The Antimicrobial Activity of Pechuel-Loeschea leubnitziae Leaf Extract and its Effect on The Expression Level of Methicillin Resistant Staphylococcus Aureus and Candida albicans Virulence-Associated Genes

The complete halt in the synthesis of new effective antimicrobial compounds is a global concern. Pathogenic microorganisms’ virulence mechanisms seem to have a significant impact on their pathogenesis. The purpose of this study was to examine the antimicrobial activity of the ethanol and methanol fractions of Pechuel-Loeschea leubnitziae leaf extract, as well as its effect on the expression level of virulence-associated genes. The extract’s fractions were evaluated for antimicrobial activity against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae (clinical), Methicillin-resistant Staphylococcus aureus, and Candida albicans ATCC 90029. The test organism’s antibiogram pattern was determined. The extracts’ attenuation effect on the target genes of the susceptible organisms was investigated employing relative quantification using RT-qPCR. The test organism’s antibiogram pattern revealed that it was drug-resistant, intermediate, and sensitive. The extracts tested positive for antimicrobial activity against Methicillin-resistant Staphylococcus aureus and Candida albicans ATCC 90029, with zones of inhibition varying from 20.33 to 29 mm. The lowest recorded MIC value was 4.688 mg/ml, while the highest was 37.5 mg/ml. In contrast to the methanol extract, the ethanol extract had a cidal action at a lower dose. The ethanol extract’s Sub-MIC (18.25 mg/ml) merely reduced the expression of the hly gene in MRSA. The MRSA virulence genes were not suppressed by the sub-MIC of methanol extract (18.25 mg/ml). Notably, the expression of als1, pbl1, and sap1 in Candida albicans ATCC 90029 was significantly attenuated when exposed to sub-MICs of ethanol extract (2,344 mg/ml) and methanol extract (9.375 mg/ml). Per the findings of this research, the leaves of P. leubnitziae could be a source of an effective antimicrobial agent in the therapy of MRSA/Candida-related disorders.


INTRODUCTION
Antimicrobial resistance is one of the greatest threats to human health globally, causing an estimated 700 000 deaths annually [1]. It is predicted that by 2050, there will be approximately 10 million deaths [2]. Antimicrobial resistance is worsening in many African and Asian countries as a result of high levels of counterfeit and substandard antimicrobials in the pharmaceutical market [3]. Antimicrobial-resistant bacterial infections kill nearly 50 000 people each year in Europe and the United States alone, with hundreds of thousands more dying in other parts of the world [4].

Antimicrobial Susceptibility Test
The agar well diffusion susceptibility method was used to evaluate the ethanol and methanol extracts for antimicrobial activity, according to the Clinical and Laboratory Standards Institute [13]. The isolates of the test organisms were sub-cultured on nutrient agar plates and incubated for 18 hours at 37 °C. The turbidity of the cultures was adjusted to 0.5 Macfarland standard (108 Cfu/ml) using normal saline. A sterile cotton swab was dipped in the standardized suspension and placed on the surface of Mueller Hinton Agar plates and allowed to diffuse for five minutes. A sterile cork borer with a 6 mm diameter was used to make the agar wells.
Exactly 50 μl of varying concentrations of each solvent extract was introduced into the wells.
The Antibiotic discs used for Escherichia coli ATCC 25922 and Klebsiella pneumoniae Candida albicans ATCC 90029 are fluconazole (100IU) and Nystatin (100IU). As a control, 5% DMSO was employed. The plates were allowed to diffuse for about 15 minutes at room temperature before being incubated at 37 °C for 18 hours. Following incubation, the plates were examined for the formation of a clear zone around the wells, which correlates with the antimicrobial activity of the tested solvent extracts. The diameter of the zones of inhibition was measured in mm using a ruler in triplicate.

Minimum Inhibitory Concentration (MIC) Analysis
The ethanol and methanol extracts' Minimum Inhibitory Concentration (MIC) on susceptible organisms was assessed using broth microdilution according to [13] approved guidelines. The dilution to obtain ten different concentrations: (600, 300, 150, 75, 37. 5, 18.75, 9.375, 4.688, 2.344, and 1.172 mg/ml). A standardized (0.5 McFarland) inoculum of 10 µl was added and stirred. MHB plus inoculum, MHB plus extract, and MHB plus 5% DMSO solution were used as negative controls. As a positive control, conventional drugs were used: ampicillin (50 g/ml) for MRSA and nystatin (100IU) for Candida albicans ATCC 90029. The plate was wrapped with a sterile parafilm and incubated for 24 hours at 37 °C. Following incubation, 20 µl of 0.05% resazurin dye was added to the wells and incubated for another 2 hours to examine colour patterns. Wells with microbial growth will turn pink, while wells without microbial growth will remain blue. The MIC was defined as the lowest extract concentration that totally inhibited microbial growth.

Minimum Bactericidal Concentration (MBC) Analysis
Sub-culturing the wells of Methicillin resistant Staphylococcus aureus that showed no colour change upon post-incubation of the microtiter plate yielded the MBC of each solvent extract. This was done by dipping a sterile inoculation loop into each well and infecting on Mueller Hinton agar plates. The plates were maintained for 18 hours at 37 °C. The MBC was determined to be the lowest concentration that showed no visible growth after incubation.

Minimum Fungicidal Concentration (MFC) Analysis.
Sub-culturing the wells of Candida albicans ATCC 90029 that showed no colour change upon post-incubation of the microtiter plate yielded the MFC of each solvent extract. This was done by dipping a sterile inoculation loop into each well and infecting on Mueller Hinton agar plates.
The plates were maintained for 18 hours at 37 °C. The MFC was determined to be the lowest concentration that showed no visible growth after incubation.

RNA Extraction and cDNA Preparation
The samples were prepared by culturing 100 µl of 0.5 Macfarland standardized (108 Cfu/ml) of MRSA and C. albicans ATCC 90029 in 4 ml of Tryptic soy broth and incubated with or without 100 µl of sub-MICs concentration of ethanol (18.25 and 2.344 mg/ml) and methanol (18.25 and 9.375 mg/ml) extracts for 16 hours at 37 °C on a shaker with 160 rpm. The cells were harvested by centrifugation of 1 ml of the suspension (13,000 × g, 5 minutes, 4 °C), and

Gene expression analysis by RT-qPCR
The primers were designed using the PRIMER3 tool version (0.4.0). The amplification specificity was validated with an in-silico PCR amplification and Sequence Manipulation Suite (SMS). The primers were synthesized by Macrogen, Europe. The details of the primers are listed in Table 1

Data analysis
All data were captured in Microsoft® Excel spreadsheets. Data from the experiment were analyzed using SPSS (20.1 version). All results were done in triplicate and presented as the mean ± standard deviation (S.D.). The MIC, MBC, and MFC were analyzed using descriptive statistics. The expression levels of the target genes were measured relative to the control sample and normalized to the expression of the endogenous reference gene (16s/ 18s ribosomal RNA gene) between samples in parallel. The relative expression level or n-fold change of target genes between treated and non-treated were calculated using the 2 -ΔΔct method according to [14]. The normalized expression levels of the genes in treated and untreated samples were compared using an unpaired two-tailed t-test at a significant level of p < 0.05. All values of nfold differential expression were plotted in a graph. Graphs were prepared using GraphPad Prism version 8.3.4 and SigmaPLot version 14.0.

Antibiotic Susceptibility Test
The AST zones of inhibition were evaluated in accordance with the Clinical Laboratory Standard Institute [13]. Amikacin, cefepime, and gentamicin were all effective against E. coli  (Figures 2 and 3).

Methicillin Resistant Staphylococcus Aureus Virulence Gene Expression Analysis
The effects of P. leubnitziae ethanol and methanol extracts on the expression of hly, sea, and icaA genes were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR), and the results are presented ( Table 6). The expression of target genes was normalized to 16S rRNA. The expression of hly, sea, and icaA of both ethanol and methanol treated samples was compared relative to the control. The fold change was analyzed using the 2^-ΔΔCT method.  Table 7). The hly gene was less expressed (0.94-fold change) compared to control when subjected to ethanol extract treatment and expressed more than the control (which is normalized to a fold change of 1) when subjected to methanol extract treatment. Ethanol extract treatment highly induced the expression of sea gene (26.66-fold change) compared to methanol extract treatment (6.17-fold change). Genes were shown to be downregulated and upregulated. Genes that were above the 0 or + sign were upregulated, whereas those that were below the 0 orsign were downregulated (Figure 10).

Candida albicans ATCC 90029 Virulence Gene Expression Analysis
The effects of P. leubnitziae ethanol and methanol extracts on the expression of als1, pbl1, and sap1 genes were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR), and the results are presented ( Table 8). The expression of target genes was normalized to 18S rRNA. The expression of als1, pbl1, and sap1 of both ethanol and methanol treated samples was compared relative to the control. The fold change was analyzed using the 2^-ΔΔCT method. The fold change ranged between 0.00001 -0.004 in the genes treated with ethanol extract (2.344 mg/ml) while ranged between 0.002 -0.003 in the genes treated with methanol extract (9.375 mg/ml) (Figure 11). The differential expression analysis showed a significant difference of all genes of the ethanol and methanol extracts treatment to that of the control. The ethanol extract treatment of als1, pbl1, and sap1 exhibited a p-value of 0.0001, 0.0001, and 0.0002, respectively. For methanol extract treatment, the genes had lower p-values of <0.0001 ( Table 9). All genes when subjected to ethanol and methanol extracts treatment were less expressed, denoting a zero-fold change compared to the control (normalized to 1-fold change) ( Figure 12). Notably, the genes subjected to both treatments showed a negative log2 which validates a downregulation (Figure 13).  Table 9. Results of unpaired two-tailed t-test on Candida albicans ATCC 90029 virulence genes.

DISCUSSION
The leaf extract of Pechuel leubnitziae was investigated in this study. The rising number of cases associated with antimicrobial resistance is currently a major public health concern [1].
Antimicrobial resistance has been attributed to the burden of managing recurrent infections, prolonged hospitalization, and an increased morbidity and mortality rate [1]. The antimicrobial activity of ethanol and methanol extracts against clinical isolates associated with antimicrobial resistance was investigated. The current study's findings revealed that ethanol and methanol extracts have antimicrobial activity, inhibiting 40% of the test organisms. The extracts were more effective against the fungus, exhibiting antimicrobial activity at all concentrations (200-600 mg/ml). The results concur with the antimicrobial activity of P. leubnitziae methanol root extract observed by [15] and the antibacterial activity reported by [16]. The findings of the study contrast to that of [15], which showed no antimicrobial activity against S. aureus and Candida albicans adopting the disc diffusion method. The variations in the results of P.
leubnitziae leaf extract could be attributed to the concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml used by [15], as opposed to the current study, which used concentrations of 200 mg/ml, 400 mg/ml, and 600 mg/ml. It could also have been due to the antimicrobial susceptibility assay used in each study. The current study has used the agar well diffusion method, which allows for high volume disperse of the test solution in the well versus the disc diffusion method used by [15], which only enables very low absorbance of the test solution by the disc. The antimicrobial effectiveness of plant crude extract is greatly proportional to the dose and strains; this could explain why the current study's findings differ from those published in a prior study, which used lower concentrations [17].
The antimicrobial activity of the ethanol and methanol extracts was verified using 5% DMSO alone, indicating that the action was exhibited by the extracts rather than the solvent employed to reconstitute the extracts. Antibacterial activity was exclusively observed in gram-positive bacteria, and without any activity observed in gram-negative bacteria. Gram-positive bacteria have a thicker cell wall that is rich in peptidoglycan and teichoic acid. Gram-negative bacteria, on the other hand, have an outer membrane and a periplasmic region rich in peptidoglycan, as well as an internal lipopolysaccharide layer. As a result, whereas compounds from P. leubnitziae leaf extract may be able to diffuse well through the outer wall of Gram-positive bacteria, they may not be able to diffuse effectively through all of the narrow channels of the Gram-negative outer membrane [18].
According to the [13] recommendation, at least half of the antibiotics used failed to achieve the required inhibitory value for reactivity, primarily against gram-positive test organisms ( Figure   1). This could underlie the selective activity of Pechuel-Loeschea leubnitziae leaf extract. The MIC of the ethanolic extract for Candida was 4.688 mg/ml and 37.5 mg/ml for MRSA, while the MIC of the methanolic extract was 18.75 mg/ml for Candida and 37.5 mg/ml for MRSA.
A high MIC value indicates poor activity, whereas a low MIC value indicates superior activity.
In the current investigation, the ethanol extract showed a lower MIC against Candida than the methanol extract, implying that it is more potent. At 37.5 mg/ml, the bacteriolytic effect of the ethanol and methanol extracts was observed. Additionally, the fungistatic and fungicidal activities of ethanol and methanol were found to be 18.75 mg/ml and 37.5 mg/ml, respectively.
At a low concentration, the ethanol extract exhibited a lethal effect, revealing preliminary evidence on its consistency as the most effective potential source of antimicrobial compounds in the treatment of microbial disorders. This observation of MIC values lines with the observations of [16] and [19]. Another study found that Streptomyces spp. SRF1 culture filtrate extract had a minimum bactericidal concentration of (0.390 mg/ml) against MRSA [20].
RT-qPCR was used in this project to assess the influence of P.  [21], who reported a reduction in hly and flu gene expression when MRSA is exposed to D-SNPs and silver nitrate. Another study, carried out by [22], discovered that Rhus javanica extract completely suppressed sea expression.
The alpha-hemolysin gene is a crucial gene from hemolysin virulence factor of MRSA. The downregulation in the expression of the hly gene when treated with sub-MIC of the ethanol extract of P. leubnitziae leaves could be indicative that it influences the bacterial defense mechanisms by lessening virulence and invasiveness [23,21]. These findings rule out the possibility of the ethanol extract having a crucial anti-virulence effect on the expression of the hly gene. The formation of biofilm and release of toxins are reported to be influenced by various conditions that are likely toxic, including an excessive amount of urea, osmolality, detergents, oxidative stress, and sub-MICs. The upregulation of sea and icaA genes, when exposed to both sub-MIC of ethanol and methanol extracts and hly gene when exposed to methanol extract, could be the pathogen's survival mechanism in response to adverse [24,21]. Thus, showing no attenuation effect of the extracts on the upregulated genes.
To allow the quantification of the expression of C. albicans ATCC 90029, als1, pbl1, and sap1 virulence genes treated with sub-MIC of P. leubnitziae ethanol extract (2.344 mg/ml), methanol extract (9.375 mg/ml), and untreated, real-time qPCR was used. The virulence of Candida albicans is attributed to several factors, including adhesion, invasion, and protease production.
These virulence factors play an essential role in the pathogenesis of C. albicans [25]. This study reported a significant downregulation in the expression of als1, pbl1, and sap1 genes when exposed to ethanol and methanol extracts. These findings are similar to the data reported by [25], who reported a suppression in sap1 and sap10 when treated with Pluchea dioscoridis.
Another study done by [26], reported a significant downregulation on the expression level of als1, als2, hwp1, SapT1, SapT2, and plb1 genes when treated with Morin. The differential expression that showed significant downregulation of C. albicans ATCC 90029 virulence genes could be due to the presence of phenolics compounds reported from quantitative analysis of P. leubnitziae leaf extract of ethanol and methanol [27]. Phenolics compounds are believed to influence the apoptotic pathway against C. albicans [28].

CONCLUSION
The antimicrobial activity of P. leubnitziae leaf extract against tested organisms affirms its traditional application in the treatment of microbial infections, per the findings of this study.
Ethanol extract had greater fungicidal activity against Candida albicans, suggesting that it could be an effective and prospective source of antifungal agents in herbal formulations. When

RECOMMENDATION
More research is needed to assess the antimicrobial activity of various solvents. It is possible to develop and evaluate antifungal gels and creams using ethanol extract. additionally, in-vivo therapeutic efficacy investigations, as well as the identification and purification of pure bioactive compounds from P. leubnitziae leaves, will be crucial for using this plant leaf extract in drug development and discovery. Molecular docking studies of bioactive compounds identified in P. leubnitziae leaves could be used to confirm the binding affinity of the compound to the protein molecules of MRSA and Candida albicans generated from various major virulence factors.

ACKNOWLEDGMENT
We would like to applaud the Pan African University of Basic Science, Technology, and Innovation for providing infrastructure, as well as Ifeoluwa Deborah Gbala for laboratory guidance all through the completion of the work. The authors are immensely thankful to the African Union for financing their research.