Antibacterial activity of the endophytic fungal extracts and synergistic effects of combinations of ethylenediaminetetraacetic acid (EDTA) against Pseudomonas aeruginosa and Escherichia coli

Leaves samples

Leaves of Thai medicinal plants, including Capsicum annuum L., Elaeagnus latifolia L., Lantana camara L., Mansonia gagei Drumm., Orthosiphon aristatus (Blume) Miq., Oroxylum indicum L., Talinum paniculatum (Jacq.) Gaertn., and Terminalia bellirica (Gaertn.) Roxb., were collected from the Walailak University botanical garden in 2022. The voucher specimens of plants are deposited at the Walailak Herbarium, Walailak University, Thasala, Nakhon Si Thammarat, Thailand, listed in Table 1. For the further experiment, only healthy plant leaves were selected. Leaves that exhibited physical damage or signs of infection were not included in the study. Leaves were then stored in Ziploc plastic bags (Huang, Zimmerman & Arnold, 2018) and preserved on ice.

Leaves preparation

The medical plant leaves were prepared according to the method by Phongpaichit et al. (2006) with minor modifications. The leaves were washed to remove any adhering epiphytes and subsequently exposed to a 70% ethanol solution for 5 min to disinfect the leaf surfaces. Cleaned leaves were cut into 0.5 cm pieces using a sterile hole puncher. The cut leaves were then subjected to surface disinfection by immersion in 95% ethanol for 1 min, followed by 3% sodium hypochlorite solution for 3 min. All samples were subsequently rinsed and dried.

Endophytic fungal isolation

Prepared leaves were placed onto Sabouraud dextrose agar (SDA) supplemented with chloramphenicol and incubated at 25 ± 2 °C for 1–3 days (Handayani et al., 2017). Fungal hyphae originating from the leaves and growing on the media were transferred to a new SDA plate. The fungal isolates were assessed for purity after incubation at 25 ± 2 °C to obtain pure cultures of endophytic fungi.

Endophytic fungi fermentation

The modified method of Phongpaichit et al. (2006) was performed to obtain the endophytic fungi extracts. Four pieces of mycelial agar plugs of endophytic fungi growth for 7 days (0.5 cm × 0.5 cm) were inoculated into culture flasks that had sabouraud dextrose broth (SDB). The culture was shaken at 200 rpm at 25 ± 2 °C for 14 days. The endophytic fungal culture was filtered to separate culture broth and mycelia. Cell-free supernatants (CFS) were concentrated by evaporation in a rotary vacuum evaporator. The evaporated extracts were dried using freeze-drying (FDU-2100; Eyela, Tokyo, Japan) at −80 °C for 36 h. The dried matter was weighed and stored at 4 °C for further analysis. The endophytic fungal extracts were dissolved in 50% dimethyl sulfoxide (DMSO). The endophytic fungal isolates are listed in Table 2.

Morphological identification of endophytic fungi

Endophytic fungi were identified by staining slides prepared from cultures with lactophenol cotton blue reagent. The stained slides were examined under a bright-field and phase contrast microscope (Sadananda, Govindappa & Ramachandra, 2014). Morphological characteristics, including hyphae, colony and medium color, surface texture, sporulation, production of acervuli, medium coloration, size, and coloration of conidia, were observed to identify the endophytic fungi (Barnett, 1960).

Bacterial strains and preparation

Pseudomonas aeruginosa ATCC27853 and Escherichia coli ATCC25922 were obtained from the American Type Culture Collection. All bacterial strains were stored in 20% glycerol stocks at −80 °C until use.

Cell lines and preparation

Human foreskin fibroblast cells (HFF-1, ATCC SCRC-1041) were obtained from Assoc. Prof. Rathapon Asasutjarit, Thammasat University Research Unit in Drug, Health Product Development and Application (DHP-DA), Department of Pharmaceutical Sciences, Faculty of Pharmacy, Thammasat University. HFF-1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 IU/mL penicillin. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ and were passaged every 3 days using 0.25% trypsin-EDTA solution to maintain exponential growth.

Agar well diffusion

Agar well diffusion assay was used to determine the antibacterial activities of endophytic fungal extracts (CLSI, 2020). The assay was performed in Mueller-Hinton agar (MHA) (Himedia, India). Overnight bacterial suspension was adjusted with Mueller-Hinton broth (MHB) to obtain a final concentration of 1 × 10⁸ CFU/mL. Bacterial suspension was seeded to MHA, and a well of 6 mm was created. Endophytic fungal extracts were added to each well at a final concentration of 1,024 µg/mL. The plates were then incubated at 35 ± 2 °C for 16–18 h. Ciprofloxacin and DMSO (AppliChem GmbH, darmstadt, Germany) were used as positive and negative controls, respectively. The inhibition zone diameter was measured in millimeters using a vernier caliper.

Determination of minimal inhibitory concentration and minimal bactericidal concentration

Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined using a modified broth microdilution method according to a modification of Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2020). Bacterial suspensions were cultured until reaching the exponential growth phase, followed by adjusting the bacterial suspension to achieve a concentration of 1 × 10⁶ CFU/mL. The crude extract concentrations were added to 96-well microtiter plates and diluted by two-fold-serial dilutions to obtain a final concentration between 512 and 2 µg/mL. The plates were incubated at 35 ± 2 °C for 16–20 h. MHB with the bacterial suspension was used as a positive control. The culture with extracts was used as a negative control. The MIC was determined as the minimal concentration of the extract present in the clear well of the microtiter plate. For MBC determination, aliquots from wells without visible growth in the MIC assay were plated onto agar media using the drop plate method (Herigstad, Hamilton & Heersink, 2001). The MBC was defined as the lowest extract concentration that resulted in no bacterial colonies on the agar plates. The assay was performed in triplicate.

Time-kill assay

Time-kill kinetics of the endophytic fungal extract against bacteria were evaluated with modifications (Sianglum et al., 2019). Briefly, crude extracts were prepared at concentrations corresponding to 1/4MIC, 1/2MIC, MIC, and 2MIC, respectively. Bacterial suspensions were then added to each well to provide a final inoculum density of 5 × 10⁵ CFU/mL and incubated at 35 ± 2 °C for 0, 1, 2, 4, 6, 12, and 24 h. Treated and untreated bacterial aliquots were diluted ten-fold, and 10 µL of each dilution was plated onto agar media. Colony-forming units (CFU/mL) were enumerated after incubation. All experiments were performed in triplicate.

Determination of fractional inhibitory concentration index

The microdilution checkerboard assay was performed to evaluate the interaction between endophytic fungal extracts and the antimicrobials (Muroi & Kubo, 1996). Endophytic fungal extracts in combination with antimicrobials in different final concentrations ranged from 1/4MIC to 2MIC, and bacterial suspension was added to 96-well microtiter plates. The plates were incubated at 35 ± 2 °C overnight. The fractional inhibitory concentration (FIC) index formula was calculated according to the following formula: FIC index = FIC_A + FIC_B = (C_A/MIC_A) + (C_B/MIC_B), where MIC_A and MIC_B are the MICs of endophytic fungal extract and antimicrobial, respectively. CA and CB are the concentrations of the endophytic fungal extract and antimicrobial in combination. The synergistic effect indicated by the FIC index < 0.5, no interaction when the FIC index is between 0.5 and 4.0. The FIC index > 4 indicated an antagonistic effect (Norden, Wentzel & Keleti, 1979). At least two independent experiments were performed in duplicate.

Growth of bacteria in the presence of endophytic fungal extracts and antimicrobials

The bactericidal effect of selected endophytic fungal extracts, including TBE1, ELE1, OIE1, TPE1, CAE2, and TBE1 in combination with EDTA against P. aeruginosa ATCC27853, was evaluated using a drop plate method (Habeeb et al., 2007). Briefly, the exponential growth phase of the bacterial culture was diluted to approximately 10⁶ CFU/mL and treated with a combination of endophytic fungal extracts and EDTA. The samples were then incubated for 12 and 24 h, respectively. The diluted samples were dropped onto nutrient agar plates for bacterial enumeration and then incubated at 35 ± 2 °C for 24 h. After incubation, the colonies were counted, and the results were expressed as the log CFU/mL against time. The experiments were performed in three biological replicates.

Molecular identification of endophytic fungi

The endophytic fungi that exhibited synergistic effects when combined with EDTA were selected for identification using molecular techniques (Raja et al., 2017). DNA amplification and sequencing of the internal transcribed spacer (ITS) region, which is widely used as a universal DNA barcode marker for fungi, exhibits a low level of intraspecific variation and a high level of interspecific variation were used for molecular identification of endophytic fungus (Crouch, Clarke & Hillman, 2005; Harnelly et al., 2022; He et al., 2017; Schoch et al., 2012). Fungal hyphae were lyophilized and ground before DNA extraction using the fungal genomic DNA kit (Geneaid, Taiwan), following the manufacturer’s protocol. The ITS region was amplified using the primers ITS5 5′ (GGA AGT AAA AGT CGT AACAAG G) 3′ and ITS4 5′ (TCC TCC GCT TAT TGA TAT GC) 3 by polymerase chain reaction (PCR) under the following conditions: 95 °C for 5 min, 31 cycles of 95 °C for 0.5 min, 57 °C for 0.5 min, and 72 °C for 1.4 min, and final extension with a 10 min at 72 °C.

Sequencing

PCR products were purified with a multiscreen filter plate (Millipore Corp, Burlington, MA, USA). A sequencing reaction was conducted using the PRISM BigDye Terminator v3.1 Cycle Sequencing Kit. Extension product-containing DNA samples were combined with Hi-Di formamide (Applied Biosystems, Foster City, USA), heated at 95 °C for 5 min, and then cooled on ice for 5 min. The prepared mixture was then analyzed with an ABI Prism 3730XL DNA Analyzer (Applied Biosystems, Foster City, USA).

Evaluation of the cytotoxicity

To determine the cytotoxic effect of endophytic fungal extracts on HFF-1 (ATCC, SCRC-1041) the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was performed (Mosmann, 1983). The 96-well microtiter plate contains HFF-1 cells at 5 × 10³ cells/well in DMEM culture media (Gibco, Grand Island, NY, USA) supplemented with 10% FBS which were incubated at 35 ± 2 °C for 24 h in a 5% CO₂ atmosphere. The endophytic fungal extracts at 0–1,000 µg/mL were added to the plates. The plates were incubated for 24 h. MTT solution at 0.5 mg/mL was added and incubated at 35 ± 2 °C in a humidified atmosphere with 5% CO₂ for 2 h. The cells were suspended in 100 µL dimethyl sulfoxide (DMSO) after discarding the culture medium. Cellular formazan production, an indicator of metabolic activity and cell viability, was quantified by measuring the absorbance at 570 nm using a microplate reader. The resulting absorbance values were then used to calculate the percentage of viable cells compared to the untreated control. The cytotoxic activity of extracts was reported as half-maximum inhibitory concentration (IC₅₀) value, which represents the concentration of the extract that inhibits cell growth by 50%. The viability percentages were calculated according to the following formula.

$$\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}}\times 100.}$$

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Holdings San Diego, CA, USA). Student’s t-test was used to compare treated and untreated groups directly. A p-value of less than 0.05 was considered statistically significant, indicating a significant difference between the compared groups.

Identification of the endophytic fungal isolates

Morphological identification was accomplished through macroscopic and microscopic observation. The colony morphology of nine endophytic fungal isolates exhibited diverse shapes, edges, elevations, colors, and textures. Each fungal hyphae shape of nine endophytic fungi showed different characteristics. The characteristics of colonies and types of hyphae and conidia shape of endophytic fungi were summarized and explained in Table 3 and Fig. 1. The endophytic fungi from C. annuum, E. latifolia, L. camara, M. gagei, O. aristatus, O. indicum, T. paniculatum, and T. bellirica were classified into different genera, including Purpureocillium sp., Trichothecium sp., Exophiala sp., Fusarium sp., Scytalidium sp., Lecythophora sp., Acremonium sp., Curvularia sp., and Alternaria sp., respectively.

Agar well diffusion assay

The preliminary antibacterial testing of test extracts against E. coli and P. aeruginosa was performed using agar well diffusion. Nine endophytic fungi cell-free supernatant at 1,024 µg/mL exhibited antibacterial activity against only E. coli. The diameter of inhibition zones of cell-free supernatants of endophytic fungi ranged from 28.20 mm to 31.15 mm, as represented in Table 4. Acremonium sp. showed the highest inhibition diameter zone with 31.15 mm against E. coli. Figure 2 shows the zones of inhibition produced by the cell-free supernatant of endophytic fungi against E. coli. No zones of inhibition were observed for the endophytic fungal extracts against P. aeruginosa.

Minimum inhibitory concentration and minimum bactericidal concentration

The MIC values of endophytic fungal extracts ranged from 32 to 64 µg/mL against E. coli and from 512 to 2,048 µg/mL against P. aeruginosa (Table 5). The lower MIC values of the extracts against E. coli compared to P. aeruginosa suggest that the extracts exhibit stronger antibacterial activity against E. coli. Among the tested fungi, Acremonium sp. isolated from O. indicum showed the lowest MIC of 32 µg/mL against E. coli, while Fusarium sp. isolated from L. camara exhibited the highest antibacterial activity against P. aeruginosa, with a MIC value of 512 µg/mL. The MBC values for all endophytic fungal extracts were more than 512 µg/mL against both E. coli and P. aeruginosa.

Time kill assay

A time-kill assay was used to evaluate the bactericidal effects of endophytic fungal extracts exhibiting effective antibacterial activity against E. coli. As shown in Fig. 3A, the untreated E. coli group gradually increased in viable cell number before 6 h, after that the bacterial cell count remained relatively stable within 24 h. Similar growth patterns were observed in E. coli treated with 1/2MIC and 1/4MIC of TPE1 extract, identified as Curvularia sp. However, E. coli treated with 4MIC and 2MIC concentrations of TPE1 extracts, identified as Curvularia sp., exhibited a slight decrease in cell number within 24 h, with a reduction of more than 3 log CFU/mL compared to the untreated group, indicating bactericidal activity. MIC value of Curvularia sp. extracts demonstrated a bactericidal effect by reducing the viable cell count to 2 log CFU/mL at 24 h compared to the untreated group, suggesting a bacteriostatic effect (Fig. 3A). A similar bactericidal effect was observed in E. coli treated with 4MIC and 2MIC concentrations of OIE1 extract, identified as Acremonium sp., with a reduction in the bacterial count more than 3 log CFU/mL compared to the untreated group within 24 h (Fig. 3B). The results indicate that the antibacterial activity of Curvularia sp. and Acremonium sp. extracts are concentration-dependent, with higher concentrations of the extracts leading to a more reduction in the number of viable bacterial colonies over time.

Effects of endophytic fungal extract and antibiotic combinations

To enhance the antibacterial activity of endophytic fungal extracts against P. aeruginosa, the combination of endophytic fungi with antimicrobials, including gentamicin, ciprofloxacin, levofloxacin, and EDTA, was investigated against P. aeruginosa. Table 6 revealed that three endophytic fungal extracts, including Purpureocillium sp., Scytalidium sp., and Fusarium sp., exhibited an indifferent effect when combined with EDTA. In contrast, synergism was observed in combinations between Lecythophora sp., Curvularia sp., Alternaria sp., Acremonium sp., Exophiala sp., and Trichothecium sp. extracts and EDTA against P. aeruginosa, as indicated by synergistic FIC index values of 0.375.

Bactericidal activity of the combination of endophytic fungi and EDTA

The combination of endophytic fungi and EDTA against P. aeruginosa, which demonstrated synergism, was selected to evaluate its bactericidal effect. A significant reduction in the number of viable P. aeruginosa cells was observed when treated with a combination of 1/8MIC endophytic fungal extracts and 1/16MIC EDTA at 12 and 24 h compared to the untreated control (p < 0.05, Student’s t-test). This combination decreased the number of viable bacterial cells by more than 3 log CFU/mL from the viable bacterial number of untreated controls at 12 and 24 h (Fig. 4). These results indicate that the combination of EDTA and endophytic fungal extracts exhibits synergistic interactions with bactericidal effects against P. aeruginosa.

Molecular identification of effective endophytic fungi

Table 7 shows molecular identification by amplified ITS region and nucleotide BLAST search of each fungal endophytic fungi. A nucleotide BLAST search using ITS sequences revealed that TBE1, OIE1, and ELE1 were identified as Purpureocillium lilacinum. P. lilacinum is a common endophytic fungus found in the site where the medicinal plant in this study was collected. The organism has the ability to adapt to various plant hosts. As a result, the same endophytic fungi were found in different plant species within the same area (Huang et al., 2008). TPE1 endophytic fungi isolated from T. paniculatum, was found to be closely related to Fusarium verticillioides. CAE2 isolate was identified as Aspergillus aculeatus. OAE1 isolated from O. aristatus belonged to Acremonium sp.

Cytotoxicity of endophytic fungal extracts

The cytotoxicity of the culture broth from endophytic fungi isolated from L. camara, O. indicum, T. bellirica, and T. paniculatum was evaluated against human foreskin fibroblasts cells, HFF-1, as shown in Table 8. The extracts which have IC₅₀ ≤ 20 μg/mL had high cytotoxicity, IC₅₀ > 20–100 μg/mL had moderately cytotoxicity, IC₅₀ > 100–1,000 μg/mL had weakly cytotoxicity, IC₅₀ > 1,000 μg/mL had inactive cytotoxicity (Nordin et al., 2018). The culture broth from endophytic fungi isolated from O. aristatus, O. indicum, and T. bellirica exhibited no cytotoxic effects, as their IC₅₀ values exceeded 1,000 µg/mL. These results indicate that these endophytic fungal extracts are safe for use. In contrast, the endophytic fungal extracts from C. annuum and T. paniculatum exhibited low cytotoxicity against normal HFF-1 cells, with an IC₅₀ value of 748.7 and 539.1 µg/mL.