Evaluating antibacterial and anticancer activity of crude extracts of bacterial endophytes from Crinum macowanii Baker bulbs

Abstract The results from this study revealed that crude extracts isolated from bacterial endophytes obtained from Crinum macowanii bulbs showed activity against both Gram‐positive and Gram‐negative pathogenic bacteria, while Acinetobacter guillouiae crude extracts displayed anticancer activity. This study aimed to isolate and characterize bacterial endophytes and their crude extracts from C. macowanii bulbs. Endophytes were isolated using validated surface sterilization techniques, followed by phenotypic and genotypic profiles of the isolates. Crude extracts were extracted from the endophytes using ethyl acetate, while methanol:dichloromethane (1:1) was used to obtain crude extracts from the bulbs. Antibacterial activity of crude extract from each endophyte was investigated against selected pathogenic strains using the broth microdilution method, and anticancer activity against U87MG glioblastoma and A549 lung carcinoma cells was determined by the MTS (3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxy‐phenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium) assay. Acinetobacter guillouiae, Pseudomonas moraviensis, Pseudomonas sp., Rahnella aquatilis, Bacillus cereus, Novosphingobium sp., Raoultella ornithinolytica, and Burkholderia tropica were successfully isolated. The crude extracts from the majority of endophytes showed antibacterial activity, ranging from 0.125 to >16.00 mg/ml against Gram‐negative and Gram‐positive pathogenic bacteria. Acinetobacter guillouiae extracts showed a high bioactive potential against U87MG glioblastoma cell lines by reducing their growth by 50% at concentrations of 12.5, 6.25, and 3.13 µg/ml. Crude extracts isolated from C. macowanii bulbs showed potential for possible drug lead against common pathogenic bacteria.


| INTRODUC TI ON
Crinum macowanii Baker, from the Amaryllidaceae family, is a bulbous plant which has been used traditionally to treat a number of different ailments (Watt & Breyer-Brandwijk, 1962). The bulb is used to treat itchy rashes, boils, acne, backache, venereal disease, inflamed sores, swelling of the body, urinary tract problems as well as to increase lactation in women and cows (Maroyi, 2016). Maroyi (2016) reported that C. macowanii has antimicrobial, antifungal, antiviral, and antiplasmodial properties. Although there are a number of studies validating the antimicrobial properties of Crinum species (Maroyi, 2016), overharvesting and overuse of medicinal plants such as C. macowanii has led to overexploitation and extinction of some of these plants (Wyk & Prinsloo, 2018). Strategies need to be deployed to find alternative methods of extracting secondary metabolites from these plants, such as using microbial sources (Chen et al., 2016;Monakisi, Esler, & Ward, 2007). An extensive literature search suggests that C. macowanii remains unexplored with regard to both its endophytes and the bioactivities of its crude extracts, including crude extracts derived from the bulb of this plant.
Endophytes are considered to be outstanding sources of bioactive natural compounds, as they can mimic the chemistry of their respective host plants and biosynthesize almost identical bioactive natural products, or even derivatives which can be more bioactive than those of their respective hosts (Rodriguez, White, Arnold, & Redman, 2009). As such, there has been an increase in the bioprospecting of novel efficacious bioactive compounds from microorganisms such as endophytes, to obtain novel bioactive products (Kusari & Spiteller, 2011;Martinez-klimova, Rodríguez-peña, & Sánchez, 2017). Bioactive secondary metabolites obtained in this way could be alternative sources of therapeutic compounds which could help eradicate problematic infections affecting the human population, for example, antibiotic resistance (Menpara & Chanda, 2013;Tidke et al., 2017).
While fungal endophytes are a source of attention in most studies, bacterial endophytes are less explored due to the small yield recoveries of crude extracts (Brader et al., 2014). Ek-Ramos et al. (2019) has reported that metabolites produced by endophytic microorganisms' act as antimicrobial and anticancer agents against human, animal, and plant pathogens and display significant potential in medical and veterinary treatments. With this in mind, the main aim of this study was to isolate and identify bacterial endophytes from C. macowanii bulbs and to explore the role of endophytic crude extracts as potential antibacterial and anticancer therapeutic agents.

| Sample collection
Fresh, healthy C. macowanii bulbs showing no apparent symptoms of disease or herbivore damage were collected from the Walter Sisulu National Botanical Garden (Roodepoort, Gauteng, South Africa, 26°05′10.4″S 27°50′41.5″E). After collection, the samples were placed in sterile polyethylene bags and transferred to the laboratory at 4°C before being thoroughly washed with sterile distilled water and used within hours of harvesting.

| Isolation of endophytic bacteria
The bulbs were surface sterilized separately using the method described by Jasim, Joseph, John, Mathew, and Radhakrishnan (2014) with slight modifications. Briefly, each bulb (approximately 10 g) was treated with 5% Tween-20 (Sigma-Aldrich) (enough to cover the plant material) and vigorously shaken for 5 min. Tween-20 was removed by rinsing several times with sterile distilled water, followed by disinfection with 50 ml of 70% ethanol for 1 min. Traces of the ethanol were removed by rinsing with sterile distilled water 5 times.
The sample was then treated with 1% sodium hypochlorite (NaClO) for 10 min and again rinsed five times with sterile distilled water.
The last rinse was used as a control, and 100 µl of this was plated on potato dextrose agar (PDA; HiMedia) and nutrient agar (NA; Oxoid).
The sample was then macerated in sterilized phosphate-buffered saline (PBS), with the outer surface trimmed out. The macerated sample was serially diluted up to 10 -3 dilution, and each dilution inoculated (using a spread plate method) in triplicate on nutrient agar. The NA plates were incubated at 30°C (IncoTherm, Labotec).
Growth was monitored periodically for 5 days. Effectiveness of the sterilization was monitored on the wash control plate, with growth indicating poor sterilization. Under such circumstances, the plates for the plant part were discarded and the sterilization repeated.
Distinct colonies were selected and subcultured on nutrient agar to obtain pure isolates. Pure bacterial isolates were preserved in 50% glycerol in a ratio of 500 µl glycerol:500 µl overnight broth culture and kept at −80°C.

| Gram staining
Pure colonies were subjected to Gram staining to establish morphological characteristics such as shape and Gram stain reaction. Gram stain slides were observed using a compound bright-field microscope (OLYMPUS CH20BIMF200) with 1,000× magnification.

| Phylogenetic analysis
The obtained sequences were screened for chimeras using DECIPHER23 and subjected to BLAST analysis using the National Center for Biotechnology Information (NCBI) database against the 16S rDNA sequence database (bacteria and archaea) to identify the closest bacterial species. Bacterial species with 98%-100% similarities were selected for phylogenetic analysis. Alignments of nucleotide sequences were performed using MUSCLE with default options.
The positions containing gaps or missing nucleotide data were eliminated. Phylogenetic trees were constructed using a neighbor-joining (NJ) method (Saitou & Nei, 1987) based on the Tamura-Nei model (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013). A total of 1,000 replications were used for bootstrap testing. All branches with greater than 50% bootstraps were considered to be significant (Soltis & Soltis, 2003). All evolutionary analyses were conducted in MEGA 7.0 (Kumar, Stecher, & Tamura, 2016). The 16S rRNA gene sequences of bacterial isolates identified in the study were deposited in GenBank (https ://www.ncbi.nlm.nih.gov/genba nk/) with the accession numbers as stated in Table 1. The assigned names of the bacterial isolates were based on the BLAST homology percentages as well as the phylogenetic results.

| Extraction of crude extracts from C. macowanii bulbs
Crinum macowanii bulbs were washed, chopped into small pieces, and air-dried at room temperature. The dried plant material was blended into a fine powder using a commercial blender. Crude extracts were obtained according to Yadav and Agarwala (2011).
Briefly, 150 g of the prepared plant material was mixed with 2 L of a 50:50 methanol:dichloromethane solution. This was allowed to shake for 3 days on a platform shaker (Amerex Gyromax) at 200 rcf.
The solution was filtered through Whatman No. 1 filter paper, and the filtrate was evaporated on a rotatory evaporator and allowed to air-dry in a desiccator.

| Extraction of crude extracts from bacterial endophytes
For each endophytic bacterium listed in Table 1, 2 L of broth was measured into a 4-L Erlenmeyer flask leaving room for aeration and autoclaved at 121°C for 15 min. Each 4-L flask was inoculated with one of the endophytic bacterium as listed in Table 1  After 7 days of cultivation, sterile XAD-7-HP resin (20 g/L; Sigma, BCBR6696V) was added to the culture for 2 hr, shaking at 200 rcf.
The resin was filtered through cheesecloth and washed three times with 300 ml of acetone for each wash. The acetone-soluble fraction was concentrated using a rotary evaporator, and a dark yellowish viscous extract was obtained, which was transferred into a measuring cylinder. Depending on the volume, ethyl acetate was added in a ratio of 1:1 (v/v). The mixture was vigorously shaken for about 10 min, decanted into a separating funnel, allowed to separate and each phase collected in a conical flask. This process was repeated until the dark yellowish viscous liquid obtained after removing the TA B L E 1 The identities and morphological characteristics of the isolated bacterial endophytes from Crinum acowanii bulbs acetone became a very light-yellow liquid. The ethyl acetate fraction was evaporated using a rotary evaporator, and the brown extract obtained was stored in an amber bottle in a cool dry place until analysis was done. The light-yellow liquid was evaporated, and no reasonable extract or further analysis was done on this substance (Maloney et al., 2009). The brown crude secondary metabolite extracts were used for antibacterial and anticancer assays.  (2001) and Sebola, Ndinteh, Niemann, and Mavumengwana (2016). The antibiotic streptomycin was used as the positive control and was prepared by weighing 0.032 mg in 1 ml of sterile distilled water while 0.1% DMSO was used as a negative control.

| Sample preparation
The crude bulb extract and crude endophytic extracts were weighed separately into empty autoclaved McCartney bottles to ensure sterility. A minimal amount of dimethyl sulfoxide (DMSO; 0.1%) was used to dissolve the crude extracts, and Mueller-Hinton (MH) broth was added to bring the volume of the dissolved crude extract to a concentration of 32 mg/ml as the stock solution.

| Microtiter plate assay
Serial dilutions were carried out using the MH broth from 16 mg/ ml down to 0.031 mg/ml, which was the lowest inhibition observed.
The experiment was carried out in five repeats using a 96-well microtiter plate. The outer wells of the plate were filled with sterile distilled water (sdH 2 O). The inoculum (100 µl) was added into each well that did not contain the sdH 2 O. The diluted crude extract samples (100 µl) were added in five wells horizontally and the concentrations decreased in vertical order from 16 mg/ml down to 0.031 mg/ml.
The plates were covered and incubated overnight at 37°C. After incubation, 10 µl of 0.02% (w/v) resazurin sodium salt dye solution was added to the wells and the resulting solution incubated for another 2 hr. On reduction, resazurin changes color from blue to pink to clear as oxygen becomes limited within the medium, indicating metabolism and the viability of bacterial cells, as well as no effect of the crude extracts on the bacteria. Any well with a known concentration showing a slight color change was used as MIC. The wells were visually inspected for color changes.  (2013) and Artun et al. (2016). Briefly, dilutions were carried out using growth media from 100 µg/ml to 3.13 µg/ml. MTS where E a is absorbance of the extract, B a is absorbance of the blank, and C a is the absorbance of the control (Handayani et al., 2018). The positive control used for all conducted tests was auranofin, as it is able to inhibit thioredoxin reductase as well as the ubiquitin-proteasome system (UPS) by targeting proteasome-associated deubiquitinase, thus inducing lung cancer cell apoptosis by selenocystine (Coussens et al., 2017;Fan et al., 2014;Roder & Thomson, 2015).

| Isolation of endophytic bacteria
From the C. macowanii bulbs, a total of eleven endophytic bacteria were isolated. As the control plates did not reveal any bacterial growth, it was concluded that the isolates reported were endophytes to the plant under study. Table 1 shows the sample code, assigned bacterial name, accession number as given by GenBank, the similarity percentage between the sample isolate, the Gram stain reaction and colony morphology (color, shape, elevation, and margin) as observed on an agar plate for each endophyte.

| Phylogenetic analysis
The BLAST search of the 16S rRNA gene sequences resulted in varying bacterial genera; the isolates were classified as seven genera, namely Acinetobacter, Pseudomonas, Rahnella, Bacillus, Novosphingobium, Raoultella, and Burkholderia ( Figure 1).

| Antibacterial evaluation of Crinum macowanii bulb and bacterial endophyte crude extracts
The lowest MIC (0.125 mg/ml) was observed mostly from

| Anticancer assays
Endophytic crude extracts showed varying activities against A549 lung carcinoma cells. However, T2 crude extracts showed a 62% reduction of lung carcinoma cells at a concentration of 25 µg/ml (Figure 2).
Acinetobacter guillouiae extract T3 also showed promising activity when tested against brain cancer cell lines, with the highest concentration, (100 µg/ml), showing minimal activity while at 6.25 µg/ ml concentration displayed a 50% reduction in the cell viability of brain cancer, compared to the other extracts (excluding extract T6; Figure 3).

| D ISCUSS I ON
Endophytic bacteria are the most unexplored yet diverse group of microorganisms with a symbiotic association with plants, and are promising sources of biologically active agents (Raghu, 2012 Sebola et al. (2016). Inhibition greater than 16 mg/ml, observed in K. pneumonia, P. vulgaris, and P. aeruginosa, was not considered to be inhibitory. The inhibition of other bacteria by C. macowanii bulb crude extracts could be due to the relationship between the plant and its endophytes, which appears to confer certain benefits such as increased resistance to disease and induced growth (Rodriguez et al., 2009).
Endophytic bacteria have been reported to produce a number of secondary metabolites such as alkaloids, steroids, terpenoids, peptides, and flavonoids with antibacterial, antifungal, and cytotoxic properties (Raghu, 2012).  properties (Zonyane, Makunga, & Vuuren, 2013). Crude extracts from B. cereus showed MIC values of between 1 and 16 mg/ml, inhibiting K. pneumonia at 1 mg/ml. Crude extract from B. cereus has been reported to possess antibacterial activity against a wide range of pathogenic microbes such as E. coli and K. pneumoniae (Kumar, Thippeswamy, & Shivakumar, 2013). This supports the findings in this study.  (Ho & Huanga, 2015). It was observed in this study that the Gram-positive bacterial species were more susceptible to the antibacterial compounds in the crude extracts than the Gram-negative bacteria. This could be attributed to the difference in the cell walls of both groups of bacteria, as Gram-negative bacteria are known to be resistant to most antibiotics due to their outer membrane, which tends to expel antibiotics from the cells by acting as a selective barrier (in contrast to that of their Gram-positive counterparts) (Delcour, 2009;Iannello et al., 2014). Greenwell and Rahman (2015), Seca and Pinto (2018) et al. (2006) where the researchers reported that after a low dose of chemotherapy, tumor tissue has a propensity to regrow, causing tumor repopulation. Hutf and Grady (1996) stated that anticancer drugs used for lung carcinoma cells have concentration-effect relationships and this could explain our results.
A puzzling finding was the increased cell viability in the UMG87 glioblastoma cell in response to the R. aquatilis crude extract.
However, UMG87 glioblastoma cell lines are known to undergo hypoxia, resulting in metabolic abnormalities such as increased uptake of glucose and acid resistance; this increased glucose uptake and high aerobic glycolysis induces proliferation of cancer cells (Jiang, 2017;Zhou et al., 2011). This would explain the high cell viability of 100% and above on the glioblastoma cell lines. Further studies are needed to elucidate this phenomenon. The activities observed from the methanol/dichloromethane crude plant extracts could be caused by artifacts as Sauerschnig, Doppler, Bueschl, and Schuhmacher (2018) mentioned that artifacts are generated by methanol during sample extraction and storage.

| CON CLUS ION
A diverse microbial community was isolated from C. macowanii bulbs, with notable inhibitory activities against Gram-positive and Gram-negative bacterial species. From these results, it can be concluded that endophytic-derived crude extracts isolated from medicinal plant C. macowanii bulbs produce potential bioactive compounds which should be explored further for their biological activities. Sisulu Botanical Garden allowed for sampling of the plant material.

CO N FLI C T O F I NTE R E S T S
None declared.