Comparison between the effect of some plant extracts and ciprofloxacin and fluconazole against some bacterial and fungal species

Introduction

Each year hundreds of thousands of people die, due to a universal resistance for antibiotics when it comes to bacteria and new strains that no longer can be treated by standard medicine the consequences being recognized as serious global health risks. In 2014, the World Health Organisation identified increased mortality from communicable disease as a critical global public health issue [1] . Increasing glyconvenient access to potent antibiotics including colistin and carbapenem, used not only where necessary but throughout the agri-food system unchecked [2,3] fuelled a huge rise in global antibiotic consumption from 2000-2015 (65%), helping create resistant bacterial strains that it is impossible or demanding (requiring doses of several other drugs together) to treat. In response to this increasing challenge, medicinal plants previously used for centuries in traditional medicine might be useful source of new antimicrobials [4,5] . The bioactive compounds present in these plants could be potential sources of resistance-breaking pharmaceutics. Despite the historical use of natural medicine for a long period, there has been little research, and their consumption is declining which highlights that caution should be applied in order to make awareness programs on herbal products with regard to plant active compounds taking into consideration. Antibiotics have revolutionized medicine, particularly while treating bacterial illnesses such Amoxil, cephalosporin and ciprofloxacin. Nevertheless, when we see the reality of increasing antibiotic resistance it is one of a number of challenges that limit our ability to develop antibacterial drugs at the same time as these key bacterial pathogens become resistant [6,7]. This situation has also reawakened the attention to traditional medicine and herbal products, due to its historical role in human health [8] .

Ciprofloxacin, a fluoroquinolone antibiotic, is FDAapproved for a number of bacterial illnesses, including as pneumonia, urinary tract infections, and several others [9] . Notably, Neisseria gonorrhea exhibits high resistance rates to ciprofloxacin, making it unsuitable as a first-line therapy for respiratory infections caused by Streptococcus pneumoniae that is responsive to penicillin. Nonetheless, patients with mixed infections or those at risk for Gramnegative infections should still take ciprofloxacin [10] . Ciprofloxacin is used off-label for Crohn's disease linked to perianal fistula and can be used to treat both acute and chronic bacterial prostatitis, according to the American Academy of Family Physicians guidelines [11] . Ciprofloxacin is a bactericidal antibiotic in the fluoroquinolone class: Ciprofloxacin is a bactericidal antibiotic belonging to the fluoroquinolone class, primarily effective against gram-negative bacilli, such as Enterobacteriaceae (e.g., E. coli, Salmonella spp., Shigella spp., and Neisseria). It functions by blocking DNA-gyrase and bacterial DNA topoisomerase, crucial for DNA replication. Ciprofloxacin is particularly potent against Pseudomonas aeruginosa, although there are reports of decreasing susceptibility, especially in hospital settings [12] . It is commonly used for infections caused by S. aureus, but increasing resistance to fluoroquinolones has prompted efforts to enhance their efficacy or find alternatives [13] . The search for natural sources has led to the extraction of drugs from traditional herbs abundant in tropical regions [14] . Fluconazole exhibits fungistatic activity against various fungi, including Candida species and Cryptococcus neoformans. It interferes with cell wall synthesis and adhesion, treating fungal infections effectively [15] . The cytochrome P-450 enzyme 14-demethylase, which transforms lanosterol into ergosterol a crucial part of the fungal cell membrane is the target of fluconazole. Fluconazole enhances cellular permeability and interferes with fungal respiration and yeast development by preventing ergosterol synthesis. In fungi, the build-up of 14-methyl sterols corresponds with the loss of sterols, contributing to its fungistatic effects [16] .

Materials and Methods

Sample collection

In this study, three medicinal plants were used: A. sativum, S. officinalis, and M. piperita. Five micro-organisms were isolated from burn patients who were admitted to Al-Fayhaa Hospital: also, two Candida species. Three medicinal plants were prepared by grinding garlic cloves, sage leaves, and mint leaves into fine powders, after which a Soxhlet extractor was used in making the plant extracts.

Identification of bacterial & fungal species

The swabs from the patients and the healthy students were first cultured on MacConkey agar and blood agar media, after which an attempt at identification of five bacterial species was made using the automated VITEK2, followed by subculture on HiCrome Candida Differential Agar for rapid identification.

Evaluation of plant extracts' antibacterial efficacy

Disc diffusion method: The disc diffusion method was used to assess the antibacterial activity of studied plant extracts. Dissolved extracts were spread over Mueller Hinton agar plates, and discs impregnated with the extracts were placed on the media.

Determination of the Minimum Inhibitory Concentration (MIC)

The Minimum Inhibitory Concentration (MIC) of plant extracts against gram negative, gram-positive bacteria, and Candida species was determined using the MHB broth micro-dilution method. Bacterial cell viability and MIC values were determined using turbidity tests. The MIC of the extracts was determined using a similar procedure, using bacteria that had been cultivated overnight suspended in sterile nutrient broth and incubated for two to four hours at 37 °C.

Results

The study assessed the antibacterial properties of the chosen herbs using seven pathogens. There were four species of bacterial pathogens were Gram negative (E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis) and one was Gram positive (S. aureus), of which were identified by VITEK device. Also two Candida species have been used which identified by HiCrome Candida medium according to colonies color; C. albicans (green) and C. tropicalis (blue) (Figure 1). The study evaluated the antimicrobial effects of selected herbs against seven pathogens, various types of Candida and both Gram-positive and Gram-negative bacteria. The findings indicated that A. sativum, S. officinals, and M. piperita shown antibacterial efficacy against every pathogen examined. Considerable antifungal efficacy was noted against C. albicans and C. tropicalis, with C. tropicalis being more sensitive. The highest activity was recorded against C. tropicalis, C. albicans, and E. coli (Table 1 & Figure 2). The study found ciprofloxacin's effectiveness against bacterial species varied compared to plant extracts, while fluconazole demonstrated high efficacy against C.

tropicalis, but C. albicans exhibited resistance (Table 2 & Figure 3).

Determination MIC and in vitro

GC-MS Analysis

The analysis of Allium sativum, an ethanolic plant extract, was conducted using gas chromatography and spectrometry for precise compound detection. GC-MS for ethanolic extract of A. sativum showed the presence of 45 peaks (Figure 4). The major peak belong to compound piperine that was found in considerable quantity (26.683%), (Table 4).

Salvia officinalis

GC-MS for ethanolic extract of S. officinalis showed the presence of 59 peaks (Figure 5). The major peak belong to compound eucalyptol that was found in considerable quantity (15.192%), (Table 5). Mentha piperita: GC-MS for ethanolic extract of M. piperita showed the presence of 54 peaks (Figure 6). The major peak belong to compound D-carvone that was found in considerable quantity (12.852%), (Table 6). As shown in (Table 1) and MIC values in (Table 3), all plant extracts inhibited both Gram-positive and Gram-negative bacteria, with significant action against the Candida species. The antimicrobial effects of A. sativum are attributed to high concentrations of piperine (26.683%), a major alkaloid known for its bioavailability-enhancing, antioxidant, and antimicrobial properties [17] . The extract showed maximum efficacy against C. tropicalis, C. albicans, and E. coli, with inhibitory zones measuring 44 mm, 40 mm, and 24 mm, respectively, consistent with findings by Aldaly [18] . The sensitivity of E. coli to piperine may be due to its highly permeable membrane, which allows piperine to alter cell wall permeability [19] . Additionally, piperine inhibits lipid accumulation in yeast, reducing cell growth [20] , and inhibits DNA and protein synthesis [21] . Additionally, it inhibits efflux pumps, contributing to bacterial resistance to antibiotics [22] . The antimicrobial activities of S. officinalis are linked to high concentrations of eucalyptol (15.192%), a bicyclic terpenoid present in various essential oils [23] . Eucalyptol's effectiveness against both Gram-negative and Gram-positive bacteria, such as S. aureus and Gram-negative strains including E. coli and P. aeruginosa, is mainly due to its ability to permeabilise cell membranes [24,25] . Given the increasing problem of antimicrobial resistance and biofilm formation among bacteria, natural compounds like eucalyptol are gaining attention for their potential to address these challenges [26] . Eucalyptol displays antibacterial properties against Gram-positive and Gram-negative pathogenic strains, including Proteus mirabilis, because of its capacity to cause cell membrane permeabilisation [27] . Its hydrophobic nature has been found to effectively combat Klebsiella pneumoniae isolates, demonstrating antimicrobial properties against bacteria with outer lipopolysaccharide membranes [28] . Carvone, a naturally occurring compound, has shown promise as an antibacterial and antifungal agent [29, 30] . It exhibits antibacterial activity against various bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) [29] , by increasing cell permeability and disrupting cell membrane integrity [29,30] . Carvone also demonstrates an antibiofilm effect against S. aureus [30] . Its antifungal properties have been studied against Candida species, mycotoxigenic fungi such as Fusarium, Aspergillus, and Penicillium, and dermatophytes such as Microsporum, Trichophyton, and Epidermophyton [31] . Like Trichophyton, Epidermophyton, and Microsporum [31] . While some studies have shown carvone's effectiveness against certain bacterial strains [29,30] , others have found no activity against Staphylococcus strains [32] . Previous research from Ethiopia focused on antibacterial activity but excluded antifungal activity and methanol in the extraction process [33-35]. However, studies from Ghana and Pakistan have reported that methanol extracts exhibited strong antimicrobial activity [33,36] .

Carvone and other natural antibacterial substances have a great deal of potential for treating infections brought on by drug-resistant bacteria [34] . Natural antimicrobial compounds offer the Global public health is seriously threatened by the rise of multidrug-resistant bacteria such as S. aureus, E. coli, and P. aeruginosa [34] . Advantage of lower resistance development compared to traditional antibiotics [23] . This raises the question of whether natural compounds could serve as viable alternatives to conventional drugs and whether they will prove effective [23] .

Conclusions

According to the study, extracts of A. sativum, S. officinalis, and M. piperita inhibit a variety of infections, such as bacteria and species of Candida, through piperine, eucalyptol, and carvone, respectively.

Recommendations

Further studies on plant extracts are needed for isolation, purification, and identification of bioactive compounds for antibiotic and antifungal activities. Further studies have to be conducted against other pathogens and also in vivo to confirm their effectiveness.

References

1. Palme BJ, Kristiansson E, Larsson DGJ. Environmental factors influencing the development and spread of antibiotic resistance. FEMS Microbiology Reviews. 2017 Oct 24;42(1):68-80.
2. Klein EY, Van Boeckel TP, Martinez EM, Pant S, Gandra S, Levin SA, et al. Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proceedings of the National Academy of Sciences. 2018 Mar 26;115(15):E3463-70.
3. Bassoum O, Sougou N, Diongue M, Lèye M, Mbodji M, Fall D, et al. Assessment of General Public's Knowledge and Opinions towards Antibiotic Use and Bacterial Resistance: A Cross-Sectional Study in an Urban Setting, Rufisque, Senegal. Pharmacy. 2018 Sep 20;6(4):103.
4. Rijeka, Croatia: IntechOpen; 2012.
5. Petrovska BB. Historical review of medicinal plants′ usage. Pharmacognosy Reviews. 2012;6(11):1-5.
6. Karpiński TM, Ożarowski M, Mrozikiewicz SA, Wolski H. Anti-candida and antibiofilm activity of selected lamiaceae essential oils. Frontiers in Bioscience-Landmark. 2023 Feb 16;28(2):28.
7. Gao C, Fan Y, Zhao F, Ren QC, Wu X, Chang L, et al. Quinolone derivatives and their activities against methicillin-resistant Staphylococcus aureus (MRSA). European Journal of Medicinal Chemistry. 2018 Sep 1;157:1081-95.
8. Adams R. Identification of Essential Oil Components by Gas Chromatography/Mass Spectorscopy. Carol Stream, Illinois, USA: Allured Publishing; 2017, p. 173-176.
9. Álvarez BJ, Ferriz SV. Increase in methicillin-resistant and ciprofloxacin-susceptible Staphylococcus aureus in osteoarticular, skin and soft tissue infections. Revista Española de Quimioterapia. 2020 Mar 24;33(02):143-4.
10. Unemo M, Lahra MM, Cole M, Galarza P, Ndowa F, Martin I, et al. World Health Organization Global Gonococcal Antimicrobial Surveillance Program (WHO GASP): review of new data and evidence to inform international collaborative actions and research efforts. Sexual Health. 2019;16(5):412.
11. Cañas HV, Soto GB, Fernández AH, Oya LA, Marí NJM, Frenich GA, et al. Antibiotic activity and concentrations in clinical samples from patients with chronic bacterial prostatitis. Actas urológicas españolas. 2017 Dec 1;41(10):631-8.
12. Rehman A, Patrick WM, Lamont IL. Mechanisms of ciprofloxacin resistance in Pseudomonas aeruginosa: New approaches to an old problem. Journal of Medical Microbiology. 2019 Jan 1;68(1):1-10.
13. El-Wafa WMA, Ibrahim YM. In vitro activity of fosfomycin in double and triple combinations with imipenem, ciprofloxacin and tobramycin against multidrug-resistant Escherichia Coli. Current Microbiology. 2020 Jan 9;77:755-61.
14. Mohamed S, Elmohamady MN, Abdelrahman S, Amer MM, Abdelhamid AG. Antibacterial effects of antibiotics and cell-free preparations of probiotics against Staphylococcus aureus and Staphylococcus epidermidis associated with conjunctivitis. Saudi Pharmaceutical Journal. 2020 Oct;28(12):1558-65.
15. Spampinato C, Leonardi D. CandidaInfections, Causes, Targets, and Resistance Mechanisms: Traditional and Alternative Antifungal Agents. BioMed Research International. 2013;2013:1-13.
16. Khosravi Rad K, Falahati M, Roudbary M, Farahyar S, Nami S. Overexpression of MDR-1 and CDR-2 genes in fluconazole resistance of Candida albicans isolated from patients with vulvovaginal candidiasis. Current Medical Mycology. 2016 Dec 1 ;2(4):24-9.
17. Nwinyi OC, Chinedu NS, Ajani OO, Ikpo CO, Ogunniran KO. Antibacterial effects of extracts of Ocimum gratissimum and piper guineense on Escherichia Coli and staphylococus aureus. African Journal of Food Science. 2009 Mar;3(3):77-81.
18. Aldaly ZTK. Antimicrobial Activity of Piperine purified from piper nigrum. Journal of Basrah Researches. 2017 Oct 15;36(5):54-61.
19. Obafemi CA, Sulaimon TO, Akinpelu DA, Olugbade TA. Antimicrobial activity of extracts and a germacranolidetype sesquiterpene lactone from Tithonia diversifolia leaf extract. African Journal of Biotechnology. 2019 Dec;5(12):1254-8.
20. Matsuda D, Ohte S, Ohshiro T, Jiang W, Rudel L, Hong B, et al. Molecular target of piperine in the inhibition of lipid droplet accumulation in macrophages. Biological and Pharmaceutical Bulletin. 2008 Jan 1;31(6):1063-6.
21. Khan IA, Mirza ZM, Kumar A, Verma V, Qazi GN. Piperine, a Phytochemical Potentiator of Ciprofloxacin against Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 2006 Feb;50(2):810-812.
22. Webber MA, Piddock LJV. The importance of efflux pumps in bacterial antibiotic resistance. Journal of Antimicrobial Chemotherapy. 2002 Dec 12;51(1):9-11.
23. Dhakad AK, Pandey VV, Beg S, Rawat JM, Singh A. Biological, medicinal and toxicological significance of Eucalyptusleaf essential oil: A review. Journal of the Science of Food and Agriculture. 2017 Sep 11;98(3):833-48.
24. Galan DM, Ezeudu NE, Garcia J, Geronimo CA, Berry NM, Malcolm BJ. Eucalyptol (1,8-cineole): An underutilized ally in respiratory disorders? Journal of Essential Oil Research. 2020 Jan 26;32(2):103-10.
25. Guzzo F, Scognamiglio M, Fiorentino A, Buommino E, D'Abrosca B. Plant derived natural products against Pseudomonas aeruginosa and Staphylococcus aureus: Antibiofilm Activity and Molecular Mechanisms. Molecules. 2020 Oct 29;25(21):5024.
26. Farhanghi A, Aliakbarlu J, Tajik H, Mortazavi N, Manafi L, Amin JG. Antibacterial interactions of pulegone and 1,8-cineole with monolaurin ornisin against Staphylococcus aureus. Food Science & Nutrition. 2022 Apr 5;10(8):2659-66.
27. Moo CL, Osman MA, Yang SK, Yap WS, Ismail S, Lim SHE, et al. Antimicrobial activity and mode of action of 1,8-cineol against carbapenemase-producing Klebsiella pneumoniae. Scientific Reports. 2021 Oct 21;11(1):20824.
28. Mun SH, Kang OH, Joung DK, Kim SB, Choi JG, Shin DW, et al. In vitro anti-MRSA activity of carvone with gentamicin. Experimental and Therapeutic Medicine. 2014 Jan 23;7(4):891-6.
29. Ding X, Chen H. Anticancer effects of Carvone in myeloma cells is mediated through the inhibition of p38 MAPK signalling pathway, ap-optosis induction and inhibition of cell invasion. JBUON. 2018;23(3):747-51.
30. Moro IJ, Gondo GDGA, Pierri EG, Pietro RCLR, Soares CP, Sousa DPD, et al. Evaluation of antimicrobial, cytotoxic and chemopreventive activities of carvone and its derivatives. Brazilian Journal of Pharmaceutical Sciences. 2017 Mar 22;53(4):53.
31. Longaray Delamare AP, Pistorello MIT, Artico L, Serafini AL, Echeverrigaray S. Antibacterial activity of the essential oils of Salvia officinalis L. and Salvia triloba L. cultivated in South Brazil. Food Chemistry. 2007 Jan 1;100(2):603-8.
32. Abew B, Sahile S, Moges F. In vitro antibacterial activity of leaf extracts of Zehneria scabra and Ricinus communis against Escherichia Coli and methicillin resistance Staphylococcus aureus. Asian Pacific Journal of Tropical Biomedicine. 2014 Aug 8;4(10):816-20.
33. Naz R, Bano A. Antimicrobial potential of Ricinus communis leaf extracts in different solvents against pathogenic bacterial and fungal strains. Asian Pacific Journal of Tropical Biomedicine. 2012 Dec;2(12):944947.
34. Novosad SA, Fike L, Dudeck MA, Bridson AK, Edwards JR, Edens C, et al. Pathogens causing centralline-associated bloodstream infections in acute-care hospitals, United States, 2011-2017. Infection Control & Hospital Epidemiology. 2020 Jan 9;41(3):313-319.
35. Donkor AM, Mosobil R, Suurbaar J. In vitro bacteriostatic and bactericidal activities of Senna alata, Ricinus communis and Lannea barteri extracts against wound and skin disease causing bacteria. Journal of Analytical & Pharmaceutical Research. 2016 Sep 2;3(1).
36. Suurbaar J, Mosobil R, Donkor AM. Antibacterial and antifungal activities and phytochemical profile of leaf extract from different extractants of Ricinus communis against selected pathogens. BMC Research Notes. 2017 Dec;10(1):1-6.