International Journal of Health and Pharmaceutical Research ISSN 2045-4673 Vol. 4 No. 1 2018 www.iiardpub.org
Antibacterial Activity of Bryophyte (Funaria hygrometrica) on some throat Isolates
Akani, N. P. & Barika P. N. Department of Microbiology, Rivers State University, Nkpolu-Oroworukwo, Port Harcourt P.M.B. 5080 Rivers State, Nigeria
E. R. Amakoromo Department of Microbiology, University of Port Harcourt Choba P.M.B 5323
Abstract An investigation was carried out to check the antibacterial activity of the extract of a Bryophyte, Funaria hygrometrica on some throat isolates and compared with the standard antibiotics using standard methods. The genera isolated were Corynebacterium sp., Lactobacillus sp. and Staphylococcus sp. Result showed a significant difference (p≤0.05) in the efficacy of F. hygrometrica extract and the antibiotics on the test organisms. The zones of inhibition in diameter of the test organisms ranged between 12.50±2.08mm (F. hygrometrica extract) and 33.50±0.71mm (Cefotaxamine); 11.75±2.3608mm (F. hygrometrica extract) and 25.50±0.71mm (Cefotaxamine) and 0.00±0.00mm (Tetracycline) and 29.50±0.71mm (Cefotaxamine) for Lactobacillus sp, Staphylococcus sp. and Corynebacterium sp. respectively while the control showed no zone of inhibition (0.00±0.00mm). The test organisms were sensitive to the antibiotics except Corynebacterium being resistant to Tetracycline. The minimal inhibitory concentration of plant extract and antibiotics showed a significant difference (p≤0.05) on the test organisms and ranged between 0.65±0.21 mg/ml (Cefotaxamine) and 4.25±0.35 mg/ml (Penicillin G); 0.04±0.01 mg/ml (Penicillin G) and 2.50±0.00 mg/ml ((F. hygrometrica extract) 0.06±0.00mg/ml (F. hygrometrica Extract) and 2.25±0.35mg/ml (Cefotaxamine) for Lactobacillus sp.; Staphylococcus sp. and Corynebacterium sp. respectively with Corynebacterium sp. being sensitive to the plant extract with an MIC of 0.63mg/ml followed by Lactobacillus sp. and Staphylococcus sp. Generally, the antibiotics proved to be more sensitive to the test organisms than the plant extract. The degree of sensitivity exhibited by the plant extracts calls for a further study to isolate and identify the active constituents to enhance its sensitivity.
Keywords: Antibacterial activity, Funaria hygrometrica extracts, Antibiotics
Introduction Funaria hygrometrica (Bonfire moss or cord-moss) is a type of water moss of the family Funariaceae which grows on moist shady, damp soil, moist walls and crevices of Rocks (Edwards and Sean, 2012). It is estimated that between 15,000 and 25,000 bryophyte species known in the world. They consist of three separate divisions, Marchantiophyta (liverworts), Anthocerotophyta (hornworts) and Bryophyta (mosses) (Goffinet and Shaw 2009). They are to be found in all ecosystems, from desert to alpine, with the exception of marine. The ecological role of bryophytes in any ecosystem is significant (Saxena and Harinder, 2004).
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International Journal of Health and Pharmaceutical Research ISSN 2045-4673 Vol. 4 No. 1 2018 www.iiardpub.org
Although bryophytes normally grow in humid habitats, they are relatively free from microbial attack and this scarcity of disease indicates that bryophytes are able to elaborate constitutive or inducible small-molecule antimicrobials (Bodade et al., 2008). In fact, bryophytes have been proven to be a rich source of antibiotics, and attempts to find potent, nontoxic, broad-spectrum antibiotics from these sources have widely been undertaken (Xie and Lou, 2009). Plants have made important contributions in the areas beyond antimicrobials such as cancer therapies. Most drugs are active analogue of the active constituents of plant extract with antimicrobial activity (Iwu et al., 1999). Plant derived antimicrobials are relatively safer than synthetic alternatives offering profound therapeutic benefits and more affordable treatment and they are effective in the treatment of infectious diseases and simultaneously mitigating against many side effects that are often associated with synthetic antimicrobials (Jockovic et al., 2008). Plant containing photoberberines and related biflavones used in traditional African system of medicine has been found to be active against a wide variety of microorganisms. Many medicinal plant of African origin have been investigated for their chemical composition and some of the isolated compounds have been shown to posses interesting antimicrobial activity especially bacteria (Basile et al., 2002). Generally, bryophytes are known to possess extremely high amounts of terpenoids, phenolics (flavonoids and bibenzyl derivatives), glycosides, fatty acids, as well as some rare aromatic compounds (Sabovljevic et al., 2010). Most throat cultures are carried out to rule out infectious diseases caused by β-hemolytic Streptococci which causes sore throat, Staphylococci, Corynebacterium diphtheriae, some Neisseria species and the primary purpose of a throat culture is to identify the specific organisms that cause sore throat especially Streptococcus pyogenes and a correct diagnosis is important to prevent unnecessary use of antibiotics and to begin treatment of Streptococcal infections as soon as possible. Besides causing sore throat it can cause Scarlet fever, rheumatic fever and kidney diseases (Oztopcu-Vatan et al., 2011). The research is carried out to determine the antibacterial activity Funaria hygrometrica on some throat organisms. The findings from this research may play a significant role in the development of new plant based antimicrobial drug to fight the increasing army of drug resistant microorganisms which has become a public health concerned.
Materials and Methods Collection of throat samples The throat samples were collected using sterile swab sticks from healthy persons in Rivers State University, and taken to the Microbiology laboratory within 2 hours of collection for analysis.
Culturing of the organisms Culturing was done as described by (Lynch 1983) using blood and chocolate agar plates. Plates were incubated aerobically and microaerophilically at 37oC for 24hours, discrete colonies were described and sub-cultured to a fresh nutrient agar plate and incubated at 37oC for 24hours for further biochemical tests.
Morphological and Biochemical characteristics of the isolates The isolates were tentatively identified based on morphological characteristics like; size, shape and Gram’s reaction. Other biochemical test includes; motility, indole, urease, catalase, Citrate, Coagulase, Oxidase, Vogues Proskauer and methyl red test (Cheesebrough, 2000). Bacterial isolates were finally identified using Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994, Cheesebrough, 2000).
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Extraction of the Bryophyte Funaria hygrometrica Funaria hygrometrica was collected from concrete floor in Rivers State University and identified by a botanist in the department of Plant Science and Biotechnology. It was sun dried for two (2) days and ground into powder form. About 10g of the powder was weighed and dispensed in 100ml full strength ethanol as solvent and after vigorous shaking was allowed for two (2) days. Then it was filtered with Whatman filter paper into a conical flask and the weight of the residue was weighed and recorded (Jones and Kinghorn, 2005; Ozturk et al., 2009)
Disc diffusion method to determine the antibacterial activity of Funaria hygrometrica extract Funaria hygrometrica disc were prepared by perforating Whatman filter paper and impregnated by the plant extract of known concentration by immersing the Whatman filter paper into the plant extract and the concentration of the plant extract was known by recording the weight of plant before and after extraction and the test organisms were streaked on the plate and the impregnated plant extract disc were placed on the nutrient agar plates, then the plates were allowed to stand for 30mins and incubated at 37oC for 24 hours. After 24hours the zones of inhibition were measured in millimeter (mm) with a rule and recorded.
Determination of the Minimal Inhibitory Concentration (MIC) of Funaria hygrometrica extract This is performed as described by (NCCLS 2008). This aims at determining the lowest concentration of the Funaria hygrometrica extract that can inhibit or kill the test organisms. About 5ml of the plant extract were dispensed in a 5ml nutrient broth to for a mixture. Then 5ml of the mixture were transferred into a new text tube containing 5ml of nutrient and the dilution continues till the six test tubes and 5ml was discarded from the last test tube. Since the stock concentration was 40mg/ml and the dilution is 1:2 dilution techniques, the concentration of the tubes gets progressively weaker by a factor of 1/2. Hence 1st tube =20mg/ml, 2nd tube =10mg/m, 3rd tube =5mg/ml, 4th tube =2.5mg/ml, 5th tube =1.25mg/ml and 6th tube =0.625mg/ml and 0.1ml of the bacterial suspension of the test organisms were added to the six test tubes accordingly and thoroughly shaken and incubated at 37oC for 24hours. Two controls were made the positive control containing 9ml nutrient broth and test organisms without extract and a negative control containing 9ml nutrient broth with plant extract without test organisms. The tubes were examined for turbidity and tubes without turbidity were recorded as the minimal inhibitory concentration of the plant extract.
Disc diffusion method and determination of MIC of the standard antibiotic The sensitivity disc of standard antibiotic was obtained and the same procedures as for the Funaria hygrometrica extract were carried out as described by (NCCLS, 2008) and the zones of inhibition were measured and recorded. The minimal inhibitory concentrations of the standard antibiotic (Tetracycline, Cefotaxamine and penicillin G) were determined from the stock concentration and the tube with no visible bacterial growth was recorded as minimal inhibitory concentration of that test organism for that particular antibiotic.
Data analysis Statistical analysis was carried out on the data which was obtained during the study. Analysis of Variance and Duncan Multiple Ranged Test were used to test for significance and means separation respectively. This is done using a computer-based Programme-SPSS version 22
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Results and Discussion Funaria hygrometrica extract were screened for antibacterial activity against some bacterial strains from the throat. The genera isolated were identified as Corynebacterium sp., Lactobacillus sp. and Staphylococcus sp. (Table 1). Result showed a significant difference (p≤0.05) in the efficacy of F. hygrometrica extract and the antibiotics on the test organisms (Tables 2 and 3).
Table 1: Colonial and Biochemical Characterization of the throat Isolates Isolates General Characteristics Probable Organism
Colonial Gram Morphology Reaction/shape
Catalase Catalase Coagulase Indole Motility Voges Proskauer Haemolysis Lipase Methyl Red Urease Oxidase
T1-MCA Creamy, raised + Large rods - - + + + - + + - - Lactobacillus smooth surface sp and edges T2-MCA Creamy, smooth + Cocci + + - - + + + + + - Staphylococcus surface and edges sp
T3-ABA Creamy, small, + Club-shaped + - + - - + + + - - Corynebacteriu raised, smooth or cocco-Bacilli m sp surface and edges
Key: += Positive, - = Negative
The zones of inhibition in diameter of the test organisms ranged between 12.50±2.08mm (F. hygrometrica extract) and 33.50±0.71mm (Cefotaxamine); 11.75±2.3608mm (F. hygrometrica extract) and 25.50±0.71mm (Cefotaxamine) and 0.00±0.00mm (Tetracycline) and 29.50±0.71mm (Cefotaxamine) for Lactobacillus sp, Staphylococcus sp. and Corynebacterium sp. respectively while the control showed no zone of inhibition (0.00±0.00mm) (Tables 2 and 3). The test organisms were sensitive to the antibiotics; Lactobacillus was most sensitive to Cefotaxamine except Corynebacterium being resistant to Tetracycline just as reported in a previous study by (Wilkinson, 2006) and the test organism were intermediately sensitive to the plant extract with Lactobacillus sp and Corynebacterium sp being more sensitive compared to Staphylococcus sp and the sensitivity of the test organism to the plant extract were due to the presence of some phytochemicals such as flavonoids, anthraquininones terpenoids which disrupt the cell membrane of microorganism just as reported by (Cushnie and lamb 2005).
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Table 2: Zones of Inhibition in diameter of the Funaria hygrometrica extract and Standard antibiotic (mm)
Treatments Bacterial Isolates Lactobaccillus sp Staphylococcus Corynebacterium sp sp
Control (broth + plant 0.00 ±0.00a 0.00 ±0.00 a 0.00 ±0.00 a extract) Extract(40mglml) 12.50 ±2.02 b 11.75 ±2.36 b 12.75 ±2.22 b
Penicillin G(10g/ml) 22.50 ±0.71 c 25.50 ±0.71 d 29.50 ±0.71 c
Tetracycline(250mg/ml) 24.50 ±0.71 c 18.50 ±0.71 c 0.00 ±.00 a
Cefotaxamine(200mg/ml) 33.50 ±0.71 d 24.50 ±0.71 d 28.50 ±0.71 c
*Means with the same alphabet across columns shows no significant difference (p>0.05)
Table 3: ANOVA table showing level of significance in Zones of Inhibition of the Funaria hygrometrica extract and standard antibiotic. Mean Sum of Squares df Square F Sig. Lactobaccillus * Samples Between Groups (Combined) 1372.417 4 343.104 165.63 .000
Within Groups 14.500 7 2.071 Total 1386.917 11 Staphylococcus * Samples Between Groups (Combined) 916.417 4 229.104 87.876 .000
Within Groups 18.250 7 2.607 Total 934.667 11 Corynebacterium * Samples Between Groups (Combined) 1691.167 4 422.792 187.907 .000
Within Groups 15.750 7 2.250 Total 1706.917 11
The results of the minimal inhibitory concentration of plant extract and antibiotics as presented in Table 4 showed a difference (p≤0.05) on the test organisms and ranged between 0.65±0.21 mg/ml (Cefotaxamine) and 4.25±0.35 mg/ml (Penicillin G); 0.04±0.01 mg/ml (Penicillin G) and 2.50±0.00 mg/ml (F. hygrometrica extract) 0.06±0.00mg/ml (F. hygrometrica Extract) and 2.25±0.35mg/ml (Cefotaxamine) for Lactobacillus sp.; Staphylococcus sp. and Corynebacterium sp. respectively with Corynebacterium sp. being more sensitive to the plant extract with the lowest MIC of 0.63mg/ml followed by Lactobacillus sp. and Staphylococcus sp. This shows that the plant extract has a greater antimicrobial effect on Corynebacterium sp than Lactobacillus sp. and Staphylococcus sp.
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Table 4: Minimal Inhibitory Concentration of the Funaria hygrometrica extract and Standard antibiotic (mg/ml)
Treatments Bacterial Isolates Lactobaccillus sp Staphylococcus sp Corynebacterium sp Control 0.00±0.00 a 0.00±0.00 a 0.00±0.00a Extract(40mg/ml) 1.25±0.00b 2.50±0.00 c 0.06±0.00a Penicillin G(10g/ml) 4.25±0.35c 0.04±0.01ab 0.15±0.07a Tetracycline(250mg/ml) 1.25±0.35 b 0.20±0.14 b 0.25±0.35a Cefotaxamine(200mg/ml) 0.65±0.21 b 0.15±0.071ab 2.25±0.35b *Means with the same alphabet across columns shows no significant difference (p>0.05)
Most of these antibiotics act by inhibiting the synthesis of bacterial cell wall (Cefotaxamine) while others make the organism sensitive by preventing the formation a functional cell wall by inhibiting the chemical cross-linkages of the bacterial cell wall (Penicillin G), some bind to bacterial ribosomes inhibiting protein synthesis (Tetracycline) (Schlegel, 1993). Many bacteria may produce an enzyme penicillinase (B-Lactamase) which can cleave the B-lactam ring of penicillin and renders the antibiotics ineffective causing the organism to be resistant to that antibiotic and majority of organisms produce this B-lactamase.
Table 5: ANOVA table showing level of significance in Minimal Inhibitory Concentration of the Funaria hygrometrica extract standard antibiotic
Sum of Mean Squares df Square F Sig. Lactobaccillus * Between (Combined) Samples Groups 21.316 4 5.329 90.322 .000 Within Groups .295 5 .059 Total 21.611 9 Staphylococcus * Between (Combined) Samples Groups 9.287 4 2.322 460.683 .000 Within Groups .025 5 .005 Total 9.313 9 Corynebacterium * Between (Combined) Samples Groups 7.360 4 1.840 36.077 .001 Within Groups .255 5 .051 Total 7.615 9
Generally, the antibiotics proved to be more sensitive to the test organisms than the plant extract. It is clear that the extract has some degree of antibacterial activity on the test organisms (Bodade et al., 2008).
Conclusion and Recommendations To halt the trend of increased emerging and resistant pathogenic microorganisms, it requires a multi-faceted approach that involves the development of new antimicrobial drugs. Using plants as the inspiration for new antimicrobials provides an infusion of novel compounds or substance for treatment of various diseases. The antibacterial activity of Funaria
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International Journal of Health and Pharmaceutical Research ISSN 2045-4673 Vol. 4 No. 1 2018 www.iiardpub.org hygrometrica studied in this work is promising which requires further investigation to be carried out on this plant. The degree of sensitivity exhibited by the plant extracts calls for a further study to isolate and identify the active constituents to enhance its sensitivity. Other chemical test should be carried out to determine the suitability for pharmaceutical use and the level of toxicity of the plant extract on human cells should be studied and test against a wide variety of Microorganisms to know whether it has a broad spectrum of antibacterial activity. The plants should also be grown axenically in the presence of oligosaccharides elicitors in other to improve its antimicrobial activity since previous study has shown that oligosaccharides elicitors can induce the production of antimicrobial substance in Bryophytes.
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