction by plant extract which that lead to kill the bacteria as known the Catalase breaks down the ROS the hyposis under anaerobic condition the ROS will be decrease so the catalase will be sufficient to break down the ROS that induced by the extract as [22], revealed Catalase (KatA) is an extremely stable enzyme that is found outside of cells. It is essential for both the full virulence and the ability to withstand peroxide in both the planktonic and biofilm states of Pseudomonas aeruginosa bacteria.

Nigella sativa and Ziziphus spina-christi extract revealed a good anti biofilm activity against some of pathogenic bacteria. It is necessary to conduct intensive researches on more isolates and other species that are pathogenic to humans to benefit from these plants as a natural treatment along with manufactured antibiotics that most pathogenic isolates are resistant to.

Declaration of Competing Interest

The authors have declared no conflict of interest

Research Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

1. Clarke JT, Warnock RCM, Donoghue PCJ. Establishing a time-scale for plant evolution. New Phytol. 2011; 192: 266-301.
2. Kessler A, Kalske A. Plant secondary metabolite diversity and species interactions. Annu Rev Ecol Evol Syst. 2018; 49: 115-138.
3. Kiari FZ, Meddah B, Tir Touil Meddah A. In vitro study on the activity of essential oil and methanolic extract from Algerian Nigella sativa L. Seeds on the growth kinetics of micro-organisms isolated from the buccal cavities of periodontal patients. Saudi Dent J. 2018; 30: 312-323.
4. Prashith KTR, Vinayaka KS, Mallikarjun N, Bharath AC, Kumar SB, Kumar RMC, et al. Antibacterial, Insecticidal and Free radical scavenging activity of methanol extract of Ziziphus rugosa Lam. (Rhamnaceae) fruit pericarp. Pharmacognosy J. 2018; 2: 65-69.
5. Belcher MS, Mahinthakumar J, Keasling JD. New frontiers: harnessing pivotal advances in microbial engineering for the biosynthesis of plant-derived terpenoids. Curr Opin Biotechnol. 2020; 65: 88-93.
6. Raghavendra HL, Vinayaka KS. Evaluation of Pericarp and Seed Extract of Zizyphus rugosa Lam. for Cytotoxic Activity. Seed, 2012; 2: 887-890.
7. Al-Charchafchi FMR, Al-Shuhumi H, Al-Meselhy S, Al-Busadi M, Al-Shuhumi H. Biological activity of seed aqueous extract of Nigella sativa (L.) on germination and seedling growth of Vigna radiata (L.). Pak J Biol Sci. 2007; 10: 4319-4322.
8. Wiegand I, Hilpert K, Hancock RE. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc. 2008; 3: 163-175.
9. Tetz GV, Artemenko NK, Tetz VV. Effect of DNase and antibiotics on biofilm characteristics. Antimicrob Agents Chemother. 2009; 53: 1204-1209.
10. Abdullah SA, Salih TFM, Hama AA, Ali SI, Hamaamin HH. The Antibacterial Property of Nigella sativa (Black seed) Oil Against Gram-positive and Gram-negative Bacteria. Kurdistan J Applied Research. 2021; 6: 156-165.‏
11. Rahman MS, Roy T. Antibacterial and Antibiofilm Activities of Nigella sativa L. Seed Extracts. Bangladesh J Microbiology. 2021; 38: 7-13.‏
12. Almatroudi A, Khadri H, Azam M, Rahmani AH, Fahd Khaleefah AK, Riazunnisa K, et al. Antibacterial, Antibiofilm and anticancer activity of biologically synthesized silver nanoparticles using seed extract of Nigella sativa. Processes. 2020; 8: 388.‏
13. Kiari FZ, Meddah B, Meddah ATT. In vitro study on the activity of essential oil and methanolic extract from Algerian Nigella sativa L. Seeds on the growth kinetics of micro-organisms isolated from the buccal cavities of periodontal patients. Saudi Dent J. 2018; 30: 312-323.
14. Chaieb K, Kouidhi B, Jrah H, Mahdouani K, Bakhrouf A. Antibacterial activity of Thymoquinone, an active principle of Nigella sativa and its potency to prevent bacterial biofilm formation. BMC Complement Altern Med. 2011; 11: 29.
15. Sofy AR, Aboseidah AA, El-Morsi ES, Azmy HA, Hmed AA. Evaluation of antibacterial and antibiofilm activity of new antimicrobials as an urgent need to counteract stubborn multidrug-resistant bacteria. J Pure Appl Microbiol. 2020; 14: 595-608.‏
16. Adonizio A, Kong KF, Mathee K. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts. Antimicrob Agents Chemother. 2008; 52: 198-203.
17. Nostro A, Roccaro AS, Bisignano G, Marino A, Cannatelli MA, Pizzimenti FC, et al. Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms. J Med Microbiol. 2007; 56: 519-523.
18. Bukar AM, Kyari MZ, Gwaski PA, Gudusu M, Kuburi FS, Abadam YI. Evaluation of phytochemical and potential antibacterial activity of Ziziphus spina-christi L. against some medically important pathogenic bacteria obtained from University of Maiduguri Teaching Hospital, Maiduguri, Borno State–Nigeria. J Pharmacognosy and Phytochemistry. 2015; 3: 98-101.‏
19. Cushnie TP, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob Agents. 2005; 26: 343-356.
20. Rabaan AA, Alhumaid S, Albayat H, Mohammed A, Fadwa AS, Mawaheb AHH; et al. Promising Antimycobacterial Activities of Flavonoids against Mycobacterium sp. Drug Targets: A Comprehensive Review. Molecules. 2022; 27: 5335.
21. Tang QL, Kang AR, Lu CX. Phytochemical analysis, antibacterial activity and mode of action of the methanolic extract of Scutellaria barbata against various clinically important bacterial pathogens. Int J Pharmacology. 2016; 12: 116-125.
22. Shin DH, Choi YS, Cho YH. Unusual properties of catalase A (KatA) of Pseudomonas aeruginosa PA14 are associated with its biofilm peroxide resistance. J Bacteriol. 2008; 190: 2663-2670.