ABSTRACT

   Amid persistent concerns over microbial foodborne illnesses and
   escalating antibiotic resistance, we introduce “NP,” a novel and
   effective broad-spectrum natural antimicrobial product derived from the
   filtered culture broth of Aspergillus oryzae grown in a food-grade
   liquid medium. NP demonstrates potent bactericidal activity against a
   range of food-borne and ESKPAE pathogens, including Staphylococcus
   aureus (including eight distinct drug-resistant methicillin-resistant
   Staphylococcus aureus strains), Listeria monocytogenes, Salmonella
   typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and
   Escherichia coli (including O157:H7) with minimal inhibitory strength
   ranging from 25% to 100%. In addition, NP exhibits robust antifungal
   activity against several human pathogenic fungi including Aspergillus
   fumigatus, Candida albicans, and the prevalent food spoilage mold
   Penicillium species, arresting spore germination and vegetative cell
   growth. Mechanistically, NP disrupts the structural integrity of
   bacterial and fungal cell membranes, increasing membrane permeability
   and leading to cell death. Furthermore, genome-wide expression analyses
   of A. fumigatus vegetative cells exposed to NP reveal the
   downregulation of genes associated with the liveness of the fungal
   cells including ergosterol biosynthesis, cell wall maintenance, and
   development, with network analysis highlighting NP’s impact on various
   metabolic pathways. Notably, NP is presumed safe and thermally stable,
   holding promise for addressing foodborne illnesses and drug-resistant
   infections through the development and widespread application of a new
   generation of antimicrobials.

IMPORTANCE

   The development of NP, a potent broad-spectrum antimicrobial, is a
   significant breakthrough in the ongoing challenge against microbial
   foodborne illnesses and the growing threat of antibiotic resistance.
   This food-grade culture broth of Aspergillus oryzae demonstrates
   exceptional broad-spectrum efficacy against a variety of harmful
   bacteria and fungi, including drug-resistant strains such as
   methicillin-resistant Staphylococcus aureus and prevalent food spoilage
   molds. NP exhibits strong bactericidal activity against various
   foodborne and ESKAPE pathogens, and strong antifungal activity against
   Penicillium species, Aspergillus fumigatus, and Candida albicans. The
   potent bactericidal and antifungal properties of NP are a result of its
   ability to disrupt microbial cell membranes leading to increased
   permeability. Furthermore, the genome-wide impact of NP on fungal gene
   expression and metabolic pathways underscores its comprehensive
   antimicrobial action, leading to transcriptomic and metabolic changes
   associated with cell death in A. fumigatus.

   KEYWORDS: foodborne pathogens, ESKAPE pathogens, antifungal activity,
   food-fermenting fungus, broad-spectrum antimicrobial, global food
   safety

INTRODUCTION

   In the past decade, there has been a surge in global concern about the
   escalating antimicrobial resistance and foodborne illness. The Centers
   for Disease Control (CDC) report on Antibiotic Resistance Threats in
   the United States ([30]1) underscores this concern, revealing an annual
   incidence of over 2.8 million cases of antibiotic-resistant infections
   in the United States alone, leading to more than 35,000 deaths ([31]1).
   In addition, the economic impact is substantial, with an estimated
   annual cost of $17 billion attributed to foodborne illnesses in the
   United States ([32]2). Recent data from the European Union (EU) present
   an alarming trend, with a significant 44% increase in reported
   foodborne outbreaks in 2022 compared to the previous year ([33]3).
   Furthermore, the rise in infections and mortality associated with
   multidrug-resistant ESKAPE pathogens (including Enterococcus faecium,
   Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
   Pseudomonas aeruginosa, and Enterobacter species) in Europe from 2016
   to 2020 underscores the urgent need for effective antimicrobial
   strategies ([34]4, [35]5). Fungal infections, particularly those
   stemming from antimicrobial-resistant species such as Aspergillus and
   specific Candida strains, pose significant challenges to global health,
   impacting over one billion individuals worldwide and contributing to
   conditions such as chronic pulmonary aspergillosis and invasive
   candidiasis ([36]6).

   Given the gravity of antimicrobial resistance, exploring alternative
   antimicrobial agents is imperative, and natural antimicrobials derived
   from microorganisms offer promising solutions across various sectors,
   including agriculture, industry, and medicine ([37]7). Filamentous
   fungi (molds) have a long-standing history of serving as a source of
   organic acids, proteins, enzymes, and small-molecule drugs, including
   antibiotics, statins, and steroids ([38]8). Many Aspergillus species
   are known for their capacity to produce antibacterial metabolites,
   prompting exploration as potential alternatives to conventional
   antibiotics ([39]9, [40]10). Of significance is the genus Aspergillus,
   which encompasses three koji molds—Aspergillus oryzae, Aspergillus
   sojae, and Aspergillus luchuensis—certified by the Brewing Society of
   Japan ([41]11, [42]12).

   In response to this urgent global concern, we have developed a novel
   food-grade broad-spectrum antimicrobial product, designated “NP”
   (Natural Product), utilizing the Generally Recognized as Safe (GRAS)
   fungus A. oryzae. NP originates from this safe fungus grown in a liquid
   medium comprising edible components followed by filtration of the
   resulting culture broth (see [43]Fig. 1a). Our findings demonstrate
   NP’s efficacy in inhibiting the growth of foodborne pathogens, ESKAPE
   pathogens, opportunistic human pathogenic fungi, and food spoilage
   fungi. Analyses of the bacterial and fungal membrane permeability,
   fungal transcriptomic responses, and examination of protein-protein
   interaction networks in actively growing Aspergillus fumigatus cells
   have led to a potential mechanism underlying the broad-spectrum
   antimicrobial properties. Our findings underscore NP’s potential as a
   viable substitute for antibiotics in treating infections, thereby
   substantially contributing to global health security.

Fig 1.

   [44]Experimental workflow depicts spore harvesting, fungal inoculation,
   and NP extraction through filtration, followed by liquid-liquid
   extraction, concentration, and reconstitution of NP-EAE for assays.
   [45]Open in a new tab

   Schematic presentation of production of NP, NP-EAE, and NP-F. The
   process of producing NP (a), NP-EAE (b), and NP-F (c) is illustrated in
   this schematic, outlining the steps involved in each production method.
   Figures were created with Biorender.com.

RESULTS

Antibacterial activity of NP-EAE and NP-F

NP effectively inhibits the growth of food-born and ESKAPE bacterial
pathogens

   To produce NP, we tested 11 different media under various growth
   conditions, as detailed in Table S7. Our results showed that culturing
   A. oryzae NRRL 3483 in TSB and BHI media provided the best
   antimicrobial activity against both S. aureus S6 (Fig. S1a) and E. coli
   K12 FRIK 2637 (Fig. S1b). However, since BHI (Brain Heart Infusion)
   contains beef heart and calf brain extracts and is not considered a
   food-grade medium, we focused on TSB. Culturing A. oryzae NRRL 3483 in
   TSB medium, which contains glucose, a pancreatic digest of casein, and
   papain digest of soybean, for 6 days at 30°C and 220 rpm, yielded a
   potent NP (Fig. S1).

   Various organic solvents were evaluated for their ability to extract
   antimicrobial compounds from NP. Ethyl acetate extract (NP-EAE)
   demonstrated significant inhibition against S. aureus S6, whereas
   toluene and BEA extracts showed no inhibition. Consequently, ethyl
   acetate was selected as the optimal solvent (Fig. S2a), highlighting
   the importance of polar compounds in NP’s antimicrobial activity. A
   dose-response assessment of NP-EAE against S. aureus S6 revealed that a
   100 µL loading volume was optimal, producing an inhibition zone of
   approximately 20 mm (Fig. S2b).

   The antibacterial effects of NP-EAE were assessed using disk diffusion
   tests against several foodborne pathogens, including S. aureus S6,
   methicillin-resistant Staphylococcus aureus (MRSA) OC11521, L.
   monocytogenes 10403S, and E. coli K12 FRIK 2637. NP-EAE exhibited
   substantial antibacterial activity, comparable to or even surpassing 5
   µg of ofloxacin or 30 µg of cefoxitin ([46]Fig. 2a). Further validation
   in Fig. S3 showed the antibacterial performance of NP-EAE through the
   growth curves of S. aureus S6, L. monocytogenes 10403S, E. coli K12
   FRIK 2637, and S. typhimurium S9. By contrast, the autoclaved medium
   without fungal inoculation did not show significant bioactivity under
   identical testing conditions. Moreover, the heat stability of NP’s
   antimicrobial compounds suggests they are resistant to thermal
   degradation. These findings reinforce that the observed bioactivity is
   due to the metabolic activity of A. oryzae, rather than any byproducts
   of the autoclaving process.

Fig 2.

   [47]Agar diffusion assays depict zones of inhibition for various
   bacteria treated with NP-EAE, OFX, and FOX, and growth curves of six
   bacterial strains treated with different NP-F concentrations over 24
   hours.
   [48]Open in a new tab

   Antibacterial activities of NP-EAE and NP-F. (a) Zone of inhibition
   caused by NP-EAE against S. aureus S6, MRSA OC11521, L. monocytogenes
   10403S, and E. coli K12 FRIK 2637. Disks were loaded with 100 µL of the
   NP-EAE (top right disk). A disk loaded with 5 µg ofloxacin (OFX 5;
   bottom left disk) and 30 µg of cefoxitin (FOX 30; bottom right disk)
   were used as the positive controls. Methanol was used as the negative
   control (C-; top left). (b) Growth curves of S. aureus S6, MRSA
   OC11521, L. monocytogenes 10403S, E. coli K12 FRIK 2637, E. coli
   O157:H7 FRIK 47, and S. typhimurium S9 in 100%, 50%, 25%, 12.5%, and 6%
   NP-F (MHB medium) at 37°C for 24 hours. The original MHB medium (0%
   NP-F) was used as the negative control. The experiment was performed in
   triplicate (n = 3).

   For a direct assessment of NP’s efficacy without the extraction
   process, NP formulated with rich nutrient medium (NP-F) was used.
   Minimum inhibitory concentrations (MICs) were determined as a
   percentage (%) of NP-F, with 50% NP-F for S. aureus S6 and MRSA
   OC11521, 25% for L. monocytogenes 10403S, and 100% for E. coli K12 FRIK
   2637, 0157:H7 FRIK 47, and S. typhimurium S9 ([49]Fig. 2b). NP-F was
   also tested against antibiotic-resistant bacteria, including MRSA
   (known for methicillin and β-lactam resistance) and ESKAPE pathogens
   (notorious for evading antimicrobials) ([50]13, [51]14). As shown in
   Fig. S4, NP-F effectively inhibited the growth of eight MRSA strains
   even at 50% concentration. In addition, 100% NP-F completely halted the
   growth of two antibiotic-resistant S. aureus strains, MW2 and 33593
   (Fig. S5a), and impeded the growth of K. pneumoniae 81-1269A (Fig.
   S5b). It also showed inhibition against P. aeruginosa strains 2368,
   3060, and 65 to some extent (Fig. S5c), as well as E. coli 1-894-1
   (Fig. S5d). This demonstrates NP’s efficacy against
   antibiotic-resistant foodborne and ESKAPE pathogens, highlighting
   significant antibacterial activity across various bacterial strains.

NP-F efficiently kills bacterial pathogens

   To determine whether NP-mediated antibacterial activity is bactericidal
   or bacteriostatic, we carried out time-kill experiments against MRSA
   OC11521, L. monocytogenes 10403S, E. coli O157:H7 FRIK 47, and S.
   typhimurium S9. In 100% NP-F (MHB liquid medium), MRSA OC11521
   exhibited a 3-log reduction within 6 hours, with no detectable live
   cells at 24 hours ([52]Fig. 3a). In addition, NP-F eradicated 100% of
   L. monocytogenes cells within 3 hours, achieving a 3-log reduction
   within 1 hour ([53]Fig. 3b). For Gram-negative bacteria, NP-F
   eliminated all E. coli cells within 24 hours and all S. typhimurium
   cells within 36 hours ([54]Fig. 3c and d). These findings demonstrate
   the effective bactericidal activity of NP-F against both Gram-positive
   and Gram-negative bacterial pathogens. Based on the rapid killing of
   bacterial cells, we hypothesize that NP effectively eliminates
   bacterial pathogens by disrupting their cell membranes.

Fig 3.

   [55]Four line graphs represent log CFU/mL over time for MRSA OC11521,
   L. monocytogenes 10403S, E. coli O157, FRIK 47, and S. typhimurium S9.
   NP-F treated groups depict significant reduction in CFU compared to the
   control.
   [56]Open in a new tab

   Time-dependent bactericidal activity of NP-F. CFU of log-phase MRSA
   OC11521 (a), L. monocytogenes 10403S (b), E. coli O157:H7 FRIK 47 (c),
   and S. typhimurium S9 (d) incubated in 100% NP-F MHB liquid medium at
   37°C for 12–48 hours. The original MHB medium was used as the negative
   control. The experiment was performed in triplicate (n = 3).

NP disrupts bacterial cell membranes

   To test the hypothesized correlation between membrane permeabilization
   and bacterial viability, we conducted experiments to determine whether
   NP’s impact on bacterial death is due to the direct disruption of the
   inner membrane of S. aureus S6 and the outer membrane of E. coli K12
   FRIK 2637. After 6 hours of NP-EAE treatment, there were significant
   increases in SYTOX Green fluorescence intensity ([57]Fig. 4a), a
   DNA-binding dye that is normally impermeable to intact membranes
   ([58]15), and NPN fluorescence intensity ([59]Fig. 4b), a neutral
   hydrophobic fluorescent probe typically restricted by the outer
   membrane ([60]16). These results suggest that the bioactive compounds
   present in NP can rapidly permeabilize bacterial cellular membranes.

Fig 4.

   [61]Increased inner and outer membrane permeability in S. aureus S6 and
   E. coli K12 FRIK 2637 treated with NP-EAE, along with a higher release
   of 260 nm absorbing materials. Flow cytometry plots confirm greater
   cell permeability in NP-EAE-treated groups.
   [62]Open in a new tab

   Potential mechanism of bactericidal activity attributed to NP. (a)
   Inner-membrane permeability of the Gram-positive bacteria S. aureus S6
   and (b) cell outer membrane permeability of the Gram-negative bacteria
   E. coli K12 FRIK after 6 hours of exposure to NP-EAE. (c) The release
   of cellular bio-materials absorbing 260 nm wavelength from bacterial
   cells after 3 hours of exposure to NP-EAE and NP. (d) Flow cytometry
   analysis of NP-EAE after 6 hours of exposure to NP-EAE, where the
   X-axis (FL3-A) represents the fluorescence intensity of the PI dye, and
   the Y-axis (FL1-A) shows the fluorescence intensity of the Syto-9 dye
   on a log scale. The highlighted yellow regions (top right corner)
   denote high-intensity PI fluorescence. Positive controls included
   Triton-X 100 (1%), while negative controls were PBS buffer and
   methanol. *P < 0.05; **P < 0.01; ***P < 0.001; ns not significant. The
   experiment was performed in triplicate (n= 3).

   Furthermore, the release of intracellular components such as potassium,
   phosphate, nucleic acids, and other cellular materials was monitored to
   confirm membrane disruption in S. aureus and E. coli. Even after just 3
   hours of NP-EAE treatment, a significant release of these intracellular
   components was observed, indicating NP’s rapid membrane-disrupting
   effects ([63]Fig. 4c). Flow cytometry was used to further evaluate the
   damaging effects of NP-EAE on the membrane. As shown in [64]Fig. 4d,
   79.0% of S. aureus and 77.6% of E. coli cells exhibited membrane
   permeabilization, compared to less than 20% in the negative control
   (methanol). These results indicate that NP induces significant
   disruption of membrane integrity in bacterial pathogens.

Antifungal activity of NP

NP effectively inhibits the growth of food spoilage and pathogenic fungi

   We investigated NP’s potential as an antifungal agent by testing it
   against the food spoilage fungus P. roqueforti and two human
   opportunistic pathogenic fungi A. fumigatus AF293 and C. albicans
   SC5314. To explore NP’s adaptability across different applications, we
   adjusted its pH from the initial 8.5 to pH3 and pH11 using 10 N NaOH
   and 6 N HCl, respectively. This adjustment aimed to mimic a range of
   environmental conditions under which NP could function as an antifungal
   agent, thus expanding its potential practicality. As shown in [65]Fig.
   5a, NP-EAE exhibited enhanced antifungal effectiveness at a
   concentration of 60×, evidenced by distinct inhibition zones
   surrounding the 13 mm disks on a PDA medium. Specifically, at pH3,
   NP-EAE showed a 28.0 ± 1.1 mm inhibition zone against P. roqueforti,
   while at the original pH (~8.5), it achieved a 29.1 ± 1.5 mm inhibition
   zone, with no inhibition observed at pH 11. For A. fumigatus, the
   inhibition zones were 31.5 ± 2.6 mm (pH 3), 26.4 ± 0.9 mm (pH 8.5), and
   17.3 ± 2.6 mm (pH 11). Regarding C. albicans, the inhibition zones were
   27.0 ± 1.7 mm (pH 3), 21.9 ± 0.8 mm (original pH), and 19.6 ± 0.8 mm
   (pH 11). These findings underscore NP’s potent antifungal activity
   across a broad pH spectrum.

Fig 5.

   [66]Agar plates test effect of NP-EAE on P. roqueforti, A. fumigatus,
   and C. albicans at different pH levels and impact of various NP-F
   concentrations on fungal growth. Growth inhibition is observed with
   higher NP-F concentrations.
   [67]Open in a new tab

   Antifungal activities of NP-EAE and NP-F. (a) Zone of inhibition
   against P. roqueforti, A. fumigatus AF293, and C. albicans SC5314
   produced by 20× and 60× equivalents of NP-EAE. NP-EAE was prepared
   after NP was adjusted to pH 3, 8.5 (original pH), and 11. A total of
   300 µL of each extract was applied to 13 mm sterile paper disks and
   placed on PDA solid medium for P. roqueforti and A. fumigatus, and YPD
   solid agar medium for C. albicans. (b) Growth of P. roqueforti at 25°C,
   A. fumigatus AF293 at 37°C, and C. albicans SC5314 at 30°C in 2 mL of
   100%, 50%, 25%, 12.5%, and 6% NP-F (RPMI liquid medium) in a 24-well
   plate over 5 days. After incubation, aliquots from each well were
   plated on PDA solid medium for P. roqueforti and A. fumigatus, and YPD
   solid agar medium for C. albicans, and CFUs were counted. The RPMI
   medium without NP-F served as the negative control. Error bars
   represent the standard deviation of three independent experiments. *P <
   0.05; **P < 0.01; ***P < 0.001.

   To evaluate the persistence of NP’s effectiveness at various dilutions,
   NP was mixed with distilled water while preserving the same nutrient
   content required for fungal growth in RPMI liquid medium and
   subsequently evaluated for its antifungal properties. As shown in
   [68]Fig. 5b, P. roqueforti, A. fumigatus, and C. albicans were exposed
   to NP-F at concentrations ranging from 25% to 100% in 2 mL RPMI liquid
   medium. After incubation, spores from the RPMI medium were collected,
   and CFUs were counted following plating on a solid agar medium. NP-F
   significantly inhibited spore germination and growth of P. roqueforti
   compared to the control (RPMI medium with 0% NP-F), with even 25% NP-F
   showing inhibitory effects. Specifically, 100% NP-F resulted in a
   substantial 2.37 log reduction in P. roqueforti growth after 5 days.
   Similarly, A. fumigatus growth and germination were significantly
   hindered by 100% NP-F, exhibiting a significant 1.98 log difference
   from the control’s growth after 5 days. In addition, 50% and 100% NP-F
   blocked the growth C. albicans, leading to a remarkable 4.19 log
   reduction compared to the growth of C. albicans in the control group
   after 5 days.

   Furthermore, spore suspensions from various Penicillium species,
   including P. roqueforti, P. chrysogenum, and P. expansum, as well as
   five strains of A. fumigatus, were applied to PDA solid medium having
   different NP-F concentrations (10%, 25%, 50%, and 100%). Remarkably,
   the application of 100% NP-F (PDA) resulted in complete inhibition of
   growth in Penicillium species, and even a modest concentration of 10%
   NP-F (PDA) significantly impeded the growth of all five strains of A.
   fumigatus (Fig. S6). These results imply that dilutions of NP could
   offer a cost-effective and practical antifungal solution.

Time-dependent antifungal activity of NP-F and safety of NP

   To determine whether NP functions as a fungicidal or fungistatic agent,
   5 × 10^5 spores of A. fumigatus and 5 × 10^5 cells of C. albicans were
   exposed to 100% NP-F RPMI liquid medium for 48 hours. The results
   revealed a 1.54 log reduction in the viability of A. fumigatus spores,
   while the control group had already undergone germination, leading to
   mycelia growth that made it challenging to count CFU ([69]Fig. 6a). In
   addition, there was a 0.88 log reduction in cell viability for C.
   albicans cells, resulting in a substantial 4.50 log difference compared
   to the control group ([70]Fig. 6b). These findings strongly indicate
   that NP-F effectively eradicates fungal spores and cells through its
   fungicidal effect.

Fig 6.

   [71]Three plots of log CFU/mL of A. fumigatus treated with 100% NP-F
   depicting reduction over 48 hours, log CFU/mL of C. albicans with NP-F
   exhibiting significant growth reduction compared to the control, and
   cell viability percentages.
   [72]Open in a new tab

   Time-dependent antifungal activity of NP-F and NP’s non-toxic nature.
   CFU of (a) A. fumigatus AF293 and (b) C. albicans SC5314 cultured in
   100% NP-F (RPMI liquid medium) at 37°C and 30°C, respectively. The
   original RPMI liquid medium was used as the negative control. (c)
   Viability of the MCF-7 cells with varying amounts of freeze-dried
   control and NP. ns not significant. The experiment was performed in
   triplicate (n = 3).

   Importantly, a toxicity evaluation of NP was conducted using the MCF-7
   breast cancer cells, which are highly sensitive to xenobiotics due to
   their rapid cell division rate. As shown in [73]Fig. 6c, when exposed
   to up to 500 µg/mL of lyophilized NP, the cells maintained high
   viability and even at 1000 µg/mL of lyophilized NP, cell viability
   remained at approximately 60%, similar to that observed in the
   non-fermented NP-producing medium containing 2.5 g/L of glucose,
   consistent with prior report on MCF-7 cells’ sensitivity to glucose
   ([74]17). These results suggest that NP is a fermented food product
   capable of effectively and safely inhibiting various bacterial and
   fungal pathogens.

NP effectively disrupts the cell membrane of A. fumigatus

   To determine whether NP interferes with the membrane of A. fumigatus,
   indicative of a fungicidal effect, an antifungal activity test was
   conducted using spores (conidia) and vegetative cells (mycelia) of A.
   fumigatus 293. NP-F completely blocked spore germination, even in the
   presence of PDB ([75]Fig. 7a). In addition, NP inhibited hyphal growth
   and led to the formation of aggregated and compacted hyphal pellets,
   distinct from the control group ([76]Fig. 7b).

Fig 7.

   [77]Conidia and vegetative cells of A. fumigatus treated with NP-F and
   NP depict significant morphological disruption. Flow cytometry confirms
   increased cell permeability in NP-EAE treated cells, followed by alamar
   blue reduction and growth inhibition assays.
   [78]Open in a new tab

   Potential mechanism of fungicidal activity resulting from NP.
   Antifungal activity of the NP-F and NP against (a) spores and (b)
   actively growing vegetative cells (mycelia) of A. fumigatus AF293.
   Microscopic images of the fungal cells of A. fumigatus after 18 hours
   of exposure. (c). Cell membrane permeabilization levels of conidia
   (left) and 24-hour grown vegetative cells of A. fumigatus (right)
   treated with control (methanol, negative control) and NP-EAE for 6
   hours were determined by flow cytometry with the membrane-permeable
   dyes Syto-9 and PI. The X-axis (FL3-A) shows the fluorescence intensity
   of the PI dye, and the Y-axis (FL1-A) shows the fluorescence intensity
   of the Syto-9 dye in a log scale. Regions highlighted in yellow (top
   right corner) show high-intensity PI fluorescence. (d) Cell viability
   of A. fumigatus by color changes (left) and the differences in
   reduction (%, right) using four different treatments were assessed at
   0, 0.5, 1, 2, 3, 4, 5, and 6 hours after inoculation. (e) Plate
   photographs of mycelia present in 1 mL of individual treatment
   groups/conditions inoculated on PDA solid medium and incubated at 37°C
   for 24 hours. PDB and Triton-X 100 (1%) were used as positive controls
   and methanol, NP-producing medium, PBS, and buffer were used as
   negative controls. The experiment was performed in triplicate (n = 3).

   Analysis of fungal cell membrane permeability showed that over 84% of
   conidia and 97% of vegetative cells of A. fumigatus 293 exhibited
   high-intensity PI fluorescence after a 6-hour treatment with NP-EAE
   ([79]Fig. 7c), indicating greater susceptibility of vegetative cells to
   the NP-EAE than conidia. Furthermore, the AlamarBlue assay, which
   distinguishes living cells by converting resazurin to resorufin and
   emitting bright red fluorescence ([80]18), has revealed that NP started
   to kill A. fumigatus vegetative cells within 30 minutes and eliminated
   live cells at 2 hours, comparable to those of 1% Triton buffer exposure
   ([81]Fig. 7d). Subsequently, samples from the AlamarBlue assay were
   plated on PDA and no fungal cell proliferations were observed following
   treatment with NP for 1 hour or longer ([82]Fig. 7e).

Genome-wide expression response and enrichment analysis of NP-treated A.
fumigatus

   To understand how A. fumigatus cells respond to NP, RNA-seq analysis
   was conducted by comparing A. fumigatus 293 vegetative cells with and
   without NP treatment, as outlined in [83]Fig. 8a. The heat map
   generated from FPKM cluster analysis based on RNA-seq data revealed
   distinct gene expression patterns between the control and NP-treated
   groups ([84]Fig. 8b). Approximately 2.71%–5.20% of DEGs were
   upregulated, and 0.05%–5.31% were downregulated in the NP-treated group
   compared to the control group ([85]Fig. 8c). Core upregulated DEGs were
   identified, with 129 genes upregulated in the NP30 group compared to
   both the C0 and C30 groups, and 67 genes common among NP15 vs C0, NP15
   vs NP15, and NP30 group, with 35 genes exclusive to the NP30 group
   (left, [86]Fig. 8d). The core upregulated 67 and 35 DEGs are listed in
   Tables S1 and S2, respectively. The core downregulated genes included
   only three genes shared by the NP30 group and both the C0 and C30
   groups, while 10 genes were downregulated in NP30 vs C0, and 517 genes
   in NP30 vs C30. Out of the 517 genes, flbD which encodes a MYB family
   developmental regulator ([87]19) is the sole downregulated DEG
   consistently identified among the NP-treated groups (Table S3). In
   addition, 328 genes were downregulated in both NP30 vs C30 and NP15 vs
   C15 (right, [88]Fig. 8d). Core downregulated DEGs in A. fumigatus upon
   NP exposure are listed in Table S4, with additional 189 downregulated
   DEGs listed in Table S5.

Fig 8.

   [89]Experimental workflow with A. fumigatus inoculation, RNA
   extraction, and sequencing. Heatmap displays gene expression changes,
   with pie charts and Venn diagrams summarizing DEGs. Cellular impacts
   and NP-affected metabolic pathways.
   [90]Open in a new tab

   Transcriptomic response of A. fumigatus to NP treatment. (a) Schematic
   representation of the experimental design for treating A. fumigatus
   AF293 vegetative cells with NP for RNA-seq sample collection. (b)
   Heatmap results from FPKM cluster analysis, clustered using the
   log2(FPKM + 1) value. Red indicates genes with high expression levels,
   and green indicates genes with low expression levels. (c) Summary of
   DEGs in A. fumigatus obtained from different treated groups; C15 vs C0,
   C30 vs C0, NP15 vs C0, NP15 vs C15, NP30 vs C0, and NP30 vs C30. (e)
   Schematic representation illustrating the transcriptomic responses of
   A. fumigatus exposed to NP. (f) Ergosterol biosynthetic pathway of A.
   fumigatus and downregulated genes affected by NP. RNA-seq was performed
   in triplicate (n = 3).

   The analysis of NP’s impact on A. fumigatus revealed significant
   insights into its mechanism of action and specific effects. [91]Table 1
   and [92]Fig. 8e present a breakdown of functional categories in A.
   fumigatus, encompassing ergosterol biosynthesis, cell wall maintenance,
   asexual development, cell stress, cell cycle/division, transport
   proteins, and DNA repair. The impairment of these critical cellular
   processes may contribute to the overall reduction in fungal growth and
   reproduction, aligning with the antifungal activity observed for NP.

TABLE 1.

   Functional classifications of DEGs in A. fumigatus on exposure to NP
   Gene ID Gene name Description log2 fold change
   Ergosterol biosynthesis
    [93]AFUA_1g11230 hmg2 HMG-CoA reductase 1.08
    [94]AFUA_6G14200 erg10 Putative acetyl-CoA acetyltransferase −2.35
    [95]AFUA_4G12040 erg7B Putative lanosterol synthase −1.66
    [96]AFUA_1G03150 erg24 Putative C-14 sterol reductase −1.61
    [97]AFUA_1G02570 erdS Ergosteryl-3beta-O-aspartate synthase −1.34
   Cell wall maintenance/remodeling
    [98]AFUA_7G08490 Putative class V chitinase 3.85
    [99]AFUA_1G17250 rodB Conidial hydrophobin 3.38
    [100]AFUA_3G00610 Putative glucan 1,4-alpha-glucosidase 2.64
    [101]AFUA_5G10520 Alpha-1,2-mannosidase family protein 2.32
    [102]AFUA_2G00930 Putative xylosidase/glycosyl hydrolase 2.31
    [103]AFUA_2G01860 Integral membrane protein 1.56
    [104]AFUA_6G02845 EutQ Putative ethanolamine utilization protein 1.45
    [105]AFUA_1G05360 CAIB/BAIF family enzyme 1.31
    [106]AFUA_3G03060 aspf34 cell wall protein PhiA, Allergen −3.17
    [107]AFUA_6G09310 Putative class V chitinase −2.48
    [108]AFUA_4G04180 chsB Putative class II chitin synthase −1.91
    [109]AFUA_6G03600 Pth11 Putative integral membrane protein −1.64
    [110]AFUA_5G06670 Integral membrane protein −1.55
    [111]AFUA_4G03240 Mp1 Cell wall serine-threonine-rich
   galactomannoprotein −1.41
    [112]AFUA_4G11720 Putative phosphatidyl synthase −1.18
    [113]AFUA_4G02970 Aqy1
   TMEM16 Putative plasma membrane channel protein, dual function calcium
   channel/ scramblase protein −1.12
    [114]AFUA_3G12330 Putative phosphatidyl synthase −1.06
   Asexual development
    [115]AFUA_1G17250 rodB Conidial hydrophobin 3.38
    [116]AFUA_1G03210 flbD MYB family conidiophore development protein
   −2.27
    [117]AFUA_2G13770 flbC Putative C2H2 zinc finger transcription factor
   −2.25
    [118]AFUA_5G06410 AmdA Putative C2H2 transcription factor −2.04
    [119]AFUA_2G08470 bud4 GTP binding protein −1.81
    [120]AFUA_3G11250 ace2 Conidiophore development regulator −1.08
    [121]AFUA_2G07550 Ark1 Putative serine/threonine protein kinase −1.07
   Cell stress
    [122]AFUA_8G00100 Putative aspartate-tRNA ligase 3.20
    [123]AFUA_8G06020 Glutamate decarboxylase 2.86
    [124]AFUA_3G02000 Putative C6 transcription factor Ctf1B-like 2.32
    [125]AFUA_5G09100 mpkC MAP kinase 2.19
    [126]AFUA_6G07040 atg5 Putative autophagy protein 1.75
    [127]AFUA_2G00910 Pfs, NB-ARC, and TPR domain protein 1.65
    [128]AFUA_6G07050 Putative Na+/H + antiporter 1.53
    [129]AFUA_4G03190 tpsC Putative alpha, alpha-trehalose-phosphate
   synthase subunit 1.43
    [130]AFUA_7G08320 Putative heat shock transcription factor 1.17
    [131]AFUA_4G01350 gprK G-protein coupled receptor −2.09
    [132]AFUA_6G13570 Putative cytochrome c peroxidase −2.03
    [133]AFUA_2G09850 Putative oxidoreductase, 2-nitropropane dioxygenase
   family −2.02
    [134]AFUA_3G09640 Putative cAMP-independent regulatory protein −1.94
    [135]AFUA_5G14060 rho4 Putative Rho-type GTPase −1.94
    [136]AFUA_7G02570 TinC NIMA-interacting protein −1.52
    [137]AFUA_4G00860 dprA Dehydrin-like protein, cell surface protein
   −1.51
    [138]AFUA_1G17370 scf1 Putative heat shock protein −1.35
    [139]AFUA_6G12180 dprB Dehydrin-like protein −1.28
    [140]AFUA_8G04920 Late embryogenesis abundant (LEA) domain protein
   −1.22
    [141]AFUA_7G04520 dprC Dehydrin-like protein −1.08
   Cell cycle/division
    [142]AFUA_7G01400 BimC Putative kinesin family protein −2.61
    [143]AFUA_2G16260 Ase1 Putative microtubule-associated protein −1.96
    [144]AFUA_8G05680 Putative serine/threonine protein kinase −1.80
    [145]AFUA_3G12820 Kinesin family protein −1.62
    [146]AFUA_3G10250 Cdc15 Putative cell division control protein −1.61
    [147]AFUA_2G04140 Spc105 Putative chromosome segregation protein −1.48
    [148]AFUA_3G12250 cdcA Putative tyrosine-protein phosphatase CDC14
   −1.45
    [149]AFUA_3G10180 HEC/Ndc80p family protein −1.45
    [150]AFUA_6G02670 nimA Putative cell-cycle regulated serine/threonine
   protein kinase, G2-specific protein kinase −1.24
    [151]AFUA_1G14730 Cdc20 Putative cell division cycle protein −1.24
    [152]AFUA_6G12980 Putative spindle pole body component −1.22
    [153]AFUA_6G08200 nimT
   Mih1 Putative M-phase inducer phosphatase
   Putative cell cycle control protein tyrosine phosphatase −1.15
    [154]AFUA_6G08530 Src1 Putative sister chromatid separation protein
   −1.12
    [155]AFUA_2G02170 smc4 Putative nuclear condensin complex subunit
   −1.07
    [156]AFUA_2G03150 Kinesin family protein −1.06
   Transport proteins
    [157]AFUA_7G00220 Putative plasma membrane hexose transporter 2.15
    [158]AFUA_3G02150 Putative MFS monocarboxylate transporter 2.13
    [159]AFUA_6G07050 Putative Na+/H + antiporter 1.53
    [160]AFUA_2G09460 Potassium transporter 1.53
    [161]AFUA_3G02890 Putative MFS sugar transporter 1.40
    [162]AFUA_6G02810 Putative low-affinity copper transporter 1.29
    [163]AFUA_6G03690 ena1 Putative P-type ATPase sodium pump −9.06
    [164]AFUA_4G01560 Putative MFS myo-inositol transporter −7.88
    [165]AFUA_4G09440 Putative P-type ATPase sodium transporter −5.85
    [166]AFUA_3G01370 Putative MFS transporter −4.76
    [167]AFUA_8G02550 Putative MFS peptide transporter −4.40
    [168]AFUA_8G05710 mfsA Putative MFS sugar transporter −3.94
    [169]AFUA_4G00800 Putative monosaccharide transporter −3.55
    [170]AFUA_3G11790 Putative galactose-proton symport −3.06
    [171]AFUA_8G06870 Putative MFS sugar transporter −2.99
    [172]AFUA_8G02010 Putative MFS sugar transporter −2.95
    [173]AFUA_1G16690 Putative MFS sugar transporter −2.75
    [174]AFUA_4G02870 Putative 2-ketogluconate transporter −2.40
    [175]AFUA_1G04780 pxa1 Putative peroxisomal ABC transporter −2.38
    [176]AFUA_1G14330 abcC Putative ABC transporter, Azole transporter
   −2.35
    [177]AFUA_7G00950 Putative MFS monosaccharide transporter −2.25
    [178]AFUA_2G12500 Putative MFS multidrug transporter −2.25
    [179]AFUA_3G01840 Putative MFS transporter −2.25
    [180]AFUA_6G03060 Putative MFS monosaccharide transporter −2.14
    [181]AFUA_4G03750 Putative phthalate transporter −2.05
    [182]AFUA_6G03720 Putative MFS allantoate transporter −2.02
    [183]AFUA_4G00150 Putative MFS maltose transporter −2.01
    [184]AFUA_5G02840 Putative MFS sugar transporter −1.88
    [185]AFUA_7G06390 MalP Putative MFS alpha-glucoside transporter, MFS
   maltose permease −1.74
    [186]AFUA_7G00780 Putative MFS monocarboxylate transporter −1.73
    [187]AFUA_6G00130 Putative MFS transporter −1.57
    [188]AFUA_8G00720 Putative amino acid transporter −1.48
    [189]AFUA_2G12550 Putative MFS multidrug transporter −1.43
    [190]AFUA_4G09150 Putative ABC multidrug transporter −1.38
    [191]AFUA_5G11980 MFS efflux transporter −1.33
    [192]AFUA_6G00440 Putative cation diffusion facilitator −1.27
    [193]AFUA_2G14590 Putative MFS monosaccharide transporter −1.16
    [194]AFUA_8G06760 Putative MFS transporter −1.09
   DAN repair
    [195]AFUA_3G12280 Translin Putative recombination hotspot-binding
   protein 1.43
    [196]AFUA_1G07000 Rad26 Putative DNA repair protein 1.13
    [197]AFUA_2G16260 Ase1 Putative microtubule-associated protein −1.96
    [198]AFUA_5G06120 Rfx1 Putative DNA damage and replication checkpoint
   protein −1.36
    [199]AFUA_3G14260 Putative mismatched base pair and cruciform DNA
   recognition protein −1.27
    [200]AFUA_4G04390 tof1 Putative topoisomerase 1-associated factor 1,
   DNA repair protein −1.14
   [201]Open in a new tab

   In particular, ergosterol, a vital component in the fungal cell
   membrane, plays a crucial role in development and growth and is a
   primary target for existing antifungal drugs ([202]20). The
   downregulation observed in genes such as ERG10, ERG7B, ERG24, and ERDS
   within the ergosterol biosynthesis pathway indicated that NP may impede
   the production of key intermediates like acetoacetyl-CoA, lanosterol,
   and 4,24-dimethylcholesta-8,24,(28)-dien-3β-ol. This parallels the
   effects seen with antibiotics like AMB and azole antifungals, which
   suppress genes such as ERG6 ([203]21) and ERG11 (CYP51A/B) ([204]22),
   respectively, targeting ergosterol biosynthesis. This indicates a
   potential blockade in the ergosterol biosynthesis pathway induced by
   NP, providing insights into its antifungal mechanism ([205]Fig. 8f).

Gene regulatory network of NP-treated A. fumigatus

   The regulatory effects of NP on A. fumigatus were investigated through
   an extensive network analysis, integrating RNA-seq data with the
   protein-protein interaction (PPI) database. The resulting network,
   comprising 22 clusters, contained 395 nodes and 1,113 edges, derived
   from 328 downregulated DEGs and 67 upregulated DEGs ([206]Fig. 9), with
   their functional enrichment detailed in Table S6. Cluster 1, comprising
   29 nodes and 258 edges, emerged as a focal point enriched in pathways
   related to unsaturated fatty acid biosynthesis, beta-alanine
   metabolism, fatty acid degradation, propanoate metabolism, and
   peroxisome function. Peroxisomes, known for their involvement in
   various metabolic processes including fatty acid breakdown and
   detoxification through hydrogen peroxide generation ([207]23, [208]24),
   were particularly prominent. These findings suggest that NP may exert
   its antifungal properties by affecting multiple enzymes involved in
   critical metabolic processes, offering valuable insights into its
   multifaceted regulatory effects on A. fumigatus.

Fig 9.

   [209]Network map of gene/protein interactions, with nodes representing
   genes or proteins and edges indicating interactions. Densely connected
   clusters suggest functional relationships, highlighting key interaction
   hubs among the genes or proteins.
   [210]Open in a new tab

   PPI in the degree-sorted network of 328 downregulated DEGs and 67
   upregulated DEGs. PPI was constructed in the STRING database and
   depicts the interactions among the shared DEGs with a confidence score
   of 0.4 with at least three nodes connected. The node colors represent
   MCL clustering with granularity parameters as 4.

DISCUSSION AND CONCLUSIONS

   Antibiotic resistance is a pressing global concern, echoing Alexander
   Fleming’s early warnings about antibiotic misuse. The emergence of
   penicillin-resistant E. coli in 1940 highlighted Fleming’s concerns
   ([211]25). A meta-analysis pooling data from various studies on
   antibiotic resistance in pathogens isolated from food revealed alarming
   statistics. Over 11% of foodborne pathogens showed antimicrobial
   resistance, with multi-drug resistant (MDR) pathogens exceeding 36%
   across all food categories. In addition, biofilm formation capacity
   among foodborne pathogens, contributing to antimicrobial resistance
   (AMR), was observed, with S. aureus exhibiting the highest propensity
   ([212]26). Despite this, there is no comprehensive international
   surveillance system for AMR. However, reports suggest over 2 million
   AMR infections occur annually in the United States, leading to around
   29,000 deaths. Similarly, in Europe, over 33,000 deaths are linked to
   AMR infections ([213]27).

   ESKAPE pathogens, part of AMR pathogens, are critical targets for new
   antibiotic development due to the decreasing efficacy of existing
   antibiotics, signaling a worrying trend ([214]5), and necessitating
   alternative strategies. Unlike the diversity of antibiotics for
   bacterial infections, antifungal options are limited primarily to
   azoles, echinocandins, and polyenes like amphotericin B ([215]6).
   Fungal infections cause about 1.5 million deaths, with over 90%
   attributed to Cryptococcus, Candida, Aspergillus, and Pneumocystis
   species, known for causing invasive fungal infections ([216]28).

   Innovative antimicrobial approaches such as engineered antibodies,
   antibiotic enhancers, and CRISPR/Cas-enabled genome editing are
   emerging, albeit with significant costs and time requirements ([217]29,
   [218]30). In addition, there is a growing consumer preference for
   minimally processed, “clean label” products, driving interest in
   natural antimicrobials perceived as safer and environmentally friendly
   ([219]31). Consequently, fermentation technology introduces
   “postbiotics,” defined as “preparations of inanimate microorganisms
   and/or their components that confer a health benefit on the host,”
   holding significant potential in antimicrobial therapy ([220]32).

   We anticipate NP’s stability and potent antimicrobial activity to make
   it a formidable broad-spectrum agent against both AMR bacterial and
   fungal pathogens. NP can eradicate MRSA within 6 hours, L.
   monocytogenes within 3 hours, E. coli O157:H7 within 24 hours, and S.
   typhimurium within 36 hours, achieving a 3-log reduction in bacterial
   counts. It also inhibits biofilm-producing ESKAPE strains like K.
   pneumoniae and P. aeruginosa, even in nutrient-rich environments. NP
   impedes spore germination and halts the growth of A. fumigatus
   vegetative cells, including clinically isolated strains CEA-10 and
   CEA-17. In addition, it shows fungistatic effects against C. albicans
   for the first 24 hours, with some cidal activity observed after 24
   hours. NP can also target food spoilage-causing Penicillium species
   such as P. roqueforti, P. chrysogenum, and P. expansum. Various NP
   application methods can be envisioned, including soaking/misting
   susceptible food products with NP, employing NP for medical purposes in
   treating skin infections or incorporating a lyophilized NP tablet into
   different food items.

   Treating fungal infections has been challenging due to the similarity
   between fungi and human hosts, both being eukaryotic organisms.
   However, RNA-seq analysis of A. fumigatus revealed downregulation of
   genes associated with ATP-binding cassette (ABC) superfamily and major
   facilitator superfamily (MFS) transporters, potentially reducing NP
   efflux ([221]33) and enhancing its antifungal efficacy. Moreover,
   examination of DEGs associated with cell wall maintenance and
   remodeling showed NP’s multifaceted impact on fungal physiology. This
   included the upregulation of genes like conidial hydrophobin B (RodB),
   pivotal for constructing the outer cell wall of conidia ([222]34), and
   chitinase activity genes essential for processes such as conidial
   germination, mycelial formation, and cell wall expansion ([223]21,
   [224]35). In addition, the downregulation of genes associated with
   conidiation, such as the MYB family conidiophore development protein
   (flbD) and C[2]H[2] zinc finger transcription factor (flbC), indicated
   a potential impact on the formation of asexual spores ([225]19). The
   modulation of genes encoding integral membrane proteins, cell wall
   proteins, and chitinase indicated a concerted effort by the fungus to
   maintain cell wall integrity under NP exposure, possibly as a survival
   strategy. Moreover, although NP likely affects membrane permeability,
   our findings suggest that its impact extends beyond mere membrane
   disruption. Unlike detergents like SDS, which cause non-specific
   membrane damage, NP appears to interact with specific cellular targets,
   leading to complex transcriptional responses. This is evidenced by the
   diverse pathways affected, indicating that NP induces a broader range
   of cellular responses. Thus, the transcriptomic changes observed are
   likely reflective of a more intricate interaction between NP and fungal
   cellular pathways, rather than being solely attributable to cell death
   or membrane disruption.

   In conclusion, this study introduces a food-grade broad-spectrum
   antimicrobial fermentate applicable to both food sources and medicine
   for pathogen control, enhancing antibiotic effectiveness, and
   addressing antimicrobial resistance. NP serves as a promising “clean
   label” solution for diverse applications across pharmaceutical and food
   industries, paving the way for safeguarding global health and food
   safety.

MATERIALS AND METHODS

Fungi and culture conditions

   For NP production, the mold strain A. oryzae NRRL 3483 was used.
   Various strains of A. fumigatus (AF293, [226]F16216, [227]F11628,
   CEA-10, and CEA-17) and Penicillium species (P. roqueforti, P.
   chrysogenum, P. expansum Pe21, and P. expansum Pe19) were employed to
   assess NP’s antifungal activity. For inoculum preparation, A. oryzae
   was cultured on potato dextrose agar (PDA) for 4 days at 30°C, while A.
   fumigatus strains and Penicillium species were cultured at 37°C for 3
   days and at 25°C for 5 days, respectively. A 0.1% Tween-80 solution was
   used to harvest asexual spores (conidia) from the PDA, followed by
   enumeration using a hemocytometer. To assess the antifungal activity of
   A. fumigatus vegetative cells, a spore suspension (10^6 conidia/mL) was
   inoculated into potato dextrose broth (PDB) and incubated at 37°C for
   18–24 hours with shaking at 220 rpm. After incubation, A. fumigatus
   mycelia were harvested by filtration through four layers of Miracloth
   (MilliporeSigma, USA). C. albicans SC5314 was used to evaluate NP’s
   anti-yeast activity, with single colonies cultured on yeast peptone
   dextrose (YPD) agar plates at 30°C overnight.

Bacteria and culture conditions

   The antibacterial efficacy of NP was evaluated against a spectrum of
   bacterial strains including L. monocytogenes 10403S, S. aureus S6, E.
   coli K12 FRIK 2637 (non-pathogenic strain), E. coli O157:H7 FRIK 47,
   and S. enterica typhimurium S9. In addition, testing extended to highly
   virulent and antibiotic-resistant ESKAPE strains, comprising two S.
   aureus strains (MW2, 33593), eight methicillin-resistant S. aureus
   (MRSA) strains (OC11521, two isolates of OC4222, three isolates of
   OC8530, 04-045, OC17042), two K. pneumoniae strains (81-1269A,
   BAA-2146), three P. aeruginosa strains (2638, 3060, 65), and two E.
   coli strains (2671, 1-894-1).

   Bacterial suspensions were prepared following established protocols
   with slight adjustments ([228]36, [229]37). Isolated colonies were
   selected from culture plates and transferred into 4–5 mL of tryptic soy
   broth (TSB) liquid medium for all bacterial strains. Cultures were then
   incubated at 37°C for 2–6 hours to reach the log phase of growth, as
   determined by the 0.5 McFarland standard, corresponding to
   approximately 1.0 × 10^8 CFU/mL when the optical density fell between
   0.08 and 0.1. Prior to the experiment, a 1:20 dilution was made by
   mixing 2.0 mL of the original suspension with 38 mL of PBS buffer,
   followed by a 1:10 dilution by adding 0.01 mL to each well to achieve a
   final concentration of approximately 5.0 × 10^5 CFU/mL.

Preparation of media for NP determination

   To identify the optimal medium for NP evaluation, several media with
   distinct compositions were prepared. These included Tryptic Soy Broth
   (TSB), Yeast Peptone Dextrose (YPD), De Man, Rogosa & Sharpe (MRS),
   Luria-Bertani (LB), M17, Mueller Hinton (MH), Brain Heart Infusion
   (BHI), Czapek (CZ), Potato Dextrose Broth (PDB), Malt Extract Broth
   (MEB), and Minimal Medium (MM). The composition of each medium (in
   grams per liter of distilled water) is detailed in Table S7. All liquid
   media were autoclaved at 121°C and 15 PSIG for 20 minutes to ensure
   sterilization.

Preparation of NP

   The full-strength culture medium for NP (TSB) was prepared by
   dissolving all ingredients (refer to Table S8) and subsequently
   sterilized under high pressure (15 PSIG for 20 minutes at 121°C). To
   initiate NP production, A. oryzae NRRL 3483 was inoculated at a final
   concentration of 5 × 10^5 conidia/mL into 250 mL Erlenmeyer flasks
   containing 150 mL of NP culture medium. The inoculated flasks were
   incubated for 6 days at 30°C with shaking at 220 rpm. Post-incubation,
   mycelia were separated from the culture broth through filtration using
   four layers of Miracloth (MilliporeSigma, USA). Subsequently, “NP,” a
   sterile, cell-free culture fermentate, was obtained by filtration
   through a 0.22-µm polyethersulfone (PES) membrane filter unit (Thermo
   Fisher Scientific, USA). The schematic representation of the NP
   production process is illustrated in [230]Fig. 1a.

Preparation and assessment of NP-EAE)

Preparation of NP-EAE via liquid-liquid extraction

   The antimicrobial activity of NP extracts, obtained via liquid-liquid
   extraction using different organic solvents, was assessed. Details
   regarding the solvents utilized are provided in Table S9, while the
   procedure for producing NP-EAE is outlined in [231]Fig. 1b. Ethyl
   acetate extracts exhibited potent antibacterial activity among the
   solvents tested, therefore referred to as “NP-EAE.”

Preparation of NP-EAE via liquid-liquid extraction

   Antimicrobial activity screening of NP extracts was carried out using
   liquid-liquid extraction with various organic solvents (listed in Table
   S9). In 50 mL conical tubes, 20 mL of NP was mixed with an equal volume
   of the respective organic solvent (vol/vol) and incubated overnight at
   25°C with shaking at 150 rpm. After centrifugation at 5,000 g, the
   organic layer was transferred to a new tube. Extracts were dried under
   gentle airflow, reconstituted in 1 mL of methanol, and filtered using a
   sterile 0.45-µm PES membrane filter unit (Thermo Fisher Scientific,
   USA). The resulting methanol solution was then tested for antimicrobial
   activity, with methanol serving as the negative control. Among the
   solvents tested, ethyl acetate extracts exhibited potent antibacterial
   activity. Subsequent experiments focused on the ethyl acetate extract,
   referred to as “NP-EAE.” A concentrated ethyl acetate extract
   equivalent to 20 times the NP fermentate, labeled as “20×,” was
   obtained through a subsequent ethyl acetate extraction on 1 mL of the
   methanol solution. This process was repeated three times, resulting in
   “60×” NP-EAE.

Agar diffusion disk test assay

   Agar diffusion disk testing, with slight modifications, was conducted
   to assess the antibacterial and antifungal activities of NP-EAE,
   following a previously described protocol based on CLSI guidelines
   ([232]36[233]–[234]38). For antibacterial activity assessment, 100 µL
   of NP-EAE was applied to 6 mm sterile paper disks, while for antifungal
   activity evaluation, 300 µL of NP-EAE was applied to 13 mm sterile
   paper disks. The paper disks were dried under the fume hood and placed
   on the surface of the TSA plates for antibacterial activity and PDA
   plates for antifungal activity. The plates were inverted and incubated
   at 37°C for 18–22 hours to cultivate bacteria. As for Penicillium sp.
   and Aspergillus sp., they were incubated at 25-37°C, for 4–6 days.
   Positive controls for bacterial testing included disks loaded with 5 µg
   ofloxacin (OFX 5; OXOID Ltd, USA), 30 µg of cefoxitin (FOX 30; OXOID
   Ltd, USA), and 10 µg Ampicillin (Sigma-Aldrich, USA). Positive controls
   for antifungal activity included amphotericin B (AMB; Sigma-Aldrich,
   USA) at a final concentration of 4 µg/mL.

Preparation and assessment of NP-F

Preparation of NP-F

   In preparation of NP-F for evaluating both antibacterial and antifungal
   activities, NP solutions were prepared by diluting NP in distilled
   water at varying concentrations (i.e., 100%, 50%, 25%, 12.5%, 6%, and
   0%) described in [235]Fig. 1c. These solutions were then used as
   solvents to produce 22 g/L Mueller Hinton Broth (MHB) contained 2.0 g
   beef extract, 17.5 g acid hydrolysate of casein and 1.5 g starch for
   antibacterial testing ([236]37), and the antifungal activity test broth
   having 10.4 g/L RPMI 1640 powder supplemented with 2% glucose and
   3-(N-morpholino) propanesulfonic acid (MOPS) at a final concentration
   of 0.165 mol/L (pH 7.0) ([237]38) or 39 g/L PDA solid medium.

Time-kill assay in NP-F liquid medium

   A modified version of the broth microdilution method was conducted to
   evaluate the antibacterial and antifungal properties of NP-F, following
   a previously described protocol based on CLSI guidelines with
   modifications ([238]36[239]–[240]38). The original nutrient medium
   served as a negative control.

Fungal growth on NP-F solid medium

   Briefly, 10 µL of spore suspensions from Penicillium species and five
   strains A. fumigatus were spot plated onto NP-F (PDA). The spore
   concentrations varied from 10^1 to 10^4 spores per spot. In addition, a
   control group with 0 spores per spot was inoculated with 10 µL of
   distilled water, as the negative control.

Membrane permeability assay

Membrane integrity in bacterial cells using fluorescent dyes

   Using an electron fluorescence spectrometer (Tecan, Switzerland), inner
   and outer membrane integrity assays were conducted with specific
   fluorescent dyes, as depicted in Fig. S7. The influx assay utilized
   SYTOX Green (Invitrogen, USA) to evaluate the permeability of S. aureus
   S6’s inner membrane, following established protocols with slight
   modification ([241]39). SYTOX Green’s excitation and emission
   wavelengths were measured at 480 and 522 nm, respectively, with 20 µL
   of 5 µM dye added to the wells. For assessing the permeability of E.
   coli K12 FRIK 2637’s outer membrane, an N-phenyl-1-napthylamine (NPN)
   assay (Sigma-Aldrich, USA) was employed. Excitation and emission
   wavelengths were set at 350 and 429 nm, respectively, with samples
   mixed with 20 µL of NPN solution (final concentration: 10 µM) as
   described previously ([242]40).

   To prepare NP-EAE samples, 100 µL of NP-EAE (reconstituted in methanol)
   was transferred into a sterile 96-well plate and dried under a fume
   hood. Subsequently, 160 µL of PBS buffer was added to the wells.
   Following this, 20 µL of bacterial cultures (~10^5 CFU/mL, obtained via
   serial dilution of overnight bacterial cultures) was added to each well
   and incubated them at 37°C for 6 hours.

Release of 260 nm absorbing materials from bacterial cytoplasm

   The release of materials such as nucleic acids from the cytoplasm was
   detected by measuring absorbance at 260 nm after 3-hour treatments
   following the procedure outlined previously ([243]41).

Flow cytometry

   To corroborate the observations on cell membrane permeability
   elucidated earlier, flow cytometry (BD Accuri, USA) was employed. After
   a 6-hour incubation of bacterial cells and fungal spores with NP-EAE
   and control, SYTO-9 and propidium iodide (PI) (Invitrogen, USA) were
   added at concentrations of 7.5 µM and 30 µM, respectively, according to
   previously outlined protocols ([244]42). Fluorescence intensities were
   measured using a green fluorescence (525/50 nm) bandpass filter for
   SYTO 9 and a red fluorescence (610/20 nm) bandpass filter for PI.

Alamarblue HS cell viability assay

   The impact of NP on the growth of A. fumigatus mycelia (actively
   growing vegetative cells) was assessed by quantifying cell viability
   and proliferation using the Alamarblue HS Cell Viability assay (Thermo
   Fisher Scientific, USA) following the manufacturer’s protocol. Five
   grams of harvested mycelia, which were cultivated for 18 hours in PDB,
   subjected to four different solutions: NP-producing fungus-free medium
   (negative control), PBS buffer (negative control), NP, and 1% Triton
   buffer (positive control), all at a volume of 25 mL. After 0, 0.5, 1,
   2, 3, 4, 5, and 6 hours of incubation, 10 µL of Alamarblue reagent was
   added to 90 µL of mycelia suspended in PDB liquid medium. Fluorescence
   was measured using a fluorescence excitation wavelength of 570 nm (with
   an excitation range of 540–570 nm) and an emission wavelength of 600 nm
   (with an emission range of 580–610 nm). The percentage reduction of
   Alamarblue reagent was determined at predefined time intervals using
   Eq. 1 according to the manufacturer’s instructions.

Mammalian cell cytotoxicity

   NP along with controls was lyophilized using a freeze dryer (Yamato,
   USA) in preparation for cell cytotoxicity test assay. A mammalian cell
   cytotoxicity assay was conducted by the Cancer Pharmacology Lab at the
   University of Wisconsin-Madison. The lyophilized samples were
   reconstituted in sterile molecular biology-grade water and were
   dispensed onto white 384-well plates (Corning 3765, Thermo Fisher, USA)
   at final concentrations ranging from 10 to 1,000 µg/mL using an Echo
   650 acoustic liquid handler (Beckman Coulter, USA). MCF7 cells (breast
   cancer cell line) were then exposed to the samples for 72 hours, and
   toxicity was assessed using the CellTiter-GLO reagent (Promega, USA)
   following the manufacturer’s protocol. Doxorubicin, a chemotherapy
   drug, served as a positive control at a concentration of 2.5 µM.

Genome-wide expression studies

Preparation of RNA samples

   Samples for RNA sequencing analysis were treated as detailed in
   [245]Fig. 8a. Following treatments at distinct time points, total RNAs
   from individual samples were extracted using RNeasy Kits (Qiagen,
   Germany) following the manufacturer’s instructions.

RNA sequencing analysis

   The total RNA samples underwent quality assessment, library
   preparation, and mRNA sequencing by Novogene company (Beijing, China).
   The quality of total RNA was validated as described ([246]43). The
   genome and gene annotations for A. fumigatus AF293 were from the
   National Center for Biotechnology Information (NCBI) website
   ([247]https://www.ncbi.nlm.nih.gov/; [248]GCA_002234985.1). The
   differentially expressed genes (DEG) level was processed using DESeq2
   (version 1.20.0). Genes with an adjusted P-value  <  0.05 and
   |log2(FoldChange)|  > 0 were considered as the threshold for
   significant differential expression.

Protein-protein interaction analysis

   A network analysis was performed by integrating the results of RNA-seq
   with the protein-protein interaction (PPI) database. Subsequently,
   known PPI information of A. fumigatus AF293 was obtained from the gene
   network database STRING (version 11.5) ([249]44). This network
   illustrates interactions among the shared DEGs with a confidence score
   of 0.4 and at least three connected nodes. Each cluster within the
   network was subjected to KEGG pathway enrichment analysis to identify
   functional enrichments ([250]45).

Appendices

   [MATH: <mstyle displaystyle="true" scriptlevel="0"><mrow><mi
   mathvariant="normal">%</mi><mtext> Alamarblue
   Reduction </mtext><mo>=</mo><mfrac><mrow><mo
   stretchy="false">(</mo><mtext> Eoxi </mtext><mn>600</mn><mo>×</mo><mrow
   ><mi mathvariant="italic">A</mi><mn>570</mn></mrow><mo
   stretchy="false">)</mo><mo>−</mo><mo
   stretchy="false">(</mo><mtext> Eoxi </mtext><mn>570</mn><mo>×</mo><mi>A
   </mi><mn>600</mn><mo
   stretchy="false">)</mo><mo>×</mo><mn>100</mn></mrow><mrow><mo
   stretchy="false">(</mo><mtext> Ered </mtext><mn>570</mn><mo>×</mo><mi>C
   </mi><mn>600</mn><mo stretchy="false">)</mo><mo>−</mo><mo
   stretchy="false">(</mo><mtext> Ered </mtext><mn>600</mn><mo>×</mo><mrow
   ><mi mathvariant="italic">C</mi><mn>570</mn></mrow><mo
   stretchy="false">)</mo></mrow></mfrac></mrow></mstyle> :MATH]
   (1)

   where;

   E[oxi600] & E[oxi570]: E of oxidized Alamarblue at 600 nm (117216) and
   570 nm (80586),

   E[red600] & E[red570]: E of reduced Alamarblue at 600 nm (14652) and
   570 nm (155677),

   A[600] & A[570]: Absorbance of test wells at 600 nm and 570 nm, and

   C[600] & C[570]: Absorbance of negative control well (media plus
   alamarblue but no cells) at 600 nm and 570 nm

ACKNOWLEDGMENTS