Production and Characterization of Novel Board-spectrum Antimicrobial 5-butyl-2-pyridine carboxylic acid from Aspergillus fumigatus nHF-01

Objectives: The present study aims to report on the production optimization, purication, and characterization of structural and functional attributes of a novel broad-spectrum antibacterial compound produced by Aspergillus fumigatus nHF-01 (GenBank Ac. No. MN190286). Materials and Methods: The culture conditions were optimized by using rigorous culture-set preparation considering various abiotic and biotic factors for a higher amount of antimicrobial production. The produced antimicrobial was solvent extracted and puried by preparative TLC and HPLC methods followed by characterization using UV-Vis, FT-IR, ESI-MS, and 1 H-NMR spectroscopy. The MIC and MBC of the antimicrobials were determined against a set of Gram-positive and Gram-negative human pathogenic bacteria. The mode of action on cellular morphology and integrity were determined by LDH and SEM studies. Its biolm-inhibition properties and synergistic activity with antibiotics were studied. The possible cytotoxic effect on human cell lines was also tested by MTT assay. The putative target site of action was evaluated through in silico molecular docking study. Results: The micro-fungus A. fumigatus nHF-01 produced the maximum antibacterial compound while grown in a combination of 2% MEB (w/v) and 4% YE (w/v) at pH 6.0 and 20 °C temperature with 100 rpm agitation for ten days. The DCM extractable crude compound has a potent growth inhibition against the target human food and topical pathogenic bacteria at a 15 mg/ml concentration and is stable up to 100 °C. The spectroscopic studies conrmed the antimicrobial compound as 5-butyl-2-pyridine carboxylic acid with MIC values from 0.069±0.0034 to 1.12±0.052 mg/ml and from 8.925±0.39 to 17.85±0.78 mg/ml; and MBC values from 8.925±0.40 to 17.85±0.776 mg/ml and from 0.069±0.0034 to 0.139±0.0065 mg/ml against human pathogenic Gram-positive and Gram-negative bacteria, respectively. A concentration of 0.139 and 17.85 mg/ml decreased the viability sharply within 15 min of the incubation period with the gradual increase in LDH activity, indicating a robust bactericidal and lytic mode of action. The time-kill kinetics study shows that at a 17.85 mg/ml dose (i.e. MBC), the compound caused zero viability of E. coli and S. epidermidis cells from the initial log CFU/ml 5.78 after 15 h of treatment. It caused a remarkable change in morphology like the formation of blebbing, notch, rupture of the entire cell walls, and entire dissolution of cell integrity at a concentration of 4 µg/ml and 129 µg/ml. It had cytotoxicity against the tested human lung carcinoma A549 cell line. It showed a notable antibiolm activity at 20 µg/ml and 4 µg/ml comparable to the standard antibiolm drug usnic acid 10 µg/ml and 64 µg/ml against E. coli and B. cereus. It had a synergistic activity with streptomycin, whereas ciprooxacin and vancomycin showed additive effects. It showed the highest binding anities with Quinol-Fumarate Reductase (1l0v), a respiratory enzyme. Conclusion: Thus, the above ndings can be concluded that the strain A. fumigatus nHF-01 produces a novel broad-spectrum antimicrobial compound 5-butyl-2-pyridine carboxylic acid with potent bactericidal activity against human food and topical pathogenic bacteria. This is the rst report of such a compound from the A. fumigatus. signicant on food on surfaces of package materials, and even inside the human a toughened potential AMCs are found ineffective in this state of the microbes. The present study revealed that the active moderate inhibitory properties on the biolm-forming E. coli and B. cereus compared to the standard antibiolm compound Usnic acid 5a). The active compound at a concentration of 4 µg/ml and 129 µg/ml showed 22.30% of biolm inhibition against B. cereus and E. coli, respectively, compared to control. The percentage of such inhibition is concentration-dependent, and it was 45.38% and 65.18% at a concentration of 517 µg/ml. The standard antibiolm drug usnic acid a notable antibiolm activity at 10 µg/ml and 64 µg/ml against E. coli B. cereus, µg/ml, and µg/ml nHF-01 active planktonic and bacterial biolm destabilization assay preformed biolm pathogens DCM stable for more than 36 months. So, the activity loss is not due to compound stability but degradative molecules' production. A similar observation Compaore et al. The pH and temperature of the culture medium are determining factors for the biosynthesis of secondary metabolites [35]. Media pH is an essential consideration for any metabolite production. Here, A. fumigatus nHF-01 at neutral to alkaline pH (8.0 -12.0) and strong acidic pH (3.0-5.0) did not produce AMC. The results indicate that a unique H + balance might induce the associated molecules or genes to produce AMC. The inuence of temperature on antibiotic production varies from strain to strain. Generally, fungi are grown at 28°C at pH 5.6-6.5. Effect of incubation temperatures and pH showed that A. fumigatus nHF-01 produced the maximum stable and effective AMC with inhibition zone (30-32 mm) in pH 6.0 at 20°C while at 28°C, 37°C and 45°C signicantly less inhibition zone (7-23 mm) was observed at the same concentration of extractable mass. Low temperature accelerated the metabolite production by the fungus while high temperature slowed down. The microscopic observation (Fig. S1b) shows that at 20°C, A. fumigatus nHF-01 had signicantly less sporulation with restricted growth, while at > 20°C, it had a puffy and velvety appearance more sporulation, no prominent vesicle was found in liquid media. This indicates that regional mycelial growth with less sporulation is ideal for this organism for secondary metabolite production. Thus, it appears that the cultural conditions of this organism are pretty unusual, as compared to other published reports [34]. However, a similar observation was reported in a marine fungus A. ustus MSF3 that produced antimicrobial compounds in 45% Sabouraud dextrose broth (SDB) with carbon (glucose) – nitrogen (yeast extract) ratio of 3:2 at 20°C temperature at seven days in solid culture [36, 37]. (Table viz. MTCC 1272, B. MTCC 411, Enterococcus faecalis MCC 2041T, Escherichia coli MTCC 723, E. coli ATCC DH5α, Klebsiella pneumonia MTCC 7407, Mycobacterium smegmatis mc 2 155, Pseudomonas aeruginosa MTCC 741, Salmonella enterica serovar Typhimurium MTCC 98, Staphylococcus aureus MTCC 96, S. epidermidis MTCC 3086, Streptococcus mutans MTCC 890, and Vibrio parahaemolyticus MTCC 451. The ATCC, MCC and MTCC strains were procured from American Type Culture Collection, USA, Microbial Culture Collection, Pune, India and IMTECH, Chandigargh, India, and maintained in the media suggested by the repository houses. The bacterial stock culture was maintained in 70% glycerol at -20°C. In addition, the strains were sub-cultured twice before experimentation. culture harvested centrifugation 7168 ×g ºC. supernatant an equal volume of n-hexane, dichloromethane (DCM), ethyl acetate solvent separating funnel; solvent phase evaporated in a rotary-vacuum evaporator (Supert, Model: PBV-7D, Mumbai, India) and used for antibacterial activity assay. The fungal biomass, extractable compound and specic activity also recorded. loaded at an initial oven temperature of 50°C (isothermal for 2 min), 7.6522 psi pressure, and a ow rate of 1ml/min for 32 min run time. The identication of the compounds was made with the spectral data match with the NIST library. The mole percentage was calculated by the formula: Mole% = Ai/Ac×100, where, Ai= peak area count of the individual compound, and Ac= cumulative peak area count of all compounds. The ESI-MS data of the HPL fraction was obtained using the Xevo G2-XS QT ESI-MS instrument (Waters Zspray TM LackSpray) connected to a capillary column. The sample was dissolved in methanol and run in a positive ion mode in the mass range of 100-400 m/z. The 1 H-NMR was carried out in the BrukerBioSpin instrument (Topspin v1.3, USA). 10 mg sample dissolved in 0.6 ml deuterated chloroform (CDCl 3 ) solvent was loaded into a 5 mm Wilmad 528-PP NMR tube. NMR was run at: Operating temperature 28°C; Proton spectra recorded at 64K; Spectral width 10.3 ppm, D1 1 second, NS 51. The chemical shifts were recorded in parts per million (ppm, δ) and the coupling constants at 600 MHz. Antibiolm assay of active compound performed in microwell plate seeded with 95 µl of cell suspension of B. cereus and E. coli (at Mc Farland standard (0.6), ~1 × 10 6 cells/ml) in the nutrient broth and 5 µl of active compound at the MIC/4, MIC/2, and MIC concentration (4, 8, and 16 µg/ml, respectively, w/v) were added in wells. The experimental setup was incubated at 37°C for 24-36 h under static conditions. Similarly, for biolm destabilization assay the 18 h preformed biolm of the strains were treated with active compound at same concentration mentioned above and incubated at 37°C for 24-36 h under static conditions. After incubation, the culture aliquote was decanted, and the wells were washed with 150 µl of distilled water to remove the planktonic cell and repeat twice. The adherent cells in wells were stained with 0.1% (w/v) Crystal violet solution (in water) to stain the polysaccharides of the biolm and kept for 15 min at room temperature. The plate was gently drained upside down on tissue paper 3-4 times and allowed drying. 125 µl of 30% (v/v) acetic acid was added to the wells and incubated for 10-15 min. OD of stained adherent bacteria was determined with a microplate reader (Microplate reader, Bio-Rad, iMark TM , USA) at a wavelength of 595 nm. Usnic acid (4, 8, and 16 µg/ml, w/v) was used as a positive control and culture only with the media served as a negative control [51]. The per cent of biolm inhibition was evaluated using the following formula. % of biolm inhibition = 100 × [(Control OD 595 nm − Test OD 595 nm)/Control OD 570 nm] [52]. bromide) based cytotoxicity assay was done on A549 (ATCC number CCL-185) mammalian alveolar cancer cell line maintained in Dulbecco Modied Eagle Medium (DMEM) supplemented with 10% fetal bovine serum with 1% Penicillin-Streptomycin at 37°C in an incubator with 5% CO 2 . After one hour, the absorbances were measured at 590 nm (Microplate reader, Bio-Rad, iMark TM , USA) [53]. All tests were performed in triplicate.


Introduction
Natural products from plants and microbial sources, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drugs discoveries due to unmatched chemical diversity [1]. Among the microbes, fungi have been a reservoir of isolation of many therapeutic drugs useful for ameliorating and curing many physiological, metabolic, and genetic diseases such as Alzheimer's disease, cancer, diabetics, hypercholesterolemia, etc. [2][3][4]. Since discovering the life-supporting drug, Penicillin, the rst β-lactam antibiotic, by Sir Alexander Fleming in the 1920s, lots of progress have been developed in the antibacterial compounds production, process development and characterization of active molecules. Due to the ever upsurge of antimicrobial resistance among the pathogenic microbes, nowadays, it is challenging to nd new effect toward HepG2 cell line (IC 50 = 9.9 µM) [15], while another strain produces ve new ergot alkaloids named fumigaclavines D-H, having a broad-spectrum of antimicrobial activity against a panel of anaerobic microorganisms with a MIC=16 µg/ml [18]. In addition to producing antimicrobial and antibio lm compounds, the endophytic strains of A. fumigatus, isolated from Moringa oleifera, exhibited excellent antiproliferative activity against different cancer cell lines; such as HCT-15, HeLa A549 and U87-MG with the IC50 values of 0.061, 0.065 and 0.072 mg ml-1, respectively [16] while A. fumigatus EFBL, isolated from Catharanthus roseus, produces potent epothilone (21.5 µg/g biomass), which has potent antiproliferative activity at IC 50 values 6.4, 8.7 and 10.21 µM, respectively against tumour cells HepG-2, MCF-7 and LS174 T by stabilizing their microtubule arrays and arresting their cellular division at the G2-M phase [19][20] also reported that a mutant strain of A. fumigatus could produce anticancer drug paclitaxel in immobilized calcium alginate gel beads. Moreover, the endophytic A. fumigatus CY018, from the leaf of Cynodon dactylon, produces two new metabolites, named asperfumoid and asperfumin, inhibitory to Candida albicans with MICs of 75.0 µg/ml [21].
In addition to endophytic species/strains, certain Aspergillus spp. are reported from deep-sea sediments or symbionts to marine animals, producing various bioactive compounds. For example, a deep-sea derived fungal A. fumigatus SCSIO produces two new alkaloids, fumigatosides E and F, showing signi cant antifungal activity with MIC at 1.56 µg/mL, and antibacterial activity against Acinetobacter baumanii ATCC 19606 with a MIC value of 6.25 µg/mL, respectively [17]. A. sydowii C1-S01-A7 produced two novel compounds, 2-hydroxy-6-formyl-vertixanthone and 12-O-acetyl-sydowinin A, which exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus and also have cytotoxic activity against both A549 and HepG2 cell lines [22]. A coral-derived fungus A. tritici SP2-8-1 produces three novel compounds, 4-methylcandidusin A, aspetritone A and aspetritone B. Among these, Aspetritone A exhibited better activities against methicillin-resistant strains of S. aureus (MRSA) ATCC 43300 and MRSA CGMCC 1.12409, and exhibited strong cytotoxic activities against human cancer cell lines HeLa, A549, and Hep G2 [23]. While the deep-sea sediment derived A. terreus PT06-2 and A. versicolor produce new compounds, terremides A and B have antibacterial activity against Pseudomonas aeruginosa and Enterobacter aerogenes with MIC values of 63.9 and 33.5 µM, and MIC values of 3.9 and 7.8 µg/mL, respectively [24]. While a halotolerant strain A. occulosus PT05-1 produces antimicrobial ergosteroids and pyrrole derivatives in a hypersaline medium having antimicrobial activity against Enterobacter aerogenes, Pseudomonas aeruginosa, and Candida albicans with MIC values of 1.6-15 lM, respectively, and showed cytotoxicity against HL-60 and BEL-7402 cells with IC50 values of 12-18l M [25].
In addition to the small molecule antimicrobial compounds, the species of Aspergillus are known to produce valuable peptides of therapeutic use. For example, an extracellular thermostable peptide (MFAP9, ∼3 kDa) puri ed from marine A. fumigatus BTMF9 exhibited strong antibacterial and antibio lm activity against Bacillus circulans (NCIM 2107) and B. pumilus with MIC and MBC values of 0.525 µg/mL and 4.2 µg/mL, respectively [26]. While the halotolerant A. sclerotiorum PT06-1 produces two novel cyclic photo-interconvertible hexapeptides, sclerotides A and B, containing both anthranilic acid and dehydroamino acid units, that have moderate antibacterial, antifungal cytotoxicity activities [27]. In addition to the direct functionalization of Aspergillus metabolites, the proteins from A. fumigatus DSM819 have been mediated in the green synthesis of silver nanoparticles (AgNPs), and its application as an antimicrobial nishing agent in textile fabrics against pathogenic microorganisms (B. mycoides, C. albicans and E. coli) exerted a signi cant antimicrobial activity [28]. Therefore, from the above literature studies, it appears that the fungal species of the genus Aspergillus play signi cant roles as model organisms in basic research and "cell factories" for producing various industrial products of human use. In this connection, the main objectives of the present study are to characterize the broad-spectrum antimicrobial compound produced by A. fumigatus nHF-01 in a submerged fermentation system and evaluate its detailed antibacterial e cacy against a large number of bacterial pathogens associated with food and topical pathogenesis.

Results
The strain Aspergillus fumigatus nHF-01: a azole sensitive strain The strain A. fumigatus nHF-01 could grow in different media like Malt extract broth (MEB), Czapek dox broth (CDB), and Cornmeal (CMB) at pH ranges from 3.0 to 10.0 and temperature ranges from 20°C to 45°C [13]. To check the health risk, an antimicrobial drug resistance study was done. The MIC of seven different antifungal drugs is shown in Table 1. The study shows that among these drugs, Luliconazole exhibited the best in vitro activity with MIC values of 0.25 µg/ml, while Fluconazole was the less active agent with MIC values of 10 mg/ml (Table S1). antibacterial compound (an average 10.5 mg/100 ml culture). In comparison, the other media produced a negligible antibacterial activity though they had a high biomass production. The suitable pH and temperature range were at pH 6.0, 20°C for ten days incubation in 100 rpm shaking ( Fig. S3). A combinational study with MEB and yeast extract (YE) showed that a mixture of 2% MEB and 4% YE (w/v) produced the highest speci c activity (Table 2; Fig. 1a-1c). Set-4 produced the highest amount of biomass among the variants, while Set-8 produced the highest extractable compound and speci c activity. The Set-7 produced a good amount of biomass but a very low extractable compound with a high speci c activity. Moreover, Set-12, Set-14, Set-15, Set-16 were found to produce a low-moderate extractable compound with no speci c activity. From the heat map (Fig. 1d), it was observed that a high concentration of YE produces poor results in terms of the extractable compound and speci c activity; according to the interest of this study, Set-4 and Set-8 was clustered together for producing nearly similar results. From surface plots (Fig. 2a to c), the interaction between MEB and YE was determined. Higher concentrations of MEB and YE at equal proportions induces higher biomass production (Fig. 2a). The highest production of the extractable compound was produced between MEB concentrations 4 to 6; YE was found to contribute less in the production of the extractable compound and was controlled mainly by the concentration of MEB (Fig. 2b). MEB concentration 4% and YE concentrations between 2 to 6% were most suitable for achieving the highest speci c activity (Fig. 2c). From the dot plots ( Fig. 2d to f) comparing predicted values with observed values, it can be observed that the models predicting response are tted quite well with the observed values and can be used for further development. Although the models tted well ( Fig.   2d to f), but none of the models (for biomass, extractable compound, speci c activity) found signi cant (at 0.05 alpha level) ( Table 3). The Rsquared and adjusted R-squared values turned out to be moderate to low; probably, a better experimental design can improve the design and Rsquared values. Model details and coe cients are given below in Table 3.  According to mole %, the GC-chromatogram showed a spectral match with 5-butyl 2-pyridine carboxylic, (mole% 5.15, RT-18.996 min) ( Table S3).
The high-resolution ESI-MS spectrum (Fig. S5 c) showed a molecular ion peak at m/z 180.1047, corresponding to the compound 5-butyl-2pyridine carboxylic acid with an empirical formula of C 10 H 13 NO 2 . The 1 H NMR spectrum (Fig. S5 [30]. Thus, the ESI-MS and 1 H NMR spectral data con rmed the potential AMC as 5-butyl-2-pyridine carboxylic acid with a molecular formula of C 10 H 13 NO 2 (Fig. S5 e).
Phytochemical screening, solubility and thermo-stability of the compound Phytochemical screening of the pure fraction showed the presence of alkaloids. Alkaloids are heterogenous natural nitrogen-containing organic compounds used to treat bacteria and serve as scaffolds for essential antibacterial drugs [31]. The thermal treatment on the compound showed that the active fraction was stable up to 100°C; however, it lost its activity at autoclave temperature. The compound had a solid appearance with a deep brownish colour and was soluble in various polar and non-polar solvents, viz. n-hexane, diethyl ether, DCM, ethyl acetate, methanol, DMSO and chloroform.
The MIC and MBC values and effect on bacterial growth, viability and cellular integrity The puri ed 5-butyl-2-pyridine carboxylic acid produced a potent antibacterial activity against the tested microorganisms.  Table 4). The effect of the active compound on growth and viability against human pathogenic Gram-positive bacteria (B. cereus and S. epidermidis) and Gram-negative bacteria (E. coli and S. enterica serovar Typhimurium) showed that at concentration 0.139 and 17.85 mg/ml, respectively it decreased the viability sharply within 15 min of the incubation period (Fig. 3a). Thus, a rapid decrease in the growth and viability of treated bacterial cells indicated that the active compounds had a strong bactericidal mode of action against the tested pathogens [32,33].
A comparative growth inhibition study with standard antibiotics, cipro oxacin, streptomycin and vancomycin at MICx2 and MICx50 doses showed that all three antibiotics caused a rapid loss of viability within 30 min of treatment (Fig. 3b). However, surprisingly the CFU/ml of the tested bacteria regained viability after 60 min. This observation indicates that the organisms either developed transient resistance to the antibiotics by changing the target site of action or enhancing the cellular transport or the e ux mechanism of the antibiotics. On the contrary, these results emphasize the potency of the new active compound from the A. fumigatus nHF-01 could be a potential lead drug in future antimicrobial therapy.
Lactate dehydrogenase (LDH) is an essential cytoplasmic enzyme of all living cells. In the presence of the pure active compound, the cellular integrity of E. coli and S. epidermidis had lost with the gradual increase in LDH activity ( Fig. 3c) from 0.3125 mU/ml to 1.25 mU/ml and 1.34 mU/ml to 1.5 mU/ml, respectively. A subsequent gradual decrease in the colony-forming unit was observed in E. coli and S. epidermidis from log 18.89 CFU/ml to log 10.23 CFU/ml and log 12.97 CFU/ml to log 4.8 CFU/ml, respectively. The positive control (sonication) showed that E. coli cells had high LDH activity of 3.375 mU/mL and S. epidermidis had 4.078 mU/mL with the lowest CFU value (2.41 CFU/ml and 1.54 CFU/ml, respectively).
The time-kill curve of E. coli and S. epidermidis showed that after 15 h of treatment, the compound at a dose of 17.85 mg/ml (i.e. MBC dose) caused zero viability from the initial Log CFU 5.78 (Fig. 3d). This indicates that the compound had a bactericidal activity with absolute lethality in the treated cells. The effect of the active compound on B. cereus and E. coli showed remarkable changes in the morphology (Fig. 4). It was found that in comparison to the untreated cells ( Fig. 4a and 4d), the treatment after 30 min caused minute cell wall to rupture (Fig. 4b, e), and at 3 h of treatment, it caused drastic changes in cell morphology like the formation of blebbing, notch, rupture of the entire cell walls, and entire dissolution of cell integrity (Fig. 4c, f). This indicates that the compound is causing lysis of the bacterial cell wall resulting in a rapid decrease in cell viability.

Antibio lm and bio lm destabilization assay
Bio lm is a complex association of microorganisms formed on solid and liquid systems. B. cereus and E. coli are found to be signi cant organisms residing on food commodities, on surfaces of package materials, and even inside the human body, forming a toughened matrix. Many potential AMCs are found ineffective in this state of the microbes. The present study revealed that the active compound showed moderate inhibitory properties on the bio lm-forming E. coli and B. cereus compared to the standard antibio lm compound Usnic acid (Fig. 5a). The active compound at a concentration of 4 µg/ml and 129 µg/ml showed 22.30% of bio lm inhibition against B. cereus and E. coli, respectively, compared to control. The percentage of such inhibition is concentration-dependent, and it was 45.38% and 65.18% at a concentration of 517 µg/ml. The standard antibio lm drug usnic acid showed a notable antibio lm activity at 10 µg/ml and 64 µg/ml against E. coli and B. cereus, which is comparable to 20 µg/ml, and 4 µg/ml of the nHF-01 active compound. So the study revealed that the active compound 5-butyl-2-pyridine carboxylic acid has the potential to inhibit both the planktonic and bio lm stages of the Gram-positive and Gram-negative bacterial strains. The bio lm destabilization assay also showed that it could destabilize the preformed bio lm of the target pathogens (Fig. 5b).
Cytotoxicity and synergistic effect of 5-butyl-2-pyridine carboxylic with different antibiotics The compound 5-butyl-2-Pyridinecarboxylic acid showed cytotoxicity against the tested human A549 cell line (Fig. 5c). The synergistic effects of 5-butyl-2-pyridine carboxylic with three conventional antibiotics (cipro oxacin, streptomycin and vancomycin) showed that all combinations demonstrated synergistic, partiality synergistic and additive effects against the tested bacteria (Fig. 5d). It showed synergistic activity with streptomycin against B. cereus, whereas it has additive effects with cipro oxacin and vancomycin. While it showed partial synergistic activity with streptomycin and cipro oxacin in E. coli, an additive effect with vancomycin (Fig. 5d).

Molecular modelling
Molecular docking between the active compound, 5-butyl-2-Pyridinecarboxylic acid, and a large number of target site acton showed (Table S4) that the three different respiratory target enzymes viz. Quinol-Fumarate Reductase (QFR, 1kf6), Quinol-Fumarate Reductase (1l0v) and quinone oxidoreductase (SQR) SdhB His207Thr mutant (2wp9) of E. coli had the highest binding a nities (Table S3). Among these, it showed the highest binding a nities with Quinol-Fumarate Reductase (1l0v) (docking score -7.1). The binding mode of the compound with these enzymes are illustrated in Fig. S6

Discussion
Antifungal drugs sensitivity of Aspergillus fumigatus nHF-01 The species of Aspergillus sp. Have been reported to have pathogenicity to human beings. Among these, azole resistance is one of the leading concerns. The present strain A. fumigatus nHF-01 is sensitive to such azole drugs; thus, the handling for large scale AMC production is less risky and safe for handling (Table S1) Optimum culture conditions for antibacterial compound production The antibacterial compound production by the strain showed that the MEB and YE triggered a high amount of AMC when grown at low temperature with mild acidic fermentation conditions. These low-cost fermenting substrates would be suitable for large-scale antibacterial compound production by A. fumigatus nHF-01. The antibacterial compound was extractable in DCM indicates the moderately polar nature of the compound, and thus, for subsequent extensive scale harvesting and puri cation DCM was used. In a study, Compaore et al. [34] reported that A. fumigatus produces fumagillin and gliotoxin optimally in a synthetic media condition (supplemented with yeast extract, lactose and other carbon sources) for 6-8 days of incubation at 37°C, pH 7.0, at 150 rpm, and extractable in acetonitrile and methanol.
The incubation period is a crucial consideration for AMC production. A. fumigatus nHF-01 produced maximum AMC at10th day with an average diameter of 19-23 mm inhibition zone at 15 mg/ml concentration, while the 8th and12th days period produced signi cantly less antimicrobial activity at the same concentration (Fig. S3). Moreover, no inhibition zones were observed on the 5th, 15th and 20th days of incubation. This indicates that the organisms' physiological status, like cell age and the media's nutritional status, might trigger the organism to produce such antimicrobial compounds. In addition, a sudden drop in antimicrobial content on the 12th day indicate that the organism could produce some degradative enzymes to impede the activity of the antimicrobial compounds. On the other hand, the compounds in cell-free supernatant or after extraction with DCM were stable for more than 36 months. So, the activity loss is not due to compound stability but degradative molecules' production. A similar observation was noted by Compaore et al. [34].
The pH and temperature of the culture medium are determining factors for the biosynthesis of secondary metabolites [35]. Media pH is an essential consideration for any metabolite production. Here, A. fumigatus nHF-01 at neutral to alkaline pH (8.0 -12.0) and strong acidic pH (3.0-5.0) did not produce AMC. The results indicate that a unique H + balance might induce the associated molecules or genes to produce AMC. The in uence of temperature on antibiotic production varies from strain to strain. Generally, fungi are grown at 28°C at pH 5.6-6.5. Effect of incubation temperatures and pH showed that A. fumigatus nHF-01 produced the maximum stable and effective AMC with inhibition zone (30-32 mm) in pH 6.0 at 20°C while at 28°C, 37°C and 45°C signi cantly less inhibition zone (7-23 mm) was observed at the same concentration of extractable mass. Low temperature accelerated the metabolite production by the fungus while high temperature slowed down. The microscopic observation (Fig. S1b) shows that at 20°C, A. fumigatus nHF-01 had signi cantly less sporulation with restricted growth, while at > 20°C, it had a puffy and velvety appearance more sporulation, no prominent vesicle was found in liquid media. This indicates that regional mycelial growth with less sporulation is ideal for this organism for secondary metabolite production. Thus, it appears that the cultural conditions of this organism are pretty unusual, as compared to other published reports [34]. However, a similar observation was reported in a marine fungus A. ustus MSF3 that produced antimicrobial compounds in 45% Sabouraud dextrose broth (SDB) with carbon (glucose) -nitrogen (yeast extract) ratio of 3:2 at 20°C temperature at seven days in solid culture [36,37].
In addition to nutrient conditions, circulatory agitation also helps aeration in the culture medium. It was found that an optimum agitation speed of 100 rpm produced a higher antimicrobial compound (0.688 mg/ml) with an average 25-30 mm inhibition zone diameter. Marked differences were observed among the agitations and non-agitation (0.38 mg/ml) for antimicrobial compound production by this organism. It is hypothesized that this fungal organism gets its optimum sheerness to spread or grow its mycelia and gain its optimum gas balance (O 2 /CO 2 ) inside the culture system to produce the metabolite optimally. It was observed that an active antibacterial compound produced by the mycelia was released in broth in shaking condition and no further extracts were recoverable from the dried mycelia. This is unique from the industrial point of view. Considering all these ndings, for antibacterial compounds production by A. fumigatus nHF-01, certain unique fermentation conditions are of the utmost need for the strain. All these parameters would guide designing the RSM model for large scale production of such antimicrobial compounds at an industrial scale. A similar culture condition-dependent metabolite pro ling of A. fumigatus with antifungal activity study done by Kang  5-butyl-2-pyridine carboxylic acid and its structure-function relations 5-butyl-2-pyridine carboxylic acid (also known as Fusaric acid, FA or 5-butyl-2-picolinic acid) and its analogues exhibited moderate antimicrobial activities [39], including growth inhibitors of E. coli [40], and act as quorum sensing [41]. It is also reported from species of Fusarium [42] and Gibberella fugikuroi [40]. Moreover, previous studies also show that it inhibits dopamine beta-hydroxylase enzymes that convert dopamine to norepinephrine and inhibits cell proliferation and DNA synthesis [41] anti-tumour activity on heme enzymes [40]. Structure-activity co-relation indicates that 2-pyridine carboxylic acid and its derivatives act as a bidentate chelating agent that effectively chelates metals in metal-containing protein complexes and enzymes required for growth replication or in ammatory response and thereby used to treat cancer. So the novelty of the present study is that the strain A. fumigatus would provide an easy biological source for large scale production of 5-butyl-2-pyridine carboxylic acid. Moreover, the compound is stable up to 100°C, superior to the novel antifungal peptide that was stable up to 70°C by A. clavatus as reported by Gargouri et al. [43]. Therefore, compared to the other antimicrobial compounds produced by Aspergillus spp., the present antimicrobial compound is more heat stable and could be used as antimicrobials in many processes involving thermal treatment up to 100 o C but not autoclaved processes.
Effect of 5-butyl-2-pyridine carboxylic acid on bacterial growth, viability and cellular integrity: Planktonic and bio lm stages The AMCs with such broad-spectrum activities is very limiting to the list of antimicrobial drugs in pharmaceutical industries. The study shows that the MIC and MBC values were more active against a broad range of food and waterborne pathogens that cause fatal food poisoning, typhoid fever, tuberculosis, and infections in scars and wounds (Table 4). So, this compound could be a new member of this list. The greater e cacy of this compound against these strains nds its application against food and topical pathogenesis. Therefore, further subsequent drug safety and molecular action studies of this compound are an utmost need for its global use. The release of LDH enzyme with content with a concomitant gradual decrease in CFU value indicates that the compound affects cellular permeability and thus rendered a quick death of the cells [32,33].
The drug e cacy is nowadays being trialled with combination mode. Very often, it was observed that co-administration of more than one active compound might enhance or reduce the drug e cacy of the lead compound. Therefore, synergistic/antagonistic study is very much essential for drug potentization. The study revealed that the compound 5-butyl-2-pyridine carboxylic acid has a synergistic effect with three conventional antibiotics like cipro oxacin which acts on bacterial topoisomerase II (DNA gyrase) and topoisomerase IV, streptomycin that binds irreversibly to the 16S rRNA and S12 protein within the bacterial 30S ribosomal subunit, and vancomycin that inhibits bacterial cell-wall biosynthesis (https://go.drugbank.com/drugs/DB00512). Moreover, these combinations would reduce the application of the antibiotic dose to cure many challenging pathogens. The literature study suggested that the combined antimicrobial effect of antibiotics and extracted metabolites increase by increasing their bonding reaction [44][45][46][47].
The fungal secondary metabolites show important biological activities like antiviral, antibacterial, activities mainly targeting different microbial proteins, like DNA-gyrase, topoisomerase IV, dihydrofolate reductase, transcriptional regulator TcaR (protein), and aminoglycoside nucleotidyltransferase. However, the metabolites acting on respiratory enzymes are very rare. The present study shows that the 5-butyl-2-pyridine carboxylic has a strong binding a nity towards the quinol-fumarate reductase (QFR), while succinate: quinone oxidoreductase (SQR, succinate dehydrogenase) and menaquinol: fumarate oxidoreductase (QFR, fumarate reductase), members of the integral membrane proteins Complex II family, that play a key role in the Krebs cycle. Hence, the molecular docking studies revealed that the compound targetting QFR of E. coli inhibits the essential respiratory enzymes, thus leading to energy depletion and cellular viability. This observation is different from the novel anthraquinone, 2-(dimethoxymethyl)-1-hydroxyanthracene-9,10-dione, isolated from A. versicolor, that had e cacy against topoisomerase IV and AmpC β-lactamase enzymes [48]. Thus, molecular docking studies also revealed a novel target site of action of the 5-butyl-2-pyridine carboxylic that could be used as a future drug and could be exploited in combating many infectious and chronic diseases.

Conclusions
The species of Aspergillus are the leading microfungi that have wide use in different industries. They produce a diverse array of potential biomolecules like antibacterial, antifungal, immunodepressants, anti-AIDS drugs, etc. However, reports on the broad-spectrum antimicrobials from this organism are very limiting. The development of new, novel and high potential antimicrobials is a global challenge due to the upsurge in multidrug resistance among the food and topical pathogens. To this critical demand, the present study reports for the rst time that A. fumigatus nHF-01 produces a novel 5-butyl-2-pyridine carboxylic antibacterial compound that has broad-spectrum antibacterial activity against human pathogenic bacteria. The compound is effective on both the planktonic and bio lm states of bacteria. The compound has absolute lethality at 15 h treatment at a dose of 17.85 mg/ml against human pathogenic bio lm forming E. coli and S. epidermidis. In addition, the compound has a strong binding a nity towards the key enzyme in the Krebs cycle and the respiratory chain resulting in rapid depletion of energy and subsequent death of cells, as seen in the growth inhibition study. It can be produced in a very low-cost media comprising 2% ME and 4% YE broth (w/v) at pH 6.0, 20°C temperature, in a ten-day shake ask condition. This robust broad-spectrum antibacterial compound could, thus, be trialled as a potent drug contributing to human endeavour in the future. Further analysis with a detailed pharmacological mechanism of action study would decipher the antibacterial action against the food and topical pathogenic bacteria.

Fungal strain and culture conditions
The micro-fungus A. fumigatus nHF-01 (GenBank Acc. No. MN190286) was cultured and maintained in Potato Dextrose Agar (PDA) at 28°C and sub-cultured in every 5-7 days interval [13]. After profuse growth and sporulation, the culture aliquots were tested for their antibacterial e cacy by the agar well diffusion method following the standard protocol [49], [13] against the human pathogenic Gram-positive and Gram-negative bacteria (Table S1)

Sensitivity of the strain towards antifungal drugs
The antifungal drug sensitivity of this strain was tested against many azole and systemic fungicide drugs (Table S1) by agar well diffusion assay and MIC was determined [13].
Screening of media and submerged culture conditions for antibacterial compound production

Optimization of cultivation conditions
After screening the primary media constituents in uencing antibacterial compound production, two critical factors, i.e. Yeast extract and Malt extract, were tested in different proportions. Therefore, the batch fermentation (100 ml) were conducted containing different % ME (0 to 8, w/v) and % YE (0 to 8, w/v) set at pH 6.0, 20°C for ten days in shake condition, and the amount of antimicrobial compounds production was recorded as described above. Approximately 1 litre of batch fermentation was conducted to harvest an ample amount of antibacterial compound for puri cation and characterization studies.

Assessment of antibacterial e cacy
The antibacterial potentiality of the crude extract (15 mg/ml) was carried out by the agar well diffusion method on nutrient agar (NA) plates following the standard protocol [13,49]. The antibacterial potency was determined as inhibition zone diameter against the targeted bacteria, as mentioned above. The experiment was repeated as triplicate trials.
Chromatographic puri cation of the active compound The analytical and preparative TLC (Thin layer chromatography) was performed using TLC silica gel 60 F 254 plates (MERCK, Germany, Analytical chromatography). The separation of the crude extract was carried out with n-hexane and ethyl acetate solvent system (9:10, v/v) and visualized under 254 nm UV light, and the Rf values were recorded. The potent antibacterial fraction was determined through TL zymogram assay by overlaying NA soft-agar seeded with sensitive bacteria. The potent antibacterial spot was marked by observing the growth inhibition zone and tallied with the Rf values. Similarly, several preparative TLC plates were run, and the potent antibacterial compound (AMC) was scraped out from the TLC plates, extracted with DCM, and evaporated to dryness. Preparative HPLC analysis of the separated fraction was carried to harvest the pure compound in a multidimensional RP-HPLC system (Water Alliance, 2695, MDLC, UV-Vis detector 2487) equipped with a C 18 column. Elution was done in a 0.5% H 3 PO4, 90% acetonitrile, and 0.1% tri uoroacetic acid as mobile phase with a ow rate of 1.0 ml/min at 30 ± 2°C and the detected at 246 nm. The fractions were collected and tested for potential antibacterial activity, as mentioned above. Several runs were conducted to harvest the pure compound for further characterization.
Chemical characterization of the puri ed active compound UV-Vis and FT-IR spectra of the active compound The UV-Visible spectrum of the HPLC fraction was carried out in a UV-Vis spectrophotometer (UV-Vis 1800, Shimadzu, Japan) using DCM as Phytochemical screening, solubility and thermo-stability studies Chemical tests for active constituents of the pure compound were carried out following Mandal et al. [33]. The thermo-stability of the compound was checked at 60°C, 80°C, 100°C in a water bath, and at 121°C in an autoclave for 30 min. The residual antimicrobial activity was assayed against the above mentioned pathogenic bacteria by agar well diffusion assay. The solubility was checked in different organic solvents, viz. nhexane, diethyl ether, DCM, ethyl acetate, methanol, DMSO and chloroform. The physical appearances and colour were also observed.
Antimicrobial potentiality studies Effect of the active fraction on growth and viability of bacterial strains The effect of the compound was studied on sensitive human pathogenic bacterial strains such as B. cereus, S. epidermidis, E. coli and S. enterica serovar Typhimurium. The 50 µl of puri ed active compound (MIC×2 dose) was added to the 210 µl of exponentially growing cells and incubated at 37°C for 33 h. The growth was monitored at OD 620 nm in a spectrophotometer (Uv-Vis 1800, Shimadzu, Japan) and viability by the decimal diluted plate-count method. For the plate count method, the treated and control cells were serially decimal diluted and 500 µl of the dilution (10 -2 to 10 -8 dilution) were plated on NA plates and incubated at 37°C. The viable colonies (CFU) were counted from respective treatment plates were counted at 48 h and calculated as log CFU/ml.

Determination of Time-kill kinetics and time-kill endpoint:
The µl of the treated culture was taken out and serially diluted at 10 -1 to 10 -4 time, spread on NA plates in triplicate, incubated at 37°C for 48 h and observed for nos. of viable colony development. The viable counts were calculated as log CFU/ml, and kill curves were plotted against incubation time. A bactericidal effect is de ned as a decrease in the logarithm value of the CFU/ml over a speci ed time [50].
Effect on cellular integrity and cell morphology: The active growing cells were treated with MIC×2 dose, and the amount of LDH in the cell-free lysate was measured for 30 min, 60 min and 480 min [33]. The sonication (Power 95%, Gap 2s, Temp 20 o C, Stroke 10 times) was done in a Probe Sonicator (PKS-250FM, USA), and the untreated cell suspension was treated as positive and negative controls, respectively. The effect on cellular morphology was studied by scanning electron microscopy (SEM). Evaluation of synergistic effects between nHF-01 active compound and antibiotics The synergy between two antimicrobial compounds is often expressed in terms of the fractional inhibitory concentration (FIC) which is expressed by the MIC of the compound in combination divided by the MIC of antibiotic acting alone. To determine the FIC, the target organisms B. cereus and E. coli was fresh cultured, harvested, and suspended in sterile NB to produce a McFarland value of 0.5. The suspension was diluted in fresh NB to achieve a nal CFU of 4×10 6 to 5×10 6 from which 10 µl was inoculated into the microwell plates and incubated at 37°C for 16-20 h. A checkerboard microdilution technique was used to examine the synergism between the antibiotics and active compounds against test organisms. All tests were carried out in a duplicated manner. The values decide the nature of the interaction like FICI < 0.5 synergy, 0.5 ≤ FICI < 1 partial synergy, FICI = 1 additive, 2 ≤ FICI < 4 indifferent, and 4 < FICI antagonism [54]. FICI was calculated as follows: FIC of nHF-01 active compound = MIC of nHF-01 active compound in combination/MIC of nHF-01 active compound alone [55]. The calculated FIC index was used to detect the nature of the interaction between the two test agents and the interaction either synergism or indifference or antagonism type.

Molecular modelling
The crystal structure of many AMC target proteins/enzymes were obtained from a protein data bank (http://www.rcsb.org). The structures were then cleaned using Autodock tools by removing heteroatoms and by adding necessary hydrogen atoms. The structure of the 5-butyl-2-pyridine carboxylic acid molecule was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Using UCSF Chimera [56], the PDB les of the 5butyl-2-pyridine carboxylic were created for docking. Autodock Vina [57] package was used for docking between the best binding sites of enzyme and proteins and 5-butyl-2-pyridine carboxylic acid. All the docked compounds were subjected to further selection for ADMET property analysis based on Lipinski's ve (Ro5) rule, and compounds with any Ro5 violations were eliminated. Ro5 includes molecular weight, lipophilicity, molar refractivity, number of hydrogen bond donors and acceptors. The Molinspiration server was used for calculating the physicochemical properties of compounds (http://www. molinspiration.com/cgi-bin/properties).

Statistical analysis
Exploratory analysis was done to nd the best media composition [58-59]. Bar plots and a heatmap clustered based on similar performance were used to understand better the effects of media composition on biomass, extractable compound and speci c activity. Response Surface Modeling (RSM) was used to determine the interaction between parameters [60]. The concentration of MEB and YE were treated as a predictor variable, biomass, extractable compound and speci c activity were treated as the response variable for creating the RSM model; details of the experimental design and results were given in Table 2. We created three models for three response variables, including all the rst order, secondorder, and interaction terms; details of the model and coe cients are given in Table 3. Predictions were made from the model and compared with observed variables. Clustering and production of heatmap were done using Orange 3 (https://orangedatamining.com/) software. The rest of the analysis, modelling and plotting were done using R 3.6.3 (Cran.r-Project.Org) software. All the codes of R were run in Rstudio environment 1.2.5042 (www.rstudio.com).

Author contributions
All authors contributed to the study's conception and design. Vivekananda Mandal performed material preparation, data collection, and analysis; wrote the rst draft of the manuscript. All authors commented on previous versions of the manuscript., read and approved the nal manuscript; Conceptualization and design of experimentation: Vivekananda Mandal; Methodology: Vivekananda Mandal did the experimentations; Prashanta Kumar Mitra did the statistical analysis and data analysis for its presentation; Narendra Nath Ghosh did the chemical characterization and in silico modelling; Formal analysis and investigation: Vivekananda Mandal; Writing -original draft preparation: Vivekananda Mandal. All the authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved; Writing -review and editing: Vivekananda Mandal and Sukhendu Mandal; Supervision: Vivekananda Mandal; Funding acquisition: No direct funding received for this research work, however, the institutions have provided necessary chemicals and infrastructural facilities to do the experiments.
Competing interests: The author(s) declare no competing interests.
Data availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.