Antimicrobial activity of endophytic fungal metabolites from breadfruit (Artocarpus altilis) leaves against multidrug-resistant Staphylococcus aureus and Klebsiella spp.

Mediterranean Journal of www.mmj.org.ly Mediterr J Med Med Sci Medicine & Medical Sciences LAP Nwaneri MGU, et al. Mediterr J Med Med Sci. 2025; 1(3): 23-30. Page 23 ORIGINAL RESEARCH article Antimicrobial activity of endophytic fungal metabolites from breadfruit (Artocarpus altilis) leaves against multidrug-resistant Staphylococcus aureus and Klebsiella spp. Miriam G.U. Nwaneri 1 , Ogochi A. Ilechukwu 1 , and Udochi A. Ugo 2 * 1 Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka 420112, Anambra State, Nigeria 2 Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria * Author to whom correspondence should be addressed Article number: 14, Received: 10-10-2025, Accepted: 09-11-2025, Published online: 15-11-2025 HOW TO CITE THIS Nwaneri MGU, et al. Antimicrobial activity of endophytic fungal metabolites from breadfruit (Artocarpus altilis) leaves against multidrug-resistant Staphylococcus aureus and Klebsiella spp. Mediterr J Med Med Sci. 2025; 1(3): 23-30. [Article number: 14]. https://doi.org/10.5281/zenodo.17611720 Keywords: Antimicrobial resistance, endophytic fungi, multidrug resistance Abstract: Antimicrobial resistance (AMR) has become a global concern due to the growing inability of medications to treat infections. Endophytic fungi produce secondary metabolites with antimicrobial properties, making them potential sources of new drugs. This study evaluates the antimicrobial activity of crude secondary metabolites produced by endophytic fungi isolated from the leaves of Artocarpus altilis against multidrugresistant Klebsiella spp and Staphylococcus aureus. Fresh leaves of Artocarpus altilis (Breadfruit) were collected, authenticated, and surface sterilized for fungal isolation. Fungal isolates were purified and fermented on rice medium at 30°C for 21 days. Metabolites were extracted using ethyl acetate and tested against MDR-Klebsiella spp and MDR-Staphylococcus aureus using agar well diffusion. Phytochemical screening was also conducted. Fungal extracts showed moderate activity against MDR-Klebsiella spp, with Aa-MR exhibiting the highest effect (MIC: 37.5 mg/mL). No activity was observed against MDR-Staphylococcus aureus. Phytochemical analysis revealed alkaloids, flavonoids, saponins, tannins, terpenoids, and glycosides. These findings suggest that fungal metabolites from A. altilis may be useful in treating infections caused by gram-negative bacteria. Introduction Microbial resistance has long been known as an imperative challenge in treating diseases caused by bacteria, viruses, fungi, and parasites. The global rise of antimicrobial resistance (AMR) has become a persistent public health challenge, resulting in the need for different antimicrobial agents [1, 2]. Antibiotic-resistant organisms in the critical priority group from the World Health Organization (WHO) include: third-generation cephalosporinresistant, carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Acinetobacter baumannii, and carbapenem-resistant Enterobacteriaceae. The priority group includes Enterococcus faecium, Staphylococcus aureus, Salmonella spp., Campylobacter, Helicobacter pylori, and Neisseria gonorrhoeae, while Haemophilus influenzae, Streptococcus pneumoniae, and Shigella spp. are classified as middle priority [3]. Endophytic fungi can produce a wide range of secondary metabolites that function as antibiotics, antifungals, antivirals, and Copyright© 2025. This open-access article is distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mediterranean Journal of www.mmj.org.ly Mediterr J Med Med Sci Medicine & Medical Sciences LAP Nwaneri MGU, et al. Mediterr J Med Med Sci. 2025; 1(3): 23-30. Page 24 anticancer agents. In addition, they enhance resistance of plants to herbivores and pathogens [4]. These make them attractive for the development of drugs [5]. Studies have shown that Artocarpus altilis (Figure 1) has anticancer, anti-ulcer, antioxidant, anti-inflammatory, anti-bacterial, and anti-atherosclerotic properties [6-9]. Their leaves are used as tea to treat high blood pressure, diabetes, and asthma [10]. Despite the rich biodiversity of breadfruit, the endophytic fungi associated with it remain underexplored. This presents a gap in our understanding of their diversity and potential to produce antimicrobial compounds. Addressing this gap is crucial for discovering new bioactive agents that could contribute to the fight against AMR. This study aims to empirically evaluate the antimicrobial activity of the crude secondary metabolites produced by the endophytic fungi isolated from the leaves of Artocarpus altilis against Multidrug Resistant Klebsiella spp and Staphylococcus aureus. Figure 1: Artocarpus altilis (breadfruit) leaves Materials and methods Test microorganism and processing: The test organisms used in this work include Staphylococcus aureus and Klebsiella spp. They were collected from the Laboratory of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. Culture media used were malt extract agar (MEA), Mueller-Hinton agar (LS BIOTECH, UK), Mac Conkey agar, mannitol salt agar, and Sabouraud agar (LS BIOTECH, UK). Nutrient Broth (LS BIOTECH) was the broth media used. The bacterial isolates were reconfirmed by sub-culturing them onto a nutrient agar plate and incubating at 37ºC for 18-24 hrs. Pure isolates were subjected to specific identification techniques. The organisms were standardized by transferring the pure isolates using a sterile wire loop into 3.0 mls of sterile nutrient broth and grown in an oxygenrich shaker water bath at 37ºC for 3.0 hrs to a cell density equivalent to the turbidity of 0.5 McFarland [11]. The susceptibility tests for the organisms were performed following the M2A6 disc diffusion method described as follows. The standardized organisms were swabbed onto a Mueller Hinton agar plate and the discs were placed on the inoculated plates and pressed firmly onto the agar plate for complete contact. The bacterial strains were tested against the following discs; ceftriaxone (30 μg), amoxicillin & clavulanic acid (30 μg), imipenem (10 μg), cefotaxime (30 μg), ceftazidime (30 μg), aztreonam (30 μg), cefoxitin (30 μg), cefpirome (30 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), gentamicin (10 μg), sulphamethoxazole-trimethoprim (30 μg), tetracycline (10 μg), meropenem (10 μg), ertapenem (10 μg), piperacillin (75 μg), nitrofurantoin (200 μg), ticarcillin/clavulanic acid (85 μg), nalidixic acid (30 μg), chloramphenicol (10 μg), tobramycin (10 μg), and cefotetan (30 μg). The plates were left for 30 min to allow for pre-diffusion of antibiotics into the agar and then incubated at 37°C for 18-24 hrs. The susceptibility of each isolate to the antibiotic disc was shown by a clear zone of growth inhibition, and the diameter of the zones of inhibition was measured and interpreted using a standard chart [12]. Collection and processing of plant materials: Fresh leaves of Artocarpus altilis were collected at the medicinal garden school of Pharmacy Agulu and were authenticated by Taxonomist from the Department of Pharmacognosy Mediterranean Journal of www.mmj.org.ly Mediterr J Med Med Sci Medicine & Medical Sciences LAP Nwaneri MGU, et al. Mediterr J Med Med Sci. 2025; 1(3): 23-30. Page 25 and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. The harvested leaves were washed thoroughly under running tap water followed by sterile double distilled water before processing. For isolation of endophytic fungi from the leaves, all the samples were subjected to three step surface sterilization, using ethanol, sodium hypochlorite and distilled water to eliminate epiphytic microorganisms. They were immersed in 70.0% ethanol for three minutes and transferred to sodium hypochlorite solution (2.0%) for five minutes before a final rinse in sterile double distilled water and then dried in the laminar flow on a sterile filter paper. The disinfected samples were cut aseptically to 1.0 cm and inoculated into previously sterilized MEA media incorporated with chloramphenicol 500 mg/l. The plates were properly sealed and incubated at 25oC for seven days while being checked for mycelium development on alternate days [13]. The isolation of pure cultures was achieved through multiple subculturing of isolates on fresh MEA. Examination of the morphological/cultural characteristics of the pure isolates was carried out by noticing visible phenotypic character keys like colony texture, pigmentation and color mycelium [13]. Fermentation and extraction of fungal metabolites: Each pure fungal isolate was grown in 1000 ml Erlenmeyer flasks containing sterilized rice medium, previously autoclaved at 121ºC at 15 psi for one hour [6]. The flasks were properly sealed and incubated under static conditions at 30°C for 21 days. Extraction of biosynthesized fungal metabolites was done using 500 ml of ethyl acetate. The filtrates were concentrated by evaporating the solvent at 50ºC using a rotary evaporator. Antimicrobial evaluation of the fungal extracts: The antibacterial assay for the fungal extract was carried out using the agar well diffusion assay described by [14, 15] with small alterations. The standardized isolates were inoculated onto sterilized Mueller-Hinton agar plates using a swab stick. A sterile cork borer was used to make five wells (6.0 mm in diameter) on each of the MHA plates. Aliquots of 80 μl of each fungal extract dilution, reconstituted in DMSO at concentrations of 150, 75, 37.5, and 18.75 mg/ml, were applied in each of the wells. Ciprofloxacin (8.0 µg/mL) served as the positive control. The cultures were incubated at 37oC for 24 hrs. The antimicrobial potential for each fungal extract was determined by measuring the zone of inhibition for each of the test organisms. Phytochemical analysis of the fungal extracts: The crude extracts were tested for the presence of various phytoconstituents like alkaloids, flavonoids, reducing sugars, saponins, proteins, tannins, amino acids, steroids, triterpenoids and glycosides using the following tests: Dragendoff’s and Wagner’s tests for alkaloids; lead acetate and alkaline reagent test for flavonoids, Fehling’s test for reducing sugar; frothing test for saponins; precipitation test for protein; ferric chloride test for tannins; ninhydrin test for amino acid; Liebermann-Burchard test for steroid; Salkowski test for triterpenoid and general test for glycosides [16-18]. Results Antibiotic susceptibility test for Staphylococcus aureus isolates. The susceptibility test carried out on different isolates of the test organisms revealed that they are multidrug-resistant (Tables 1 and 2, Figure 2). Isolation of endophytic fungi and extraction of metabolites. A total of 14 endophytic fungal isolates were obtained from the leaves of A. altilis (Figure 3). Nine originated from the leaf blade and five originated from the midrib. The number of endophytic fungi in the leaf blade was significantly higher than that in the midrib of the leaves, indicating that the distribution of endophytic fungi differs among the different parts of the leaves. After further sub-culturing, three pure isolates were obtained from the leaf blade while a single isolate was obtained from the midrib (Figure 4). The yields of crude fungal metabolites obtained from various fungal isolates showed that AaLb3 recorded the highest yield of 2.42 g, while Aa-Lb1 had the lowest yield of 1.00 g (Table 3). Mediterranean Journal of www.mmj.org.ly Mediterr J Med Med Sci Medicine & Medical Sciences LAP Nwaneri MGU, et al. Mediterr J Med Med Sci. 2025; 1(3): 23-30. Page 26 Table 1: Antibiotic susceptibility test for Staphylococcus aureus isolates Isolate code Antibiotics Used / Inhibition zone diameter (mm) Status AUG IMP OFX CIP LBC CTX CRO CXM ZEM GN ERY AZN H10 0 0 20 16 0 0 0 0 0 20 0 10 MDR H29 0 0 30 0 34 0 0 0 0 22 0 26 MDR H35 0 0 9 0 14 0 0 0 0 0 0 0 MDR H38 0 0 0 28 34 0 0 0 0 20 0 26 MDR H48 0 0 16 22 0 0 0 0 0 12 0 0 MDR Key: AUG-amoxicillin clavulanate (30 mcg); CTX-cefotaxime (25 mcg); CRO-ceftriaxone sulbactam (45 mcg); ZEM-cefixime (5 mcg); LBC-levofloxacin (5 mcg); CIP-ciprofloxacin (5 mcg); IMP-imipenem/cilastatin (10/10 mcg); CXM-cefuroxime (30 mcg); OFX-ofloxacin (5 mcg); ERY-erythromycin (15 mcg); GN-gentamicin (10 mcg); AZN-azithromycin (15 mcg) MDR-multidrug resistant Table 2: Antibiotic susceptibility test for Klebsiella spp Isolate code Antibiotics used / Inhibition zone diameter (mm) Status OFX LBC AUG IMP ACX CTX CXM CRO ZEM GN NF NA K32 0 28 0 0 0 0 0 10 0 24 14 0 MDR K28 18 0 0 0 0 0 0 0 27 17 0 17 MDR K43 25 21 0 0 10 0 0 10 0 20 10 0 MDR K31 20 0 0 0 0 0 0 0 0 22 0 15 MDR K38 0 30 0 0 0 0 0 12 14 0 16 0 MDR Key: AUG-amoxicillin clavulanate (30 mcg); CTX-cefotaxime (25 mcg); CRO-Ceftriaxone sulbactam (45 mcg); ZEM-cefixime (5 mcg); LBC-levofloxacin (5 mcg); NA-nalidixic Acid (30 mcg); NF-nitrofurantoin (30 mcg); IMP-imipenem/cilastatin (10/10 mcg); CXM-cefuroxime (30 mcg); OFX-Ofloxacin (5 mcg); GN-gentamicin (10 mcg); ACX-ampiclox (10 mcg) MDR-multidrug resistant Figure 2: Antibiotic susceptibility test of the test microorganisms Figure 3: The Endophytic fungi isolated from the midrib and leaf blades of A. altilis Figure 4: The 4 purified fungal isolates from the midrib and leaf blade A. altilis Mediterranean Journal of www.mmj.org.ly Mediterr J Med Med Sci Medicine & Medical Sciences LAP Nwaneri MGU, et al. Mediterr J Med Med Sci. 2025; 1(3): 23-30. Page 27 Table 3: The yield of the fungal metabolites from the different isolates Fungal isolate Yield (g) Aa-Mr 2.33 Aa-Lb2 2.10 Aa-Lb1 1.00 Aa-Lb3 2.42 Key: Aa-Artocarpus altilis; Lb-Leaf Blade; Mr-Mid rib Determination of the antibacterial potential of the metabolites: Tables 4 to 7 revealed the antibacterial potential of crude endophytic fungal extracts against the multi-drug resistant Klebsiella spp. and Staphylococcus aureus. Phytochemical analysis of the crude fungal extract: The phytochemical analysis of the leaves showed the presence of alkaloids, flavonoids, saponins, tannins, glycosides, steroids, and terpenoids. None contained reducing sugars (Table 8). Table 4: Antibacterial activity of Aa-Lb3 against MDR test organisms Concentration (mg/mL) Test organisms / inhibition zone diameter (mm) K28 K32 K38 K43 H10 H35 150 3.0±0.0 4.0±0.0 3.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 75 0.0±0.0 2.0±0.0 3.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 37.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 18.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 CIPRO (8µg/mL) 12.0±0.0 15. 0±0.3 17.0±0.0 8.0±0.0 11.0±0.0 5.5±0.2 Key: Aa-Artocarpus altilis; Lb-Leaf blade; K Klebsiella spp; H-Staphylococcus aureus Table 5: Antibacterial activity of Aa-Lb2 against MDR test organisms Concentration (mg/mL) Test organisms / inhibition zone diameter (mm) K28 K32 K38 K43 H10 H35 150 4.0±0.0 8.0±0.0 5.0±0.0 3.0±0.0 0.0±0.0 0.0±0.0 75 2.0±0.0 4.0±0.0 4.0±0.0 2.0±0.0 0.0±0.0 0.0±0.0 37.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 18.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 CIPRO (8 µg/mL) 12.0±0.0 15.5±0.3 17.0±0.0 8.0±0.0 11.0±0.0 5.5±0.2 Key: Aa-Artocarpus altilis; Lb-Leaf blade; K-Klebsiella spp; H-Staphylococcus aureus Table 6: Antibacterial activity of Aa-Lb1 against MDR test organisms Concentration (mg/mL) Test organisms / inhibition zone diameter (mm) K28 K32 K38 K43 H10 H35 150 8.0±0.0 9.0±0.0 8.0±0.0 5.0±0.0 0.0±0.0 0.0±0.0 75 4.0±0.0 5.0±0.0 6.0±0.0 2.0±0.0 0.0±0.0 0.0±0.0 37.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 18.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 CIPRO (8 µg/mL) 12.0±0.0 15.5±0.3 17.0±0.0 8.0±0.0 11.0±0.0 5.5±0.2 Key: Aa-Artocarpus altilis; Lb-Leaf blade; K-Klebsiella spp; H-Staphylococcus aureus Mediterranean Journal of www.mmj.org.ly Mediterr J Med Med Sci Medicine & Medical Sciences LAP Nwaneri MGU, et al. Mediterr J Med Med Sci. 2025; 1(3): 23-30. Page 28 Table 7: Antibacterial activity of Aa-Mr against MDR test organisms Concentration (mg/mL) Test organisms / inhibition zone diameter (mm) K28 K32 K38 K43 H10 H35 150 7.0±0.0 8.0±0.0 11.0±0.0 6.0±0.0 0.0±0.0 0.0±0.0 75 6.0±0.0 5.0±0.0 5.0±0.0 3.0±0.0 0.0±0.0 0.0±0.0 37.5 4.0±0.0 3.0±0.0 3.0±0.0 2.0±0.0 0.0±0.0 0.0±0.0 18.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 CIPRO (8µg/mL) 12.0±0.0 15.5±0.3 17.0±0.0 8.0±0.0 11.0±0.0 5.5±0.2 Table 8: Phytochemical analysis of the crude fungal extract Key: Aa-Artocarpus altilis; Mr-Mid rib; K-Klebsiella spp; H-Staphylococcus aureus Aa-MR: Crude fungal extract obtained from the midrib of Artocarpus altilis leaves; Aa-LB: Crude fungal extract obtained from the leaf blade of Artocarpus altilis; + = present; - = absent Discussion The isolation of 14 fungal strains from the leaf blade and midrib of A. altilis indicates the diverse presence of endophytic fungi in the plant. The higher fungal prevalence in the leaf blade suggests it may provide a more conducive microenvironment for fungal colonization, due to differences in nutrient availability or surface area compared to the midrib. This aligns with findings from the previous study [20], where distinct fungal distributions were observed across various plant tissues including the roots, stems and leaves. The variability in yields highlights differences in metabolite production capacities among the isolates. Factors such as genetic differences and substrate utilization efficiencies could explain this variation. Similar observations were reported in studies by [21], where endophytic fungi exhibited diverse metabolic profiles and extractive yields depending on isolation sources and growth conditions. The susceptibility testing confirmed the multidrug resistance of Klebsiella spp. and Staphylococcus aureus isolates. Such resistance is a significant public health concern and supports the necessity of exploring alternative antimicrobial agents, including bioactive compounds from endophytic fungi [2, 22, 23]. The fungal extracts demonstrated moderate activity against MDR-Klebsiella spp. but exhibited no activity against MDR-Staphylococcus aureus at the tested concentrations. The antibacterial efficacy exhibited by Aa-MR against Klebsiella spp. suggests that their metabolites possess significant potential for combating these gramnegative bacteria even at relatively low concentrations, though their inactivity against MDR-Staphylococcus aureus, a gram-positive strain may indicate a narrower spectrum of activity. The differential efficacy observed across extracts is consistent with the previous study [19], which noted that fungal metabolite activity can vary significantly depending on both fungal strain and bacterial target. This is in line with various works which reported that endophytic fungi isolated from various plant tissues demonstrated activities against known bacterial pathogens [23-28]. This is a novel report on the antimicrobial activity of an endophytic fungi extract isolated from A. altilis. The observed antibacterial activity of fungal extracts from A. altilis can be attributed to the presence of Phytoconstituents Test Aa-MR Aa-LB Aa-LB1 Aa-LB2 Aa-LB3 Alkaloids Dragendorff’s/Wagner’s + + + + Flavonoids Magnesium Ribbon Test + + + + Saponins Frothing + + + + Tannins Ferric Chloride + + + + Steroids Salkowski + - - + Terpenoids Liebermann–Burchard + + - - Glycosides Keller-Killiani + - + - Reducing Sugars Benedict - - - - Mediterranean Journal of www.mmj.org.ly Mediterr J Med Med Sci Medicine & Medical Sciences LAP Nwaneri MGU, et al. Mediterr J Med Med Sci. 2025; 1(3): 23-30. Page 29 various bioactive phytochemical constituents such as alkaloids, flavonoids, saponins, tannins, glycosides, steroids, and terpenoids. None of the extracts contained reducing sugars, possibly indicating the fungi's preference for producing more complex, bioactive compounds. This is a promising development, though; there is a need for further exploration of this extract. Moreso, MDR Klebsiella spp. infections are challenging to treat with conventional antibiotics, and the demonstrated efficacy of these fungal extracts could serve as the basis for developing new therapeutic agents. Conclusion: The current findings uncovered novel strains from Artocarpus altilis leaves that produce bioactive compounds with antimicrobial properties which could lead to the development of new antimicrobial agents for effective treatments against some gram-negative pathogens. References 1. Aljeldah MM. Antimicrobial resistance and its spread is a global threat. 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International Journal of Molecular Sciences. 2021; 22: 959. doi: 10.3390/ijms 22020959 Acknowledgements: The authors are thankful to the Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Awka, Anambra State, Nigeria. Authors’ contributions: MGUN conceptualized and designed the study. OAI collected data. MGUN & UAU contributed to data analysis. MGUN & UAU drafted the manuscript. All the authors read and approved the final form of the manuscript. Conflict of interest: The authors declare the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethical issues: The authors completely observed ethical issues, including plagiarism, informed consent, data fabrication or falsification, and double publication or submission. Data availability statement: The raw data that support the findings of this article are available from the corresponding author upon reasonable request. Author declarations: The authors confirm that they have followed all relevant ethical guidelines and obtained any necessary IRB and/or ethics committee approvals. Generative AI disclosure: No Generative AI was used in the preparation of this manuscript.