Antimicrobial Effect of Geophila Obvallata (Schumach) Didr. Leaf Extracts Against Some Medically Important Bacteria and Fungi

Background: The increase in synthetic drug resistance by pathogenic microbes has led to the development of plant-based antimicrobial drugs that are more reliable and non-lethal to human health at increased dosage. Methods: The antibacterial and antifungal potential of Geophila obvallata extracts were tested on clinical isolates (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Streptococcus pyogenes, Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus) using standard techniques. Results: The zones of inhibition were shown to increase with increasing concentrations of the extracts. Inhibition was higher in Gram positive bacteria (9.10 to 31.00mm in 40mg/mL concentration) than Gram negative bacteria (3.50 to 27.00mm in 40mg/mL concentration), while the fungal isolates had the least zones of inhibition (2.83 to 25.00mm in 40mg/ml concentration). The minimum inhibitory concentrations (MIC) were lowest in the methanol extract than aqueous extract. Simillarly, MIC for bacteria (Bacillus subtilis) and fungi (Aspergillus fumigatus) were 0.3 and 2.0mg/mL respectively. Methanol extract had higher antibacterial and antifungal effect than aqueous extract. Ciprooxacin, used as control for bacteria had the highest inhibitory activity (33.67mm) when compared to that of the highest concentration of plant extracts administered. Also, ketoconazole gave the highest zones of inhibition (32.33mm) on the fungi isolates compared to those of the extracts. The performance of the methanol extract of 40mg/mL of Geophila obvallata in the inhibition of Bacillus subtilis was not signicantly different from that of Ciprooxacin. Conclusion: The ndings in this study therefore validate the antimicrobial effect of Geophila obvallata leaf extracts as well as its possible application in medicine. in even strokes obtain a uniform growth pattern across the entire surface of the plate. This was achieved by rotating the plate 90 degrees followed by 45 degrees with continuous streaking, and nally by streaking round the diameter of the agar. The 7 mm wells were lled with different volumes of the stock concentration of the extracts corresponding to 40, 30, 20 and 10 mg/mL concentrations. The same quantity of sterilized distill water, standard drugs (1 µg/mL of Ciprooxacin for bacteria plates and 10 µg/mL of Ketoconazole for fungal plates) served as controls setup for the experiment. The plates were left to stand for 1 hour on a work-bench to allow diffusion of the extracts before incubating bacterial plates overnight at 37ºC and the fungal plates at room temperature (25±2 o C) for 72 hours. The diameter of clear zone was observed and measured in mm (millimeters). The experiments including controls were done in triplicates and the mean zones of inhibitions calculated.


Introduction
The increasing occurrence of multi-drug resistant strains of infectious bacteria and fungi is a major threat to public health worldwide. The distribution of these resistant strains in the population has rendered most antibiotics and major last -resort drugs ineffective in combating infections or diseases (Mandal et al., 2009;Bhatia and Narain, 2010). Microbial resistance to antimicrobial agents increases the chance of hospitalization and in most cases death (Winstanley, 1997). In recent times, the indiscriminate use of antibiotics in most developing and developed countries of the world has led to reports of drug resistance to pathogenic microbes in humans (Gupta et al., 2017). As a result of this rapid spread and development of resistance microbial strains, the need for the discovery of novel antimicrobial agents that are effective alternatives to synthetic and hazardous chemical drugs cannot be over-emphasised. This has led to the exploration of plants and plant based products as novel remedies in the treatment of disease (Basualdo et al., 2007;Mandal et al., 2010). Plant based antimicrobials (extracts) have been recognised as valuable alternatives in the treatment of most microbial infections compared to synthetic antibiotics due to their stability, non-lethality and e cacy (Iwu et al., 1999;Alam et al., 2009). Currently, more than 30% of the antimicrobial agents produced in modern pharmacology are obtained directly or indirectly from plants and their extracts (Murugesan et al., 2011). The World Health Organization (WHO) has a rmed Preliminary phytochemical pro ling The bioactive constituents present in the methanol and aqueous extracts of Geophila obvallata were previously screened for avonoids, polyphenols, alkaloids, tannins, saponins, steroids, terpenoids and cardiac glycosides (Iserhienrhien and Okolie, 2018), according to the methods described by Sofowora, (1993).
Microbial culture A total of 9 clinical isolates were obtained from stock cultures after appropriate permission was sought from the Department of Pharmaceutical Microbiology laboratory, Faculty of Pharmacy, University of Benin, Nigeria. The referenced microorganisms include: S. aureus, K. pneumoniae, P. aeruginosa, E. coli, B. subtilis, S. pyogenes, C. neoformans, C. albicans and A. fumigatus. Prior to use, the test microorganisms were authenticated and sub-cultured from stock into sterile nutrient broth (for bacteria) and Sabouraud Dextrose broth (for fungi) and incubated overnight at 38 o C for 24hours for bacteria and at room temperature (25±2 o C) for 72hours for fungi. After incubation, overnight broth culture was adjusted to 0.5 McFarland standard to give an inoculum size of approximately 1.5 x10 8 cfu/mL and a further one in hundred serial dilution (1:100) using normal saline solution to yield approximately 10 6 cfu/mL.

Antimicrobial assay of plant extracts
Antimicrobial susceptibility test was carried out using agar-well diffusion method (Vinothkumar et al., 2010). Wells of 7 mm in diameter were made into previously seeded Mueller Hinton / Sabouraud agar plates using a amed (sterile) cork borer. Prior to seeding, the turbidity of the overnight broth culture of each isolates were adjusted to match 0.5 McFarland turbidity standard and diluted (1:100) to give approximately 1x10 6 cfu/mL microbial suspension. Sterile swab sticks were then dipped into the standardized microbial suspension and gently streaked on the surface of the agar plates in even strokes to obtain a uniform growth pattern across the entire surface of the plate. This was achieved by rotating the plate 90 degrees followed by 45 degrees with continuous streaking, and nally by streaking round the diameter of the agar. The 7 mm wells were lled with different volumes of the stock concentration of the extracts corresponding to 40, 30, 20 and 10 mg/mL concentrations. The same quantity of sterilized distill water, standard drugs (1 µg/mL of Cipro oxacin for bacteria plates and 10 µg/mL of Ketoconazole for fungal plates) served as controls setup for the experiment. The plates were left to stand for 1 hour on a work-bench to allow diffusion of the extracts before incubating bacterial plates overnight at 37ºC and the fungal plates at room temperature (25±2 o C) for 72 hours. The diameter of clear zone was observed and measured in mm (millimeters). The experiments including controls were done in triplicates and the mean zones of inhibitions calculated.

GC-MS analysis of methanol extract
The analysis was done with GCMS-QP2010SE (Shimadzu, Japan). Helium served as carrier gas at a column ow rate of 3.22mL/min with a split ratio of 5:1.
1 μl of sample was injected into the column at an injection temperature of 250.00 °C and detection temperature was 290 °C. The oven starts at 60 °C while the hold time is for 2 mins, this is maintained at 10 °C /min to 290 °C without holding. Total running time was 21 mins. The detector was a ame ionization detector (FID). Detected peaks were compared with NIST 11s, library to verify the names and molecular weights of the samples constituents.

Data analyses
Data collected during this study were analyzed using MINITAB 16 and represented as mean ± standard deviation. Fishers Pairwise Comparison (FPC) of ANOVA at a signi cance level of P < 0.05 was used to test the variability in susceptibility of the microorganisms towards the extractives.

Results
The in vitro experiment conducted showed that the best antimicrobial effect on K. pneumonia, P. aeruginosa, E. coli, S. aureus and S. pyogenes were seen in the methanol (30 and 40 mg/mL) extract group when compared with the aqueous extract group. The zones of inhibition on culture plate of the isolates at 30 and 40 mg/mL of the methanol extract were measured as 25.67 and 27.00 mm, respectively for K. pneumonia, 21.33 and 24.00 mm, respectively for P. aeruginosa, 25.67 and 26.00 mm, respectively for E. coli, 24.33 and 27.33 mm, respectively for S. aureus, 27.67 and 29.33 mm, respectively for S. pyogenes ( Table 1). Indicating that both concentrations of the methanol extracts of Geophila obvallata performed very well in limiting the growth and metabolic activities of the isolates compared to the aqueous extract and the control setup for the experiment (DH 2 O). Cipro oxacin (CIP) a synthetic drug, performed better than all other groups (Table 1). However, the performance of the methanol extract at 40 mg/mL of Geophila obvallata in the inhibition of the microbe B. subtilis was not signi cantly different (P > 0.05) from that of the synthetic drug Cipro oxacin.     The results displayed in Tables 3 and 4 Table 5). Table 5 shows the bioactivities of the reported phyto-compounds.  In this study, the crude aqueous and methanol extracts of Geophila obvallata showed appreciable inhibitory activity against both gram negative bacteria (K. pneumonia, P. aeruginosa, E. coli) and gram positive bacteria (S. aureus and S. pyogenes, B. subtilis) especially at higher doses. However, the methanol extracts at a dose of (40 mg/mL) showed the best growth inhibitory performance, especially against the gram positive microbe B. subtilis, which was not signi cantly different from that of the control (Cipro oxacin). The antibacterial effect highlighted in the crude methanol extract may be due to (1)  Simillarly, the methanol extract also showed signi cant growth inhibitory properties against the radial mycelia growth of fungal isolates (C. neoformans, C. albicans, and A. fumigatus) especially at higher doses (40 mg/mL) compared to the aqueous extract. Also, the group treated with 40 mg/mL of the methanol extract produced similar effects like the synthetic drug (Ketoconazole) on the pathogens C. albicans (31.67  In conclusion, the crude aqueous and methanol leaf extracts of Geophila obvallata showed exceptional concentration-dependent antibacterial and antifungal activity against selected clinical isolates. However, the methanol extract at a dose of 40 mg/mL showed the best antimicrobial effect against the tested isolates. These ndings con rm the antimicrobial potency of the plant extract and give credence to the orthodox use of the medicinal plant as a suitable alternative to synthetic antimicrobial agents.

Declarations
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.9734/ajrb%2F2018%2Fv3i229823 Ethics approval and consent to participate: The experimental protocol and methods were performed in accordance with the relevant guidelines and regulations.
Consent for publication: Not applicable.
Availability of data and materials: The datasets supporting the conclusions of this article are included within the article and its additional le.
Competing interests: The authors declare that they have no competing interests.
Funding: There was no funding.
Authors' contributions: Okolie Ngozi participated in the conception, design of the study, data analysis and drafted the manuscript. Iserhienrhien Lucky carried out the experiments and data analysis. Etaware Mudiaga assisted with the data analysis and helped to draft the manuscript. All authors read and approved the nal manuscript.