Assessment of Antibacterial Efficacy of Callistemon viminalis (Sol. ex Gaertn.) G. Don against Some Isolates Obtained from Urinary Tract Infections

1. Introduction

Callistemon viminalisis is a member of the Myrtaceae family, which belongs to the Plantae Kingdom and has several species (34 species). Some of the most famous species include Metrosideros viminalis (Sol. ex Gaertn.), C. viminalis (Sol. ex Gaertn.) G. Don, and Melaleuca viminalis (Sol.ex Gaertn.) Byrnes ( 1 ). The flowers are cylindrical and resemble bottlebrush. The common name of the C. viminalis is the weeping bottle brush. It is native to Australia and temperate regions. In its natural habitat, it grows in the form of shrubs or small trees reaching four meters in height ( 2 - 4 ).

Undoubtedly, urinary tract infections (UTIs) have become more dangerous due to bacterial resistance to antibiotics, which has led to the emergence of strains that are resistant to most antibiotics and cause multiple infections ( 5 ). The increasing use of antibiotics in various medical, industrial, and agricultural fields has led to the emergence of a phenomenon called "multi-drug resistance" which refers to the resistance of bacteria to many classes of antibiotics. This increases the risk of infections and reduces the chances of recovery ( 6 ).

WHO ( 7 ) has reported that medicinal plants can meet community needs and provide primary healthcare in case of the aggravation of the problem of antibiotic resistance. This is due to their effective substances that are capable of making a difference and providing hope for the future. Secondary metabolites produced by medicinal plants can act as bacteriostatic and bactericidal against "multi-drug resistant" microorganisms and are regarded as a good precursor for the synthesis of new antibiotics and drugs for controlling infectious diseases, principally from the causative agent bacteria of UTIs ( 8 - 10 ).

Humans in all civilizations knew the importance of medicinal plants and were guided to the treatment of diseases by experience; hence, herbal medicine is the oldest known method of treatment. However, this study aimed to examine the antibacterial effects of constituents obtained from C. viminalis leaves by using three organic solvents, such as Ethanol, Ethyl acetate, and Hexane to prevent the growth of the UTI isolates in Iraq.

2. Materials and Methods

2.1. Materials of Plant

The fresh leaves of C. viminalis were collected from different regions of Hillah City, Iraq, in March 2020. The leaves were classified according to the taxonomic features ( 11 ), and plant materials were prepared and kept according to McClure ( 12 ).

2.2. Callistemon viminalis Components Extraction

The C. viminalis leaves were extracted by using three organic solvents, such as Ethanol, Ethyl acetate, and Hexane as previously described by Handa, Khanuja ( 13 ). The extractions were performed by a method of digestion according to Handa, Khanuja ( 13 ). The stock solution of 200 mg/mL was prepared in 10% of Dimethylsulfoxide. A Millipore filter (0.22 µm) was used for the sterilization of all the extracts used in this study. Afterward, the extracted samples were stored in the refrigerator at -20 °C ( 14 ).

2.3. Antibacterial efficacy

Agar well diffusion method was applied to evaluate the antibacterial effects of the C. viminalis dried leaves extracted by Ethanol, Ethyl acetate, and Hexane solvents at three different concentrations of 50, 100, and 200 mg/mL against the urinary tract infection bacterial isolates ( 15 ). A 6-mm diameter Cork borer was used to make wells in agars. Control negative was made by adding 10% Dimethyl sulfoxide to wells and meropenem antibiotic was used as a control positive treatment (Table 1).

2.4. Bacterial Pathogenic Isolates

Isolates of UTIs were obtained from Microbiology laboratories in different hospitals within the boundaries of the municipality of Hillah-Iraq (Table 2).

2.5. Statistical analysis

The experiments were based on a completely randomized design. An analysis of variance and least significant differences at P≤ 0.05 was performed by using SPSS software (version 16.0), and the results were expressed in the form of mean±standard deviation.

3. Results

The antibacterial effects of phytochemical complexes separated from C. viminalis leaves by using various sorts of organic solvents, such as Ethanol, Ethyl acetate, and Hexane, against urinary tract bacterial isolates, are represented in tables 3, 4, and 5. The outcomes displayed that the three organic solvents extracts of ethanolic, Ethyl acetate, and hexane of C. viminalis leaves exhibited significant decline (P≤0.05) in the growing of urinary tract bacteria, compared to the negative control. The antibacterial properties of C. viminalis were tested at a concentration of 50, 100, and 200 mg/mL, compared to dimethylsulfoxide (10%) as the negative control and meropenemas as a positive control.

The results showed that the inhibitory growth effects arise significantly (P≤0.05) at a concentration of 200 mg/mL for all the extracted materials by using all the solvents except for K. pneumonia. The K. pneumonia was not inhibited by the C. viminalis leaves extracted materials. The recorded data of the current study revealed that ethanolic C. viminalis extract was significantly superior to the meropenem when applied to Proteus sp. pathogenic bacteria with an inhibition diameter of 20±1.0, compared to 15±0.0 in the meropenem-treated group. The results also revealed that the constituents extracted by an ethanolic organic solvent at a concentration of 200 mg/mL exhibited similar influences on E. coli.

There were no significant changes (P≤0.05) between the C. viminalis extracts and the meropenem as the inhibitory belt reached up to 20±1.0 mm in diameter in the ethanolic C. viminalis crude extract, compared to the meropenem (20±0.0 mm) once utilized for the E. coli pathogen.

The C. viminalis also exhibited significant inhibition in the culture of P. aeruginosa isolates where the diameter of the inhibition increased to 15.33±0.57 mm at the concentration of 200 mg/mL, compared to the negative control. Nevertheless, this inhibition was not significant in comparison with the diameter of the inhibition produced by the meropenem antibiotic where the meropenem was significantly superior to the ethanolic plant extract when applied to P. aeruginosa. Moreover, K. pneumonia showed complete resistance to all concentrations used in this study (Table 3).

The results of the current study revealed that there were significant (P≤0.05) declines in UTIs bacterial isolates growth with the increasing concentrations of constituents obtained by Ethyl acetate organic solvent, compared to the negative control group as represented in table 4. In a similar direction, the results of the Proteus sp. isolate culture revealed that constituents extracted by Ethyl acetate solvent at a concentration of 200 mg/mL had lower inhibitory effects (14.66±0.57) compared with meropenemas (15±0.0) (P≤0.05). In contrast, E. coli, P. aeruginosa, and K. pneumonia exhibited complete resistance to the different concentrations of Ethyl acetate used in the study.

In addition, the hexane extract was the least effective among the solvents used in the study, as both Proteus sp. and K. pneumonia showed complete resistance to all concentrations. However, the meropenem antibiotic was significantly superior to the hexane extract although the inhibition diameter increased to 15±0.0 when applied to E. coli and P. aeruginosa (Table 5).

4. Discussion

Development of resistant pathogenic bacteria to drugs in humans, animals, and crops as well as the unwanted side effects of these drugs have encouraged scientists to investigate different medicinal plants to fight bacterial infections. In contrast to the chemical drugs and antibiotics, the medicinal plant is also characterized by being an integrated part of pharmacy that contains more than one effective substance which works synergistically with each other in treating the disease. All these combined factors and the possible synergistic effects have attracted great attention to the exploration of new safe plant-derived drugs, especially, in light of the high global poverty rates due to wars, suffocating economic crises, and high prices for chemical treatments.

There is no doubt that the effective compounds extracted from medicinal plants remain one of the important, if not the most important, sources in the fight against diseases, especially in light of the aggravation of the problem of the resistance of a microorganism to antibiotics. Medicinal plants are also less harmful in terms of side effects, compared to chemical drugs. Constituents are separated from different active parts of numerous medicinal plants, such as (Lactuca serriola leave, Lepidium sativum leaves, Myrtus Communis leaves, Cassia senna leaves, Ricinus communis leaves, Cassia didymobotrya leaves, Melia azedarach leaves, Dianthus caryophyllus flowers bud, and Salvia hispanica seeds), possess the ability of antibacterials for controlling several pathogenic microorganisms isolated from different clinical samples ( 16 - 24 ).

Hussein, Naji ( 25 ) reported that constituents separated from unicellular primitive plants, like Chlorella Vulgaris possess the ability of antibacterial counter to pathogenic bacteria. Kamal, Hussein ( 26 ) used phytochemical compounds separated from Hibiscus sabdarifa for controlling E. coli and Proteus sp. Kamal, Al-Kaim ( 27 ) used constituents extracted from Ficus carica L. for controlling E. coli and Pseudomonas aeruginosa. AL-Masoodi, Hussein ( 28 ) used phytochemical compounds extracted from Boswellia carteri and Curcuma longa for controlling Fusarium sp. isolated from seeds of corn.

Hussain, Hussein ( 29 ) used terpenoids compounds extracted from Carthamus tinctorius L. against Aspergillus species isolated from stored medicinal plant seeds. Secondary metabolites represented by Alkaloids and Flavonoids compounds separated from M. Communis leaves are considered a worthy source for controlling pathogenic microorganisms segregated from hemodialysis fluid specimens ( 30 ).

Leaves of C. viminalis have an appreciable level of total phenolic and flavonoids content ( 31 ). Phytochemical compounds extracted by Methanol and Ethyl acetate solvents from the leaves of C. viminalis have antibacterial effects against pathogenic bacteria ( 32 ). Phytochemical compounds extracted from leaves of C. viminalis are used for Molluscicidal activity against Snails ( 33 ). Unlike P. aeroginosa, S. aureus, and S. pyogenes, Congener and Tormentic acid extracted from C. viminalis have antibacterial effects ( 34 ).

Essential oils extracted from C. viminalis have antibacterial effects against Listeria monocytogenes and antifungal activity against Aspergillus flavus ( 35 ). Essential oils also exhibited strong antibacterial activity against S. faecalis, S. aureus, B. cereus, and S. macrcesens ( 36 ). Lastly, the antibacterial effects of C. viminalis might belong to Constituents and their influence on the synthesis of proteins, DNA, RNA, polysaccharides, and disturbance in the permeability of cell membranes or preventing the efflux pump to work properly.

Based on the findings of the current study, the constituents found in the C. viminalis plant are very effective against microorganisms isolated from the urinary tract. Therefore, they can be recommended to be used against other pathogenic isolates as well as fungi and insects. Moreover, they can be used for the detection of these Constituents by using Gas chromatography and Mass spectrometry.

Constituents extracted from C. viminalis (Sol. ex Gaertn.) G. by utilizing Ethanol and Ethyl acetate are regarded as respectable sources for controlling isolates of Urinary Tract Infections.

Authors' Contribution

Study concept and design: S. H. R. and H. J. H.

Acquisition of data: S. A. K.

Analysis and interpretation of data: N. M. S. and H. J. H.

Drafting of the manuscript: S. A. K.

Critical revision of the manuscript for important intellectual content: H. J. H.

Statistical analysis: S. H. R. and H. J. H.

Administrative, technical, and material support: S. H. R. and H. J. H.

Conflict of Interest

The authors declare that they have no conflict of interest.

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