Antimicrobial activity

The crude extracts of the putative endophytic bacterial strains were assayed for antimicrobial activity against pathogenic strains (Gram-negative strains Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Klebsiella oxytoca ATCC 13182; Gram-positive Staphylococcus aureus NCTC 6571 and Bacillus cereus ATCC 10876). Among the five endophytic bacteria, only four except Staphylococcus sp. LCP showed antimicrobial activity.

Pseudomonas sp. SSRN1 and Enterobacter sp. SSRP1 were considered as the most active strains as they both had a moderate activity against Staphylococcus aureus. A wide zone of inhibition was obtained by Pseudomonas sp. SSRN1 and Enterobacter sp. SSRP1, followed by Lysinibacillus sp. HSRN then lastly Bacillus sp. LRP (Table 4.1). The results of ANOVA indicates that p value was less 0.05 (p <0.05) therefore there was a significant difference between all five bacterial endophytes antimicrobial activity.

Endophytic bacteria have potential to produce novel natural compounds with antibacterial and antifungal activity (Christina et al., 2013). Bacterial endophytes (Psedumonas sp. and Bacillus sp.) isolated from Plectranthus tenuiflorus have shown great antimicrobial activity against some human pathogenic strains such as Salmonella typhi, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae Proteus mirabilis, Candida albicans (EI-Deeb et al., 2013). Furthermore, Enterobacter sp. isolated from Raphanus sativus L also showed antibacterial activity against a few human pathogenic bacteria including E. coli, S. enteritidis, S. sonnei, S. typhimurium, P. aeruginosa, S. flexneri and B. cereus (Seo et al., 2010). Pseudomonas species have proven to possess antimicrobial compounds called

0

51 ecomycins and pseudomycins (Christina et al., 2013).Secondary metabolites from C. molle were also reported to possess antimicrobial activity (Kaleab et al., 2006; Fankam et al., 2015).

It is evident from the current study that the isolated bacterial endophytes also have antibacterial activity with a broad antibacterial spectrum. Thus, the bacterial endophytes with antibacterial activity from the current study can play a part in inhibiting plant pathogens growth in plant host. In addition, potential applications such as drug discovery and biocontrol use in agriculture can arise from these bacterial endophytes and necessitates further investigations.

Medium compositions, incubation temperature, and degree of aeration can affect quantity and production of secondary metabolites produced by endophytes (Dos Santos et al., 2015).

In this study nutrient broth was used for production of secondary metabolites by the bacterial endophytes. Kumar et al. (2012) reported that media containing yeast and fructose showed higher antimicrobial activity. Luria broth contains higher grams (5 g) of yeast extract than nutrient broth (2 g). Thus, higher antimicrobial activity could be achieved if secondary metabolites were grown in Luria broth. Luria broth is recommended for bacterial growth because of its nutritional capacity and it also permits fast growth (Sezonov et al., 2007).

52 Table 4.1: Antimicrobial activity of Endophytic bacteria from C. molle

Pathogenic

(+) weak activity, (++) moderate activity, (+++) strong activity, (-) no zone of inhibition *(weak activity: 0- 2mm, moderate activity: 3-5mm, strong activity: 6-8mm), value of significant is at p < 0.05

4.4 Conclusion

Endophytic bacteria are promising source of biologically active compounds and can serve as a producer of novel antimicrobial compounds. C. molle host different kinds of endophytic bacteria some of which showed antibacterial activity against the tested pathogenic microorganisms. The study showed increased antimicrobial activity against Pseudomonas sp.

SSRN1 and Enterobacter sp. SSRP1. This indicates the pharmaceutical potential of secondary metabolites produced by bacterial endophytes from C. molle.

53

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CHAPTER FIVE:

Extraction and Characterisation of Secondary Metabolites from Combretum molle and its

Bacterial Endophytes*

*This chapter will be submitted to 3 Biotech, titled ‘Extraction and Characterisation of Secondary Metabolites from Combretum molle and its bacterial Endophytes (2017)’ and was still under preparation on submission of the dissertation. It will be written and prepared by Diale, MO¹, Ubomba-Jaswa, E² and Serepa-Dlamini, MH¹.

1 First co-author

2 Second co-author

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Abstract

Plants are associated with a diverse community of microorganisms known as endophytes which are known to produce the same bioactive compounds as the host plant. Endophytes produce various classes of secondary metabolites including alkaloids, tannins, steroids, flavonoids and saponins. Many endophytes are known to produce novel pharmaceutical bioactive compounds with antioxidant, antifungal, antibacterial, antitumor, antiviral, anti-inflammatory and immunosuppressive potential. Phytochemical constituents of C. molle plant parts and endophytes crude extracts were analysed for alkaloids, flavonoids, steroids, saponins and tannins. The crude extracts were further analysed using LCMS quadrupole time of flight. The presence of tannins and flavonoids were found in both C. molle plant parts and endophytes crude extract. Most of metabolites identified were flavonoids.

Keywords: Endophytes, Medicinal plants, Combretum molle, Phytochemical compounds

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5.1 Introduction

The search for novel bioactive secondary metabolites synthetised by microorganisms remains a vital area to explore in order to manage the increasing demand for treatment of diseases (Rao et al., 2015). Natural resources such as plants and microorganisms have played significant role in drug discovery (Ji et al., 2009). Endophytes are known to produce secondary metabolites and majority of these metabolites have been discovered in plants (Brader et al., 2014). These metabolites are grouped into two, viz primary and secondary metabolites.

Primary metabolites are involved in the growth and development of the species, and some examples include chlorophyll, common sugars and proteins are primary compounds, in contrast secondary metabolites are compounds produced by species, not directly involved in growth and its development but may infer ecological function, examples include but not limited to terpenoids, alkaloids and phenolic compounds (Wadood et al., 2013).

Secondary metabolites obtained from endophytic microbes possess antibacterial, antioxidant, antidiabetic, antifungal, and antitumor properties (Bhoonobtong et al., 2012).

Endophytic bacteria metabolites are less investigated as compared to endophytic fungi and few reports shows bacteria as great potential for bioactive compounds (Christina et al., 2013).

Some of the natural products from endophytic bacteria include Econmycin, Pseudomycins, Munumbicins and Xiamycins which are antibacterial, antimycotic and antiplasmodial drugs and 4-arylcoumarins an antitumor bioactive compound (Miller et al., 1997; Rafat et al., 2011, Christina et al., 2013).

Combretum species are known to be rich in triterpenoids and phenolic compounds (Eloff et al., 2008). Uzor et al. (2013) reported that endophytic fungi isolated from Combretum dolichopetalum produced phenolic and triterpenoids compounds. The compounds isolated from C. dolichopetalum and its endophytic fungi were similar. To the best of our knowledge this the first study to report on secondary metabolites of endophytic bacteria isolated from C.

molle.

Metabolite identification is increasingly becoming an important for early phases of drug discovery for reaching a conclusion as to whether or not a drug candidate needs further development (Lan et al., 2013). Liquid Chromatography mass spectrometry (LCMS) is an ideal method for separation and detection of semi-polar secondary metabolites in plants such as

59 phenolic acids, alkaloids, phenylpropanoids, saponins, flavonoids, glucosinolates, polyamines (Simsek, 2014; Moco et al., 2016). Using LCMS technique, hundreds to thousands of compounds can be detected in organisms (Sawada et al., 2013). High resolution mass spectrometer such as quadrupole time-of-flight is advantageous as it provides an opportunity to detect large number of known and unknown parent ions available in an extract (Zhu et al., 2013; Mocco et al., 2016). We report here on the phytochemical analysis and LC-MS QTOF of crude extracts of C. molle and its associated bacterial endophytes.