Effect of endophytic fungi Daldinia eschscholtzii against multidrug resistant pathogens
Abstract
This study aimed to explore the in vitro antibacterial potential of endophytic fungus isolated from the roots of Polianthes tuberosa against multidrug-resistant pathogens. Fungal isolates were screened for their antibacterial activities by disc diffusion, agar plug diffusion, MIC, and MBC method. GC-MS was carried out to determine the crude extract's chemical profile. The highest antagonistic effect in the disc diffusion method was seen in the ethyl acetate extract of PTR3, with an inhibitory zone ranging from 14-18 mm. Therefore, the potent isolate was identified as Daldinia eschscholtzii. The maximum inhibition of 18 mm and 20 mm was observed against MRSA (ATCC 43300) (ATCC 700699), followed by VRE (14 mm). The MIC and MBC were between 3.12-25 μg/mL and 6.25-50 μg/mL, respectively. 1,2-Benzene dicarboxylic acid and diheptyl ester were the primary chemical constituents present in the extract. These findings confirm that D. eschscholtzii PTR3 crude extract shows antibacterial activity.
Introduction
Natural products are most likely to afford a wide range of difficult-to-synthesize structures, making them the prospective sources of novel antibiotic classes (Bate et al., 2020).
Fungi are the most often isolated endophytes. Opportunities exist for retrieving unknown fungi, as most of the plants endophytes have not yet been studied (Strobel and Daisy, 2003). Endophytic fungi are well-known biological agents that live inside the plant's internal tissues and cause no harm to their hosts (Khan et al., 2021). Endophytes have a mutualistic relationship with their host plants by receiving protection and nutrition from a host and providing increased resistance to pathogens, herbivores, and abiotic stresses. Depending on the growth phase of the fungus and host, host defense response and environmental factors, some endophytic fungi can become plant pathogens (Hema et al., 2015). They are the reservoir of various phytochemicals like alkaloids, quinones, terpenoids, tannins, steroids, phenolic acids, and saponins (Verma et al., 2022). Endophytes-derived secondary metabolites can inhibit the mechanism of resistance by incapacitating pathogenic invasion (Manganyi and Ateba, 2020). Thus, the compounds derived from endophytes exhibit a broad range of activities in human diseases, including immunomodulatory, anti-diabetic, antifungal, antioxidant and anti-cancer (Verma et al., 2022). The bioactive metabolites produced by the fungal endophytes were divided into four groups, including terpenes, alkaloids, polyketides and non-ribosomal peptides based on their biosynthetic pathway and chemical structures (Khan et al., 2021). In ethnobotanical aspects of plant protection, conservation and sustainable use, researchers have been interested in isolating microbes associated with plants. Endophytic fungi meet this demand and serve as the best substitute source of compounds with a range of bioactivity (Verma et al., 2022).
The ethyl acetate extracts obtained from Pestalotiopsis sp. Eef-9, Chaetomium sp. Eef-10 and Lophiostoma sp. Eef-7 of Eucalyptus exserta exhibited potent antibacterial activitiy against some pathogenic bacteria (Ababutain et al., 2021). Cyperaceae plants harbor numerous endophytes that produce antibacterial metabolites active against both Gram positive and, to a lesser extent, Gram negative bacteria (Ratnaweera et al., 2018). There is report of antimicrobial effects of endophytic fungi from E. chrysantha. (Zhang et al., 2015).
Due to the increased demand for alternative therapy for infectious diseases, the present research focused on isolating and identifying endophytic fungi from the Polianthes tuberosa plant. It belongs to the family of Amaryllidaceae and is an ornamental plant (Sundar and Arunachalam, 2022). The antibacterial activities, including Kirby Bauer disc diffusion, minimum inhibitory and bactericidal concentration and time-kill study of the endophytic fungal extracts against Gram-positive resistant pathogens, were investigated.
Materials and Methods
Isolation and identification of endophytes
P. tuberosa plant materials were collected and bought in a polythene bag to the laboratory from the Vellore District. The collected samples were washed thoroughly in running tap water to remove the surface adhere dust or soil particles. Surface sterilization was processed under the laminar hood using ethanol (70%) for 30 sec, followed by 3-5 min washing in 1% sodium hypochlorite solution. Later, washed two to three times under sterilized distilled water. The segment was placed aseptically on a potato dextrose agar medium and incubated for a week at 27 ± 2°C. Mycelial tips were subcultured on potato dextrose agar to produce pure colonies and stored for further analysis. Fungal isolates were morphologically identified by colony characteristics such as hyphal features, reproductive structures and spores arrangement. Lacto phenol cotton blue staining was carried out for microscopic identification. 18S rRNA ITS molecular sequencing identification was carried out for the molecular identification of the fungus (Roopa et al., 2015).
Mycelial growth rate
The isolated endophytic fungus was cultured and incubated at 27 ± 2°C. After 7 days, the mycelial colony diameter of all three fungi was measured every 3 days to determine the growth characteristics of the fungal isolates (Techaoei et al., 2020).
Preparation of biomass filtrate of active fungus
Fungal endophytes were grown in potato dextrose agar (500 mL) in a 1L Erlenmeyer flask at 27 ± 2°C for 21 days. The fermented broth of each isolate was thoroughly blended with solvents of different polarities, such as hexane, dichloromethane, ethyl acetate and n-butanol, sequentially in equal volumes. The obtained crudes were dried and the resultant dissolved in dimethyl sulfoxide to prepare a stock solution of 1 mg/mL and used for antibacterial studies (Palanichamy et al., 2018; Elghaffar et al., 2022).
Kirby Bauer disc diffusion assay
The endophytic fungal isolates that exhibited a broader antibacterial spectrum in the initial screening were taken further for secondary investigation by following the disc diffusion protocol. The test bacteria were uniformly seeded on the surface of Mueller-Hinton agar. About 6 mm sterile discs loaded with different extract concentrations (25, 50, 75, 100 µg/mL) prepared from the stock solution were placed on the medium. Inoculated plates were incubated overnight at 37°C and measured for inhibition zone to evaluate their antibacterial action. Dimethyl sulfoxide (5%) and oxacillin (1 µg) were included in the plates as a negative and positive control (Katoch et al., 2017; Marcellano et al., 2017). In addition, the activity index for each extract was calculated (Singh and Kumar, 2012).
Activity index = Inhibition zone of the sample / Inhibition zone of the standard
Box I: Agar Plug Diffusion Assay
The agar plug diffusion method was primarily used to determine the antibacterial activity.
Requirements
Muller Hinton agar, Potato dextrose agar, Tryptic soy broth, Brain heart infusion broth, Test pathogens (methicillin-resistant Staphylococcus aureus-MRSA (ATCC 43300), (ATCC 700699), Vancomycin-resistant Enterococci-VRE (ATCC 51299), S. aureus (ATCC 25923), S. aureus (MTCC 3160) and Enterococcus faecalis
Procedure
Step 1: The isolated fungal colonies were grown on the PDA medium at 27 ± 2°C for 7 days
Step 2: From the actively growing culture, 1 cm (diameter) and 4 mm (thickness) agar plugs were bored and transferred onto the MHA medium spread with the test microbes
Step 3: The plates were sealed and initially kept overnight at 4°C to allow the diffusion of bioactive compounds
Step 4: Post incubation, the plates were incubated at 37°C for 12 hours. The clear zone around the agar plugs was measured
Reference
Taufiq and Darah, 2018
References (Video)
Rattanasuk et al., 2021; Semerci et al., 2020; Krishnan et al., 2019
Minimum inhibitory concentration
The two-fold micro broth dilution method recommended by CLSI was used to determine the minimum inhibitory concentration (MIC) of ethyl acetate crude extracts that displayed the highest antagonistic activity. First, 50 to 0.39 µg/mL extract concentration was prepared from the stock solution. Then, Muller Hinton broth (100 µL) and extract (different dilution) were added to the 96-well microtiter plate. About 10 µL of bacterial inoculum was added and the setup was incubated at 37°C overnight. The lowest concentration was recorded with no visible bacterial growth (Ohikhena et al., 2017).
Minimum bactericidal concentration
The different turbidity sample (6.25 and 3.12 μg/mL concentration) from the MIC microtiter plate was used to determine the minimum bactericidal concentration (MBC) value. The samples were streaked on the Mueller-Hinton agar medium and incubated at 37°C for 18 hours. Post incubation, the lowest concentration showing no visible growth of bacteria was noted as the MBC of the extract (Ohikhena et al., 2017).
GC-MS analysis
Ethyl acetate derived from PTR3 containing different metabolites was subjected to GC-MS analysis. The study was carried out in Perkin Elmer Clarus 680 equipped with Clarus 600 mass spectrometer that was fitted with 30 m, 0.25 mm ID, 250 µm df Elite-5Ms capillary column. Initially, the GC oven was maintained for 3 min at 55º, ramp 10°C/min - 300°C for 6 min. Helium is used as a carrier with a flow rate of 1 mL/min. The mass transfer line and source temperature are set at 240°C. Turbo mass version 5.4.2 is used for spectral studies. The obtained unknown spectrum was compared with known spectrum components stored in the NIST library 2008 for structure identification (Liu et al., 2021).
Results
Isolation and antibacterial screening of endophytic fungi
Three endophytic fungi were isolated from the root part and coded PTR1, PTR2 and PTR3 (Figure 1). Each of these isolates showed different morphological and microscopic features. Then the isolates were subjected to antibacterial potential against the selected resistant pathogens. The isolated PTR3 had a remarkable inhibitory ability against the pathogens developing a clear zone in primary screening. Further, the crude extracts obtained from PTR3 were tested by the agar disc diffusion method.
Figure 1: Endophytic fungal colony characteristics in PDA plates PTR1 (A), PTR2 (B), PTR3 (C)
Table I shows the antibacterial activities exhibited by the active fungal crude extracts against the tested bacteria at 100 µg/disc concentration. All four solvent crude extracts prepared from the potent isolate demonstrated promising antagonistic effects. Of them, ethyl acetate extract exhibited potent inhibition against the test pathogens.
Furthermore, it revealed the highest inhibition zone (IZ) of 20 mm with activity index (AI) 3.3 and (IZ 18 mm; AI 3) against MRSA (ATCC 700699) and (ATCC 43300), which was 2 to 3 times higher than the positive control Linezolid. Moreover, for VRE (IZ 14 mm; AI 1.4) zone was recorded (Table I). Thus, it indicates the isolate was a promising source for a novel antibiotic agent.
Table I: Average inhibition zone and activity index of PTR3 ethyl acetate extract (100 µg/mL)
| Pathogens | Average inhibition zone (mm) | |||||||
|---|---|---|---|---|---|---|---|---|
| Hexane | Dichloromethane | Ethyl acetate | n-Butanol | |||||
| ZOI | AI | ZOI | AI | ZOI | AI | ZOI | AI | |
| MRSA (ATCC 43300) | 6.5 | 1.1 | 15 | 2.5 | 18 | 3 | 10 | 1.7 |
| MRSA (ATCC 700699) | - | - | 12.5 | 2.1 | 20 | 3.3 | 6.8 | 1.1 |
| S. aureus (ATCC 25923) | 13 | 0.8 | 17 | 1.1 | 19 | 1.2 | 11 | 0.7 |
| S. aureus (MTCC 3160) | 14 | 0.9 | 16 | 1.0 | 20 | 1.3 | 17 | 1.1 |
| VRE (ATCC 51299) | - | - | 10.2 | 1.0 | 14 | 1.4 | - | - |
| ZOI means zone of inhibition; AI means activity index | ||||||||
Morphological and molecular identification of the PTR3 isolate
The 18S rRNA sequencing identified the potent fungus as Daldinia eschscholtzii and submitted it to GenBank (accession No. OP800285). Figure 2 shows the phylogenetic tree and morphological SEM study of PTR3.
Figure 2: LCB stained at 40x (A) and 100x (B), SEM (C), Phylogenetic tree (D) generated by maximum likelihood of Daldinia eschscholtzii PTR3
Evaluation of MIC and MBC of active extract
The D. eschscholtzii PTR3 ethyl acetate extract that showed the maximum antagonistic effect was subjected further to determine their MIC and MBC. The extract showed a MIC of 6.25 and 3.12 µg/mL, an MBC of 50 and 25 µg/mL against two MRSA (i.e., ATCC 43300, ATCC 700699), respectively (Table II). Whereas, against VRE, the MIC and MBC were 12.5 and 50 µg/mL.
Table II: Minimum inhibitory and minimum bactericidal concentration of PTR3 ethyl acetate extract
| Test pathogens | Concentration (µg/mL) | |
|---|---|---|
| MIC | MBC | |
| MRSA (ATCC 43300) | 6.25 | 50 |
| MRSA (ATCC 700699) | 3.12 | 25 |
| S. aureus (ATCC 25923) | 3.12 | 6.25 |
| S. aureus (MTCC 3160) | 3.12 | 12.5 |
| VRE (ATCC 51299) | 12.5 | 50 |
| Oxacillin | - | - |
| Vancomycin | 25 | - |
| MIC means minimum inhibitory concentration; MBC means minimum bactericidal concentration | ||
Growth rate of mycelia
The radial growth of the PTR3 fungus was more rapid when compared with PTR1 and PTR2 isolates, representing that nutrient-rich media could enrich mycelial density and the growth rate (data not shown).
GC-MS of potent extract
As seen in Figure 3, 1,2-benzene dicarboxylic acid and diheptyl ester were the primary compound present in two different retention times of 19.3 and 19.8 min with an area percentage of 24.1% and 19.2% respectively.
Figure 3: Chromatogram analysis of ethyl acetate extract
Discussion
The present study demonstrated significant antibacterial activity of D. eschscholtzii isolated from P. tuberosa against drug-resistant pathogens.
The previous study reported that at 1 mg/mL concentration, ethyl acetate crude extracts of D. eschscholtzii isolated from the plant Musa paradisiaca displayed substantial antibacterial activity against P. aeruginosa (5 mm), E. coli (7 mm) and B. subtilis (3 mm). It also inhibited extended spectrum beta lactamase, a resistant bacterial strain of E. coli, with a 4 mm zone of inhibition (Victor et al., 2020). The ethyl acetate extract obtained from D. eschscholtzii of Psidium guajava has antibacterial activity against B. subtilis, S. aureus, E. faecalis, P. aeruginosa, E. coli, P. aeruginosa and P. vulgaris. E. faecalis was the most sensitive to the extract, with an inhibition zone of 15.7 ± 0.6 mm and a MIC of 1.25 mg/mL (Chutulo and Chalannavar, 2020). The broth ethyl acetate extract of D. eschscholtzii FK511P from Gorgonian Annella sp. inhibited Gram-positive bacteria S. aureus and MRSA with 8.3 ± 0.5 and 11.8 ± 1.0 mm inhibition, respectively, and Gram-negative E. coli with 9.0 ± 0.5 mm inhibition. Furthermore, mycelia methanol extract inhibited S. aureus and MRSA with inhibition zones of 8.1 ± 1.0 and 17.1 ± 1.2 mm, respectively, and E.coli with an inhibition zone of 12.1 ± 1.6 mm (Kandou et al., 2021). Nodulisporin H and 8-O-methyl-nodulisporin F, two new polyketides isolated from the ethanolic extract of the mangrove-derived fungus D. eschscholtzii HJ004, demonstrated moderate antibacterial activity against S. aureus, MRSA and Bacillus cereus, with MIC ranging from 6.25 to 12.5 μg/mL. The compound 5-hydroxy-2-methoxy-6,7-dimethyl-1,4-naphthoquinone had antibac-terial activity against B. cereus with a MIC value of 12.5 μg/mL (Liao et al., 2019). Endophytic D. cf concentrica and its volatile metabolites from Olea europaea have shown antibacterial efficacy against plant pathogenic fungi. Similarly, D. sacchari metabolites from sugarcane in South India displayed antibacterial action against fungus and Gram-positive bacteria (Chutulo and Chalannavar, 2020).
Previous studies reported that 1,2-benzene dicarboxylic acid, mono(2-ethylhexyl) ester isolated from leaves of Stevia rebaudiana showed antifungal activity against S. sclerotiorum with an inhibition zone of 29 mm after 48 hours. Similarly, the compound 1,2-benzene dicarboxy-lic acid, (2-ethylhexyl) ester produced by the marine Burkholderia cepacia showed potential antibacterial activity against Aeromonas hydrophila, Edwardsiella tarda and Vibrio ordalii (Verma et al., 2014). 1,2-Benzene dicarboxylic acid, bis (2-ethyhexyl) ester and 1,2-benzene-dicarboxylic acid, dibutyl ester derived from ethyl acetate fraction of Alternaria sp. isolated from Salvadora persica showed antimicrobial and antioxidant activity (Elgorban et al., 2019). And also, 1,2-benzene dicarboxylic acid and dioctyl ester isolated from the soluble sub-portion of ethyl acetate extract of the Nauclea latifolia unripe fruits displayed antibacterial activity against Gram-positive bacteria.
This is the first report on endophytic fungi D. eschscholtzii isolated from a root of P. tuberosa and their antibacterial activity.
Conclusion
This study shows the significant antibacterial activity of the endophytic fungi D. eschscholtzii isolated from the root of P. tuberosa against antibiotic-resistant pathogens.
Acknowledgement
The authors thank Vellore Institute of Technology's management for providing the facilities to carry out this study.
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