Invitro Antimicrobial Activities of Mitracarpus scaber Against Some Common Bacteria of Aquatic Origin

Some plants have been reported to be of medicinal values and reserve some antimicrobial properties. One of such plants is Mitracarpus scaber and its effect on bacterial growth is evaluated. The study aimed at evaluating the phytochemical analyses and antimicrobial potentials of Mitracarpus scaber against aquatic bacteria. Leaves of Mitracarpus scaber and phytochemical analysis and antimicrobial investigation of ethanolic and aqueous extracts of the leaves were carried out against bacteria isolated from diseased Catfish from various farms. The phytoconstituents detected include saponins, tannins, flavonoids, tarpenoids, steroids, anthraquinones and alkaloids in both aqueous and ethanolic extracts. The ethanolic extract had zones of inhibition similar to that of standard antibiotics (enrofloxacin) across all tested microbes. The lowest minimum inhibitory concentration of ethanolic extract of M. scaber was against Bacillus sp with 10mg/ml while the highest was 85mg/ml against Staphylococcus species. The results of the assays showed promising evidences that M. scaber is a potential antibacterial agent against aquatic microbes. However, further studies are recommended to fractionate its constituents and determine the in vitro and in vivo anti-microbial activities and the exact mechanism of action of the constituents.


INTRODUCTION
One of the clogs in the wheel of advancement of global aquaculture is disease outbreak, especially of Bacterial origin (Ajadi et al., 2018). Many of these bacteria are normal flora, but under stress or favourable conditions, they multiply, invade and spread to various tissues and organs (Marian M. Cahill, 1990).
Aeromonas, Vibrio, Staphylococcus and Bacillus species are among the common bacteria of aquatic origin. Others include Pseudomonas, Streptococcus, Escherichia, Edwardsiella and Renebacterium (Austin and Austin, 2012). Aeromonas hydrophila is an opportunistic bacterium and is reportedly ubiquitous in the aquatic environment with high tendency to cause diseases in fresh water species such as African cat fish (Anyanwu et al., 2015). Various species of Vibrio that cause vibriosis in fish are among the commonest fish bacterial pathogens that cause severe clinical signs and high mortality rate (Ina-Salwany et al., 2019).
More often than not, the immediate measure taken to stem the situation is the use of antibiotics. This approach, however, when indiscriminately employed, results in antibiotic resistance and drug residue (Chuah et al., 2016). Apart from decreased production, as a result of decreased efficacy of these agents both chemotherapeutically and prophylactically, the widespread administration of these drugs may lead to the development of resistant human pathogens (De La Pena et al., 2009).
The use of phytogenic agents has been explored as a better and safer alternative to the menace of antimicrobial resistance. Medicinal plants have been incorporated into preventive and control measures against disease outbreaks and profound successes have been reported (Menanteau-Ledouble et al., 2015;Karaskova et al., 2015;Yang et al., 2015). These plants contain chemicals that are categorised as primary or secondary metabolites. The primary metabolites are the common proteins, sugars, amino acids, purines and pyrimidines of nucleic acids and chlorophyll. Secondary metabolites include the remaining /chemicals which are produced from the primary metabolites. These include alkaloids, Phenolics, terpenoids, steroids tannins, and volatile oils (Okwu et al., 2010).
Mitracarpus scaber is one of the tropical plants that have high medicinal values and their use in traditional medicine have been widely explored on terrestrial animals (Alli-Emmanuel et al., 2003) as well as in humans (Ekpendu et al., 1994). Mitracarpus scaber is a perennial plant, abundant in African soil and is used for the treatment of bacterial, fungal and parasitic infections in terrestrial animals (Sanogo et al., 1996;Cimanga et al., 2004).
Leaves of M. scaber have been extensively used in traditional medicine in the tropics for the treatment of headaches, toothaches, skin infections, amenorrhea, dyspepsia, hepatic diseases, venereal diseases and leprosy (Bisignano et al., 2000). The aerial part of M. scaber are used in the treatment of skin diseases such as superficial fungal diseases, produced as lotion and skin ointment (Sanogo et al., 1996). The plant contains active compounds that are responsible for the antifungal, antiinflammatory and anti-parasitic activities (Ekpendu et al., 1994;Cimanga et al., 2004). There is, however, dearth of information on the use of this plant against bacterial infections of aquatic origin.
In this study, the phytochemical analyses and antimicrobial potentials of M. scaber against aquatic bacteria including Aeromonas, Bacillus, Vibrio and Staphylococcus spp were evaluated.

Plant collection
Leaves of M. scaber were collected from premises of Lagos State University staff quarters, Nigeria. They were authenticated at the Department of Plant Biology, University of Ilorin. A voucher specimen was deposited and voucher number was issued as UILH/002/558. The leaves were washed with clean water, air dried for two weeks at room temperature and pulverized into powdery form for extraction.

Aqueous and Ethanoic Extraction
280g of powdered M. scaber was extracted by maceration at room temperature in 140ml each of 98% ethanol and distilled water for 72 hours according to Mungole et al. (2010). The extracts were stored at 4°C until needed for use. The percentage yield of extract was calculated (table 2).

Phytochemical Screening
Qualitative phytochemical analysis of each extract was done according to standard established procedures. 1 g of each ethanolic and aqueous extracts of M. scaber was dissolved in 99 ml ethanol and water respectively to obtain 1% (w/v) stock concentration. The resultant extracts were subjugated to phytochemical screening according to the previously described method (Krishnaiah et al., 2009). Saponins, tannins, flavonoids, cardiac glycosides, tarpenoids, steroids, anthraquionones and alkaloids were screened for.
Test for tannins 0.5 g of powdered sample of M. scaber was boiled in 20 ml of distilled water in a test tube and then filtered. 0.1% FeCl3 was added to the filtered samples and observed for brownish green or a blue black colouration, which showed that the plant contained presence of tannins.

Test for saponins
2 g of powdered samples of M. scaber was boiled together with 20 ml of distilled water in a water bath and filtered.10 ml of the filtered sample was mixed with 5 ml of distilled water in a test tube and shaken vigorously. The product was then mixed with 3 drops of olive oil and observed for the formation of emulsion, which suggested the presence of saponins.

Test for flavonoids
A few drops of 1% NH3 solution was added to the extract of M. scaber in a test tube. A yellow colouration was observed indicating the presence of flavonoid.
Test for terpenoids 5 ml of extract of M. scaber was mixed with 2 ml of CHCl3 in a test tube. 3 ml of concentrated H2SO4 was gently added to the mixture to form a layer. An interface with a reddish brown coloration formed indicated the presence of terpenoids.
Test for cardiac glycosides 5 ml of extract of M. scaber was mixed with 2 ml of glacial CH3CO2H containing a drop of FeCl3.The resultant mixture was gently added to the 1 ml of concentrated H2SO4 so that the concentrated H2SO4 was underneath the mixture. a brown ring colouration indicated the presence of the cardiac glycoside constituent.
Test for anthraquionones 5 ml of M. scaber was mixed with organic solvent and filtered. 2ml of NaOH was added. A pink or violet colour in the base layer indicated the presence of anthraquionones.

Bacterial Isolation
The bacteria used for the antimicrobial analyses were obtained from samples (gills, kidneys and skin) collected from diseased African cat fish from various farms in Kwara state, Nigeria. The samples were analyzed at the Veterinary Microbiology laboratory, University of Ilorin, Nigeria according to method of Fagbemi et al. (2009). Briefly, 1 g of each sample was pre-enriched in 9 ml of buffered peptone water (Oxoid, Hampshire, UK) incubated at 37 0 C for 24 hours. Pre-enriched sample (1ml) was then inoculated into 9 ml of nutrient broth (Oxoid, Hampshire, UK) and incubated overnight at 37 0 C. The turbid nutrient broth was subsequently inoculated unto 5 % sheep blood agar (Oxoid, Hampshire, UK) incubated aerobically at 37 0 C for another 24 hours. The resultant colonies were purified on nutrient agar (Oxoid, Hampshire, UK) and then subjected to Gram staining and biochemical characterization including oxidase, catalase and coagulase tests (Cowan and Steel, 2002). The isolates were kept in 20 % glycerol broth at -20 0 C for future use.

Antimicrobial Screening
The bacteria (Aeromonas, Vibrio, Staphylococcus and Bacillus) were subcultured from the stock into freshly prepared nutrient agar (Oxoid, Hampshire, UK). 1.0 g of each of aqueous and ethanolic extract of M. scaber was dissolved in 10 ml of respective solvent. A concentration of 100mg/ml was obtained to investigate the antimicrobial activities of M. scaber extracts (Joshi et al., 2009).
Mueller-Hinton agar (Oxoid, Hampshire, UK) was used for the antimicrobial screening according to Hudzicki (2009). The agar was seeded with the test microbes to evenly cover the surface of the prepared plates with the inoculum (0.5 Mac Farland dilution equivalence of approximately 1.5 x 10 8 CFU mL -1 ). The plates were allowed to dry and standard cork borer of 6 mm in diameter was used to bore holes (Fagbohun et al., 2013). Each hole was labelled according to the extract to be dispensed in it. 0.1 mL of each extract was introduced into the respective well with sterile syringe. Enrofloxacin and ethanol were used as controls. The plates were aerobically incubated for 24 hours at 37 0 C after which the zones of inhibition of growth were observed and measured using a transparent ruler. The zones of inhibition of the extracts were measured and compared with that of standard antimicrobials CLSI (2012a).
Minimum inhibitory concentration (MIC) of the ethanolic extract of M. scaber was determined using broth macrodilution method as previously described (Balouiri et al., 2016). Two-fold dilutions (10, 20, 40, 80 and 160 mg/mL) of the extract was prepared in nutrient broth (Hi-media, New Delhi, India) for each of the isolates. 2 mL of each dilution was dispensed in different test 2021 tubes. Well isolated colonies of each isolate were inoculated into each tube and the turbidity of each tube was adjusted to 0.5 % Mac Farland standard against a nephelometer (Oxoid, Hampshire, UK). The tubes were incubated with minimum agitation at 37 0 C for 24 hours. The MIC of each isolate was taken as lowest concentration of the extract that completely inhibits growth of the isolates in tubes as indicated by clear broth when viewed by unaided eye (CLSI 2012b).
Because of the economic importance of Aeromonas species in fish production, and its high occurrence and distribution in this study, the need to further characterize them molecularly targeting 16S rDNA sequence for their identification became necessary. However, considering the economic challenge during the study, only two of the isolates were randomly selected and shipped to International Institute for tropical agriculture (IITA) Ibadan, Nigeria, for molecular identification.
DNA extraction was carried out according to method described by Ahmed et al. (2017) and PCR assay using 16S rDNA primers (16SR: GTGCCAGCAGCCGCGCTAA, 16SR: AGACCCGGGAACGTATTCAC) was done according to protocol described by Wagner et al. (2008). Partial genome sequencing of the amplified product was done at Bioscience laboratory of IITA, Ibadan. The obtained 16S rDNA sequences was compared with sequences available in GenBank database of National Center for Biotechnology Information (NCBI) using the algorithm BLAST program (Wagner et al., 2008;Thomas et al., 2014). MEGA version X was used to conduct phylogenetic analyses of the isolate and those obtained from NCBI (Wagner et al. 2008). Consensus sequence from the test isolate was generated using Bioedit and sequences of eight Aeromonas and seven Pseudomonas (outgroup) strains used in the phylogenetic tree were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/) and a phylogenetic tree was constructed. The tree was constructed using the maximum likelihood program with the Tamura-Nei model and Boot strapping 1000 times (Wang et al., 2014).

RESULTS
The phytochemical screening revealed the presence of high concentration of steroids and terpenoids in ethanolic extract while anthraquinones and saponins were much concentrated in aqueous extract (Table 1). The percentage yield of ethanolic and aqueous extracts were 3.6% and 2.3% respectively.   Table 2 shows percentage yield of the constituents, table 2.1 -2.3 (flavonoid, alkaloid and saponin respectively). The bacteria isolated from diseased African catfish from various farms within Kwara state, Nigeria were Aeromonas, Vibrio, Staphylococcus and Bacillus (Table 3) Aeromonas spp was the most isolated bacteria present in all the farms. On phylogenetic analysis, the test isolates (presumptive Aeromonas species) associated genetically with different strains of Aeromonas especially, the strain SG3 with accession number MT409620.1 (Ajadi 1) and strain RB 5 with accession number 396436.1 (Ajadi 2). However, they did dissociate from the outgroup (Pseudomonas species) ( fig. 2).   (Table 4). All the bacteria show similar rate of susceptibility to the ethanolic extract as to that of the control (enrofloxacin). Table 5 shows that the lowest MIC of ethanolic extract of M. scaber was against Bacillus sp with 10mg/ml and the highest was 80mg/ml against Staphylococcus sp. The values of inhibitory zones included the 6mm of each of the agar well.

DISCUSSION
Globally, the menace of drug resistance due to abrupt use of antibiotics is alarming and the need to stem this malady is expedient. Phytogenic agent is one of the panaceas to consider as a way of proactive measure. One of the many tropical plants that are traditionally used for medicinal purposes is M. scaber. It was revealed in this study, that plant extracts (aqueous and ethanol) contained secondary metabolites including saponins, tannins, flavonoids, terpenoids, steroids anthraquinones and alkaloids in varying degrees, this is in agreement with earlier study by Abere et al. (2007).
These metabolites and several other aromatic compounds are responsible for the antimicrobial activities of the plant (Opoku et al 2015). Previous studies have reported the antimicrobial activities exhibited by these phytoconstituents including tannins (Kolodziej et al., 1999); flavonoids (Cottiglia et al., 2001;Elsohly et al., 2001), tarpenoids (Braca et al., 2000) and anthraquinones (Houghton et al., 2000). Saponins have been reported to have antimicrobial activity and hence, it's an essential bioactive component of plant involved in plant disease resistance (Godswill et al., 2010;Hossain et al., 2013).
The activities of M. scaber reported in this study could probably be related to the presence of these phytoconstituents in the plant. In comparison, the ethanolic extract was seen to be more active against the tested bacteria than the aqueous extract and has similar activity with Enrofloxacin. This could be due to the insolubility of the phytoconstituents in water or denaturation of the active compound by hot water in the process of extraction (Opoku et al., 2015). This is an indication that ethanol would be a better solvent for plant extraction for antibacterial analyses (Irobi et al., 1994).
The use of enrofloxacin as a positive control is due to the fact that most isolated organisms from fish are susceptible to the antibiotic (Zhang et al., 2010) and that Enrofloxacin is one of the antibiotics generally in use for the treatment of infection in fish production in Nigeria (Abalaka et al., 2013). However, the incessant and indiscriminate use of chemotherapeutic agents results in less efficient disease control and subsequent antibiotic resistance and eventual drug residue (Chuah et al., 2016). Earlier studies have reported the antimicrobial, anti-inflammatory, antifungal and analgesic effects of M. scaber (Owolabi et al., 2013;Bisignano et al., 2000;Sanogo et al., 1998;Ekpendu et al., 1994;Irobi et al., 1994). Ali-Emmanuel et al., 2003 reported its effectiveness against bovine dermatophilosis.
Extracts of M. scaber possess active ingredients that are effective bactericidal (Adeshina et al., 2019). This makes the plant to be essential in trado-medicine for the treatment of various diseases such as infectious dermatitis, candidiasis, toothache and venereal diseases among others that are caused by several bacteria and fungi. The concentration of ethanolic extract of M. scaber to inhibit bacterial growth is lowest (10mg/ml) against Bacillus sp and highest (85mg/ml) against Staphylococcus sp. Therefore, in this study, the ethanolic extract of M. scaber is most potent against Bacillus sp and least against Staphylococcus sp. Aeromonas and Vibrio spp were moderately inhibited.

CONCLUSION
Extracts of M. scaber showed moderate antimicrobial activities against test organisms including Aeromonas, Bacillus, Vibrio and Staphylococcus isolated from diseased African catfish. The results of the antimicrobial assay showed promising evidence that the plant (M. scaber) is a potential antibacterial agent against aquatic microbes. Further in vivo study is expedient to substantiate the antimicrobial potentials of this extract against these bacteria of aquatic origin.