Endophytic Fungi from Alstonia boonei De Wild and Greenwayodendron suaveolens (Engl. and Diels) Verdc. subsp. Suaveolens Possess Inhibitory Activity against Pneumonia Causing Bacteria

This study evaluated the antibacterial efficacy of methanolic extracts of isolated endophytic fungi from stem barks and leaves of Alstonia boonei De Wild and Greenwayodendron suaveolens (Engl. and Diels) Verdc. subsp. Suaveolens against Klebsiella pneumoniae ATCC 43816, Haemophilus influenzae ATCC 49247, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 35218, responsible for causing pneumonia. The endophytic fungi were isolated and characterized in the Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), and Czapek Dox Agar (CDA) media. The fungi and their methanolic extracts were tested for in vitro antibacterial potential by antagonistic assay for endophytic fungi against bacterial pathogens and microdilution method. The phytochemical screening of extracts was carried out according to the colorimetric and precipitation methods to reveal the presence of secondary metabolites. The results showed that 24 macroscopically and microscopically distinct endophytic fungi were isolated, identified, and stored. These endophytic fungi possessed antibacterial activity against the selected bacterial strains with inhibition zones ranging from 7.00 to 25.00 mm. The endophytic fungi GS15 and AB24 have presented the inhibitions zones of 20.33 mm and 25.00 mm, respectively, and these were better than the ones obtained for Levofloxacin®. The endophytes with inhibition zones greater than 10 mm were used for extraction of their secondary metabolites. The endophytic fungi extracts showed antibacterial activity with the minimum inhibitory concentrations (MICs) ranging from 6.25 × 10−4 to 2 × 10−2 g/L and the minimum bactericidal concentrations (MBCs) ranging from 2.5 × 10−3 to 2 × 10−2 g/L. The endophytic fungi GS15 extract was the most effective extract; it showed bactericidal effects on the tested bacterial strains. The phytochemical screening of the extracts revealed the presence of secondary metabolites classes, responsible for causing the obtained antibacterial activity. Thus, the endophytic fungi methanolic extracts from A. boonei and G. suaveolens have the potential to inhibit the growth of bacteria responsible for nosocomial pneumonia.


Introduction
Pneumonia is a lower respiratory tract infection caused by several infectious agents, including viruses, bacteria, and fungi [1]. However, pneumonia caused by bacterial infection has a rapidly progressive clinical course, which often becomes complicated by inflammation of the lungs, multilobular involvement, and lung abscesses [2,3]. As the third deadliest infectious disease worldwide after tuberculosis and hepatitis B, it is also the leading cause of infectious deaths in children worldwide, but it is most prevalent in South Asia and sub-Saharan Africa. It accounted for 922,136 and 808,694 deaths in children below the age of five years in 2015 and 2017, respectively, representing up to 15% of all deaths of children below five years of age [4]. Hence, the pneumonia causing bacteria represents the leading cause of infant morbidity and mortality recorded in low-income countries [5] accounting for 16.5% deaths among children aged 0 to 59 months in Cameroon [6,7]. In France, pneumonia is also responsible for 30% of deaths among adults over the age of 65 years. Appropriate antibiotic therapy involves Amoxicillin ® /Clavulanic ® acid and Levofloxacin ® for patients with pneumonia [8]. However, this treatment is relatively expensive in low-income countries; meanwhile, the pneumonia causing Gram-negative bacteria is responsible for microbial resistance [9]. erefore, this scenario necessitates exploration of the various possibilities for treatment of this disease [10]. Different therapeutic strategies have already been implemented for treatment of this disease; among which use of medicinal plants offers an inexhaustible source of drugs [11].
Alstonia boonei De Wild belongs to the Apocynaceae family. About 110 species of this genus grow alongside the American tropical region. Preparations using the leaves, seeds, stem barks, and roots of different plants from the Apocynaceae family have been largely used in traditional medicine and some plants of Alstonia genus have been widely used as a febrifuge to treat malaria and other skin problems. Alcoholic extracts of the stem barks of A. boonei (to a lesser degree of the leaf ) showed a broad-spectrum activity against both Gram-negative and Gram-positive bacteria, as well as fungi [12].
According to Lissambou et al., Greenwayodendron suaveolens (Engl. and Diels) belonging to the Annonaceae family has long been used as food and herbal medicine in Central Africa, especially as powdered supplement [13]. e genus Greenwayodendron is traditionally used for its antimalarial, antimenorrhagic, and antidysenteric properties [14]. is genus is a rich source of biologically active secondary metabolites with antimicrobial [15,16], analgesic [17], antimalarial [18], and anthelminthic [19] activities.
For centuries, plants have served as medicinal bioactive compounds source against many forms of disease. In contrast, during the last few years, microorganisms associated with plants rather than the plants themselves have been shown to offer products with high therapeutic potential [20]. All microorganisms (bacteria and fungi) that inhabit asymptomatically, at least for one period of their life cycle, the internal plant tissues beneath the epidermal cell layers may be considered as endophytes [21]. eir role is to improve plant's access to nutrients and produce special substances, mainly secondary metabolites and enzymes which are responsible for plant adaptation to abiotic stress [22]. During the long coevolution of endophytes and their host plants, endophytes have adapted themselves to their special microenvironments by genetic variation, including uptake of some plant DNA into their own genomes. is could have led to the ability of certain endophytes to biosynthesize some "phytochemicals" originally associated with the host plants [23]. Of the endophytic microorganisms, fungi have been isolated the most [24] as they have been proved to be the promising sources of biologically active products with antioxidant, immunosuppressive, antiproliferative, anti-inflammatory [23], and antibacterial activities [25]. Furthermore, considering the exponential evolution of antibiotic resistance in human pathogens in recent years, it has become inevitable to search for more efficacious antibiotics. us, the antimicrobial activities have been evaluated for a variety of metabolites biosynthesized by endophytic fungi [10,24] and there is an increasing effort to characterize, identify, and evaluate the potential biological effects of endophytic fungal extracts isolated from medicinal plants [21]. However, the endophytes of A. boonei and G. suaveolens have not yet been characterized. e present study evaluated the antibacterial potential of the methanolic extracts of endophytic fungi of stem barks and leaves of Alstonia boonei De Wild and stem barks of Greenwayodendron suaveolens (Engl. & Diels) Verdc. subsp. Suaveolens on the growth of several bacterial strains responsible for nosocomial pneumonia.

Isolation and Cultivation of Endophytic Fungi.
e plant samples were washed under running tap water, and sterilization of leaves surfaces was achieved by subsequently soaking them in a series of solutions as follows: sterile distilled water for 2 min, ethanol 70% for 2.5 min, sodium hypochlorite 2.4% for 4 min, and ethanol 70% for 1 min, and finally they were rinsed with sterile distilled water for 3 times [26,27]. ey were dried in sterile absorbent paper. e last washing water was plated on Petri dishes containing Potato Dextrose Agar (PDA). e success of surface sterilization method was confirmed by the absence of any microbial growth on the culture media plated with the last washing water. e sterilized leaves were cut into 5 mm segments using a sterilized knife. Ten parts of each plant segments were placed in Petri dishes (9 cm) containing PDA supplemented by 0.5% of chloramphenicol and incubated at 28 ± 2°C [26]. Regular observations were carried out from the second day onwards for a period of 3 to 4 weeks for fungal growth. e fungal growth from internal tissues was checked for purity and transferred to fresh culture slants and then stored at 4°C for further study [27].

Identification of Endophytic Fungi.
Identification of endophytic fungi associated with leaves or stem barks segments of plants was done based on their morphological and taxonomic properties (macroscopically and microscopically) on PDA, Sabouraud Dextrose Agar (SDA), and Czapek Dox Agar (CDA) media at 28 ± 2°C [28][29][30][31][32][33][34]. e macroscopic characteristics observed were color and surface colonies (granular, such as flour, mounting, slippery), texture, zonation, growth area, the lines of radial and concentric, reverse color, and exudate drops. e microscopic examinations of the vegetative thallus, fruiting bodies, and spores were carried out with methylene blue and Congo red reagents. e species identification was done according to the methods described earlier [35][36][37][38].

Extraction of Secondary Metabolites of Endophytic Fungi
2.4.1. Fermentation. Four endophytic fungi previously purified with inhibition zones higher than 10 mm on the tested bacteria were chosen for this purpose. us, some mycelial agar plugs from these endophytic fungi culture were cultivated into 500 mL culture flask containing 250 grams of sterile rice. e culture was incubated at still condition at room temperature under dark condition for 3 to 4 weeks [39].

Antagonistic Assay for Endophytic Fungi against
Bacterial Pathogens. Antagonistic activities of twenty-four endophytic fungi were tested for their antibacterial activity [40]. e test bacteria grown on liquid Mueller-Hinton medium for 24 h; the concentration was adjusted at 1 × 10 6 cells/mL [41]. e bacteria (100 μL) were inoculated on the Petri dishes containing solid Mueller-Hinton medium. Afterwards, the disks of agar (Ø 6 mm) of each endophytic fungus young strain (5 to 7 days) were inoculated and placed at equidistant. e plates remained incubated at 37 ± 2°C for 24 h. Levofloxacin ® (Sigma) (100 μg/mL in dimethyl sulfoxide (4%; v/v)) was employed as positive control. All the experiments were carried out using a completely randomized design (CRD), with three repetitions [42].
e presence of zone clearance on agar plates was used as an indicator for the antibacterial activity of endophytic fungi under investigation. e strains, which showed the maximum zone of clearance, were chosen for further study. e presence of these inhibition zones on agar plates was used as indicator of bioactive potential of endophytic fungi strains [43,44].

Microdilution Assay of Endophytic Fungi Extracts (1) Determination of Minimum Inhibitory Concentration (MIC).
e geometric serial broth microdilution method was carried out according to the Microplate Alamar Blue Assay (MABA) described previously by the Clinical and Laboratory Standards Institute. A stock solution was prepared by diluting the respective sample in dimethyl sulfoxide (4%; v/v). Stock solution was then added to Mueller-Hinton broth to reach final samples concentrations ranging from 2 × 10 − 2 g/L to 1.95 × 10 − 5 g/L. Bacterial inocula (1.5 × 10 8 CFU/mL) were added to each dilution. Each 96well microtiter plate was incubated at 37°C for 24 h. Positive control consisted of Levofloxacin ® at 2.5 × 10 − 4 g/L, negative control contained no drugs, and blank contained neither inoculum nor drug. e concentration of dimethyl sulfoxide in the assay was kept at 5% to ensure that its effect on bacterial growth can be maintained as minimal. Upon incubation periods, 40 μL of 0.02% resazurin was added to individual wells and the plates were reincubated for additional 30 min and checked for color change. Change in resazurin color from blue to pink indicated reduction of the indicator due to bacterial growth. e MIC was defined as the lowest concentration of samples at which the microorganisms did not demonstrate growth [45].
(2) Determination of Bactericidal Effect. e Minimal Bactericidal Concentrations (MBC) of promising methanolic extracts of endophytic fungi were assessed by subculturing MIC test microtiter plates on Mueller-Hinton medium. e MBC was considered as highest dilution or lowest concentration at which no growth occurred in the medium. All the experiments were done in triplicate [45].
Evidence-Based Complementary and Alternative Medicine e antibacterial effect was deemed bactericidal or bacteriostatic depending on the ratio of MBC to MIC. If MBC/MIC is lower than four, the antibacterial effect of endophytic fungi extracts is bactericidal and bacteriostatic when MBC/MIC was higher than four [46].

Phytochemical Screening of Endophytic Fungi Extracts.
Preliminary phytochemical screening of secondary metabolites of methanolic extracts of endophytic fungi was carried out according to methods described by Harborne and Evans [47,48].

Statistical Analysis.
All experiments for antagonistic assays were conducted in triplicate and values of inhibition zones expressed in mm as the mean ± SD. Variations in means were analyzed using one-way analysis of variance (ANOVA) and means were statistically significant if p < 0.05.

Isolation and Identification of Endophytic Fungi.
A total of 83 endophytic fungi were isolated from A. boonei and G. suaveolens and grouped into 24 fungi isolates. It was found that 16 fungi isolates were identified, belonging to four different genera: Aspergillus (12.5%), Fusarium (37.5%), Neoscytalidium (8.33%), and Acremonium (8.33%). ese genera belong to Deuteromycota division, more precisely to the Hyphomycetes class. ey were divided into hyaline hyphomycetes (Moniliaceae order) with Aspergillus spp., Fusarium spp., Acremonium spp., and dark hyphomycetes (Dematiaceae order) with the only genus Neoscytalidium spp. being identified. However, the remaining 08 endophytic fungi could not be identified due to their issues related infertility (Table 1).
Some morphological and microscopical characteristics of isolated endophytic fungi are represented in Figure 1.
Isolates of the genus Aspergillus appeared with bright colors, which made their identification easy. ree species of this genus were identified in this study. e mycelial colonies of the isolated species are either powdery with black color (Aspergillus sp. 1) and green in PDA and SDA media but orange-yellow in CDA medium (Aspergillus sp. 3) or downy with black color in PDA and white in CDA and SDA media (Aspergillus sp. 2). ese isolates were also characterized by a septate thallus and an unbranched conidiophore with varying lengths and shapes, ending in a bulge or vesicle bearing phialides or sometimes separated by metules, which is characteristic of the genus called Aspergillus head. e colonies of isolates of the Fusarium genus were downy or cottony with different colors ranging from pinkish white for Fusarium sp. 1 and 2 to brown in PDA for Fusarium sp. 3 with other colors such as pink or yellow for Fusarium spp. 4, 7, 8, and 9, especially in SDA and CDA media. Microscopy presented septate vegetative thallus with cluster or chain arrangement of microconidia, fusiform macroconidia, curved and quite pointed at the tips of the phialides, and the early presence of chlamydospores as seen on Fusarium sp. 2, justifying their belonging to the Fusarium genus. e colonies of isolates of the Acremonium genus were woolly in SDA and CDA and very poor in filaments or even hairless in PDA. Different colors were obtained like white and pink in SDA medium with a slow growth rate for Acremonium sp. 2 and a rapid growth rate for Acremonium sp. 1, as well as milkywhite and white in PDA and CDA media. Microscopy presented a vegetative thallus made up of septate filaments, fine, cylindrical phialides at their tips, and cylindrical conidia grouped together in clusters. e colonies produced by the genus Neoscytalidium were milky-white fluffy in PDA and CDA media or woolly beige for Neoscytalidium sp. 2 and hairless brown for Neoscytalidium sp. 1 in SDA media. Microscopically, the hyphae are septate and hyaline; others are larger and strongly pigmented dissociating into arthroconidia. However, fungi isolates such as 17, 18, and 19 were unidentified as they did not produce sporing structures on PDA, SDA, and CDA media but presented dirty white cotton growth that darkened with age differently in each isolate. Microscopy presented septate and dark hyphae in fungi 17 and 18 differently from fungus 19, which presented septate and hyaline hyphae.

Antagonistic Potential of Endophytic Fungi.
In this investigation, four endophytic fungi (GS 15 , AB 24 , AB 38 , and AB 83 ) were selected for determination of inhibition parameters (MIC and MBC) after testing the 24 fungi isolates  38 , and AB 83 ) presented an antibacterial activity, with the inhibition zones ranging from 10.33 ± 0.57 mm against E. coli to 25.00 ± 1.00 mm against H. influenza. is was the most sensitive bacteria compared to those selected endophytic fungi with inhibition zone ranging from 20.33 ± 0.57 mm for AB 24 to 25.00 ± 1.00 mm for GS 15 ; the data obtained was compared to Levofloxacin ® with an inhibition zone of 19.05 ± 0.82 mm as in Table 2.

Phytochemical
Screening. e extracts of the selected endophytic fungi (GS 15 , AB 24 , AB 38 , and AB 83 ) have been used for several phytochemical tests to determine the presence of different types of bioactive compounds in them. e results presented in Table 4 indicated the presence of 10 secondary metabolites families in the methanolic extracts: alkaloids, phenols, polyphenols, tannins, saponins, flavonoids, anthocyanins, coumarins, terpenoids, and sterols. Unlike other classes of secondary metabolites that were ubiquitous to all four methanolic extracts, tannins were found exclusively in AB 38 methanolic extract of Neoscytalidium sp. 2.
e obtained results are similar to those of Abdel-Motaal et al. [49] who isolated endophytic fungi of Neoscytalidium, Aspergillus, Acremonium, and Fusarium genera from Hyoscyamus muticus, an Egyptian plant belonging to the Apocynaceae family same as A. boonei. In contrast, the study of Tolulope et al. [50] reported that the isolation of A. boonei endophytic fungi harvested in Nigeria contained three characterizing genera (Aspergillus, Microphoma, and Trichoderma).
ese results could therefore justify the hypothesis that endophytic fungi isolated from plants belonging to the same genus or family belong to the same genera. is is the case of A. scholaris and Hyoscyamus muticus according to the studies of Mahapatra and Banerjee and Abdel-Motaal et al., respectively, which indicated the presence of 19 genera with four similar genera (Neoscytalidium, Aspergillus, Acremonium, and Fusarium) in agreement with our study [49,51]. We noticed that the colonizing endophytic fungi from A. boonei and G. suaveolens in our study have low genera diversities.
In addition, the genus Fusarium was the predominant genus isolated from A. boonei and G. suaveolens with a frequency of 37.5%. Indeed, it is one of the genera most associated with higher plants. Fusarium is among the most common fungi in terrestrial ecosystems. ey are found in cultivated soils in temperate and tropical regions [37], and their predominance among the fungi isolated in this study corroborates with the study of Ilyas et al. [52] who explained that several fungi of the genus Fusarium were plant colonizers. In addition, A. boonei was harvested in tropical areas, where soils are favorable for the growth of species of this genus.

Antagonistic Potential of Endophytic Fungi.
e antagonistic potential of endophytic fungi (GS 15 , AB 24 , AB 38 , and AB 83 ) allowed obtaining clearance zones on agar plates against H. influenzae, P. aeruginosa, K. pneumoniae, and E. coli which are greater than 10.33 mm on the bacterial strains tested. e present inhibition justifies the antibacterial activity of these endophytic fungi strains on the bacteria responsible for pneumonia, which could be due to the presence of bioactive substances in the mycelial discs of these endophytic fungi [53]. However, other studies have shown that endophytic fungi isolated from plants could have antibacterial activity, so they would resist invasion and inhibit the bacteria inducing the diseases in humans through the production of inhibitory substances belonging to several structural classes like alkaloids, peptides, steroids, terpenoids, phenols, quinines, and flavonoids [24,54,55]. Our results corroborate with an earlier report in which the endophytic fungi studied belonged to Aspergillus, Acremonium, and Trichosporon genera isolated from Artemisia absinthium. ese endophytic isolates showed antibacterial activity against P. aeruginosa ATCC27853 and E. coli ATCC25922 strains [56]. Although the tested strains responsible for pneumonia are naturally resistant due to their "Gram-negative" character, the obtained results show that the bioactive compounds secreted by these endophytic fungi would have the capacity to penetrate the external membranes surrounding the wall of Gram-negative bacteria made up of covering lipopolysaccharides by the phenomena of diffusion of lipophilic compounds. Another phenomenon, which could also explain the results obtained, is the diffusion of active substances on the agar, which depends on the nature of the diffusing substances excreted by the endophytic fungi, their concentrations, their solubility, and pH of the culture medium [57]. e high antibacterial activity revealed by endophytic fungi isolated from A. boonei and G. suaveolens is related to their ability to produce bioactive substances with medicinal properties [58]. Evidence-Based Complementary and Alternative Medicine

Antibacterial Activity of Endophytic Fungi
Extracts. e determination of the inhibition parameters (MIC and MBC) of methanolic extracts is shown by their MICs values of 6.25 × 10 − 4 to 2 × 10 − 2 g/L for H. influenzae, P. aeruginosa, E. coli, and K. pneumonia strains. e highest antibacterial activity was obtained with the methanolic extract of Aspergillus sp. 1 that presented a bactericidal effect on all tested strains. is could be linked to a synergistic or additive effect of the secondary metabolites present in the methanolic extract. In addition, the mean bactericidal effect of this extract on the strains tested is in line with data reported by Amadi and Adeniyi, as well as those reported by Indira et al. [59,60] who demonstrated the antimicrobial activity of the genus Aspergillus. e reduction mechanisms of bacteria or fungi growth of the Aspergillus genus (Aspergillus terreus, Aspergillus flavus, and Aspergillus fumigatus) endophytes may possess higher concentration of alkaloids (fumiclavin A and B and, fumigaclavin B). In addition, most of these complex structures of fungi metabolites inhibit cell division and glucose transport [59,61]. e data obtained in the present study were different from those of Kalhouche and Meziane [62] showing the MIC value of Aspergillus sp. 1 extract against E. coli to be 100 mg/mL. is difference may be because the Aspergillus strain used to inhibit the E. coli growth was not isolated from a medicinal plant. Hence, the production of secondary metabolites may not have been directed to any phytoprotective activity of plants. e methanolic extracts of AB 24 , AB 38 , and AB 83 exhibited MICs ranging from 10 − 2 to 2 × 10 − 2 g/L. Indeed, the phytochemical screening of these extracts revealed the presence of phenols, flavonoids, and coumarins whose antibacterial effect could have been inhibited or neutralized by other compounds present in these extracts at higher concentrations [63]. ese bioactive secondary metabolites would have the ability to penetrate the lipopolysaccharides of the wall of the Gramnegative bacteria tested and inhibit their growth by mechanisms of action that allowed them to inactivate bacterial enzymes, modify the structure of the active site, and induce intracellular accumulation of bioactive metabolites [64]. e bioactive compounds found in the methanolic extracts of endophytic fungi of A. boonei and G. suaveolens are responsible for the antibacterial activity and may have a synergistic action [65].

Phytochemical Screening of Endophytic Fungi Extracts.
e phytochemical screening of the methanolic extracts of endophytic fungi isolated from A. boonei and G. suaveolens has shown that all the extracts contain alkaloids, polyphenols, flavonoids, terpenes, and sterols. e presence of these secondary metabolites in the endophytic fungi could justify their antibacterial effect. Previous studies have shown that phytochemical compositions of A. boonei and G. suaveolens stem barks extracts were similar to those in the present study with the presence of alkaloids, phenols, polyphenols, flavonoids, anthocyanins, saponins, terpenoids, and sterols [66,67]. ese studies permitted highlighting the phytomolecular mimicry of endophytic fungi extract from A. boonei and G. suaveolens. It appears that the phytochemical compositions of the endophytic fungi methanolic extracts of these plants would be similar to those of plant extracts [66,67]. erefore, to verify this hypothesis, it would be necessary in a further study to make additional quantitative and qualitative analysis, especially molecular and genetic levels between endophytic fungi and the host plant.

Conclusion
In this study, the endophytic fungi from A. boonei and G. suaveolens exhibited antibacterial activity against some of the human pathogenic bacteria responsible for nosocomial pneumonia.
is activity was greater in the methanolic extract of GS 15 endophytic fungal isolated from G. suaveolens, a plant that has never been listed for its antipneumonic potential. e present study helped to valorize the use of endophytic fungi from A. boonei and G. suaveolens in drug discovery process. However, a molecular identification of isolated endophytic fungi from these plants is necessary for further investigations.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.