Overview on antibacterial metabolites from terrestrial Aspergillus spp

ABSTRACT Medicines developed from natural sources are a frequent target for the research and discovery of antimicrobial compounds. Discovering of penicillin in 1928 was a motive to explore of nature as a source of new antimicrobial agents. Fungi produce a diverse range of bioactive metabolites, making them rich source of different types of medicines. The purpose of this paper was to review studies on antibacterials from terrestrial Aspergillus published exclusively during 1942–2018, with emphasis on their antibacterial activities, structures, and mechanisms of action if present. According to the results from different studies in the world, large number of compounds and extracts showed different activities against different bacterial species, including Gram-positive and Gram-negative bacteria. The most prominent result was that of the compound CJ-17,665, isolated from A. ochraceus, showing good activity against multi-drug resistant Staphylococcus aureus, which is well-recognised to be one of the most important current public health problem. These findings may motivate scientists to undertake a project that may result in the development of novel antibacterial drugs from terrestrial-derived Aspergillus spp., although further toxicity assays (in vivo) must be performed before their application.


Introduction
Fungi represent one of humanity's oldest domesticated organisms and are responsible for the production of some of industrial (e.g. alcohol), nutritious (e.g. bread), and medically useful (e.g. penicillin) products (Kavanagh 2011). From the beginning until now, the humankind has always been faced with a problem of spreading of bacterial infections. The kingdom fungi (Mycetae) is well-known as a valuable source of diverse bioactive compounds of therapeutic importance since the discovery of penicillin in 1928 by Alexander Fleming (Fleming 1929;Clutterbuck et al. 1932) from Penicillium notatum. The treatment of bacterial infections is increasingly complicated by the ability of bacteria to develop resistance to antibacterial agents (Levy and Marshall 2004). Therefore, an urgent need to exploit new classes of antibacterial agents with a novel mechanism of action is required. The increasing need for new antibacterial agents able to control emerging diseases or resistant strains of bacteria inspired a number of research groups to explore the terrestrial and marine environments for new antibacterial compounds (Ng et al. 2015). Filamentous fungi represent an important group of microorganisms known to synthesise a vast diversity of bioactive molecules that are traditionally termed secondary metabolites or natural products (Calvo et al. 2002;Horgan and Murphy 2011;Jansen et al. 2013;Bills and Stadler 2014). Secondary metabolites are biologically active organic compounds that are not required for normal growth, development, or reproduction, but provide a competitive advantage to the producing organism. Secondary metabolites are used as medicines, flavourings, pigments, and recreational drugs (Craney et al. 2013). The discovery of penicillin was a real revolution in medicine and the research interest to explore new antibacterial agents from microorganisms is continued. Many intensive studies, mainly on terrestrial-derived and marine-derived fungi, displayed that fungi are a rich source of unique bioactive substances. A large number of antibacterial metabolites are identified in many species of fungi throughout the world and some have been approved as drugs, such as cephalosporins and fusidic acid (Newton and Abraham 1955;Wo et al. 1962). Most fungi-derived pharmaceuticals have been sourced from Ascomyceteous fungi, such as Aspergillus, Penicillium, and so on, whereas perhaps fewer pharmaceuticals, certainly in an industrial context, have been derived from Basidiomycetous fungi and others (Bala et al. 2011). However, more than 30% of isolated metabolites from fungi are from Aspergillus and Penicillium (Bérdy 2005).
Aspergillus species were the most diverse and common fungal species found in the environment. One explanation for the high number of compounds reported from this genus is that Aspergillus spp. are salt tolerant, fast growing species, and are easily obtained from many substrates (Bugni and Ireland 2004). Secondary metabolites isolated from species of the genus Aspergillus have continually attracted the interest of pharmacologists due to their broad array of biological activities and their structural diversity (Cai et al. 2011;Wang et al. 2017). Aspergillus have proven to be a prolific source of secondary metabolites with interesting biological activities, including antibacterial activity (Perrone et al. 2007;Xu et al. 2015). The aim of this review is to present the antibacterial metabolites or extracts described from terrestrial Aspergillus spp., which may have pharmaceutical importance as antibacterial agents. In this review, it is noticed that antibacterial compounds were presented in chronological order. In addition, it is worth mentioning that we have collected all relevant information as far as we can.

Assessment techniques of antibacterial activity of fungal metabolites
To assess the antibacterial activity of fungal extracts or pure compounds, several bioassays are well known and commonly used, such as agar disc diffusion method, agar well diffusion method, microdilution method, and a method with incorporation of the extract in the culture medium and determination of bacterial colonies. Thus, the results of antibacterial activity are expressed in different units. In agar disc diffusion method, the extract is incorporated into discs at different concentrations. The clear or inhibition zone developed around each disc was measured and the antibacterial activity is represented by inhibition zone diameter (IZD) (CLSI 2012). The principle of the agar well diffusion method is the same as that of the agar disc diffusion method, but the extract, in fixed volumes, is placed in wells rather than discs (Valgas et al. 2007). Microdilution method comprises microdilutions of the extract in liquid medium using microplates to determine the values of minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), which also known as the minimum lethal concentration (MLC), or a concentration that inhibit 50% of the bacterial growth (IC 50 ) (Soothill et al. 1992). With respect to the fourth method, the extract is incorporated in the culture medium and the resultant colony forming units (CFU) are determined. Other bioassays, such as flow cytofluorometric and bioluminescent methods are not widely used because they require specified equipment and further evaluation for reproducibility and standardization (Balouiri et al. 2016). For many details, the above assessment techniques were reviewed by Balouiri et al. (2016).

Antibacterial metabolites from terrestrial Aspergillus spp
Many species of Aspergillus are active producers of many products of industrial and pharmaceutical importance (Mehta et al. 2017;Wang et al. 2015). Recently, a growing number of Aspergillus spp. fungi, have been reported to produce novel bioactive compounds, such as butenolide derivatives as anti-inflammatory agents ), aurasperone H as anticancer (Li et al. 2016), asperchondols A and B as antibacterials (Liu et al. 2017), aspergivones A and B with anticancer and antibacterial activities (Ma et al. 2017b), and aspochalasin B and D as antibacterials (Ratnaweera et al. 2016). In this section, we discuss bioactive metabolites of Aspergillus spp., isolated from terrestrial environments, that shown antibacterial activities and could provide further cues for clinical trials. Table 1 summarises antibacterial findings reported on the preclinical antibacterial activity of the compounds and extracts from Aspergillus spp. Wilkins and Harris (1942) examined 100 species of fungi for the production of bacteriostatic or bactericidal substances. About 40% of the Aspergillus strains yielded antibiotics. A. fumigatus mut. helvola yielded particularly active filtrates, apparently superior to the bacteriostatic substances produced by Penicillium. The tested bacteria were E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa.
A year later, Chain et al. (1943) subsequently isolated a crystalline antibiotic against Gram-positive bacteria from Aspergillus filtrates, which was named helvolic acid (1), and the name being derived from the variety of A. fumigatus yielding the best product.
A penicillin-like substance was isolated from A. giganteus (Wehm) by Philpot (1943), which she named gigantic acid. Later, Florey et al. (1944) isolated the antibiotic claviformin (patulin) (2) from a solution produced by A. giganteus (Wehm). For many years, claviformin was originally used as an antibiotic against Gram-positive and Gram-negative bacteria, but due to its toxicity to humans and animals, it was reclassified as a mycotoxin during the 1960s (Bennett and Klich 2003).
In 1944, Soltys (1944) extracted a product from A. fumigatus called aspergillin (3). This product is inactive against staphylococci but inhibits the growth of Mycobacterium tuberculosis even when tested in high dilutions. He concluded that this antibacterial is nontoxic for experimental animals and is sufficiently stable to withstand boiling for one hour.
A compound, named as CJ-17,665 (9), was isolated from the fermentation broth of A. ochraceus (CL41582), which isolated from a soil sample collected in Venezuela. This compound showed inhibitory effect against multi-drug resistant S. aureus (MDRSA), S. pyogenes, and E. faecalis, with MIC values of 12.5, 12.5, and 25 μg ml −1 , respectively (Sugie et al. 2001). The chemical structure of this compound contains a diketopiperazine and an indole N-oxide moiety.
The chloroform extracts of A. fumigatus, isolated from a soil sample collected in Pantanal, Brazil, showed antibacterial activity against S. aureus and M. luteus, with IZDs of 20.7 and 39.3 mm, respectively. The antibacterial activity detected for the chloroform extract of the culture broth incubated without the pool of bacteria was lower than that detected for the extract with the pool. Benzathin penicillin G was used as positive control, with IZDs of 33 and >40 mm for S. aureus and M. luteus, respectively. The active compounds were isolated from the broth of the culture grown in the presence of pooled bacteria and identified as 3,4-dimethoxyphenol (10) and 1,3,5-trimethoxybenzene (11) (Furtado et al. 2002).
It is well known that kojic acid (21), produced by various fungi including Aspergillus spp., is reported to possess antibacterial activity (Wilkins and Harris 1942;Marwaha et al. 1994). In a study conducted by El-Aasar (2006), A. flavus, A. glaucus, A. oryzae, A. parasiticus, and A. tamirii, isolated from a soil, were cultured for kojic acid production. The antibacterial activity of this compound was tested against six bacterial pathogens including Gram-positive and Gram-negative bacteria. The results revealed that kojic acid has activity against all the tested bacteria, with MIC values of 176-285 μg ml −1 . Ceftazidime (MIC = 14-34 μg ml −1 ) and nitrofurantoin (MIC = 65-105 μg ml −1 ) were used as positive controls.
Six alkylitaconic acids, designated tensyuic acids A to F, were isolated from the culture broth of A. niger (FKI-2342) isolated from a soil collected at Ooura Tensyudou, Nagasaki, Japan. Only tesyuic acid C (22) showed antibacterial activity against B. subtilis, with IZD of 10 mm, at a concentration of 50 μg disc −1 (Hasegawa et al. 2007).
Neosartorin (29) was isolated from the endophytic fungus A. fumigatiaffinis, which isolated from the plant Tribulus terrestris (Zygophyllaceae) collected in Uzbekistan. Compound 29 inhibited the growth of a broad spectrum of Gram-positive bacteria, while the tested Gram-negative species were not affected. MIC values from4 to 8 μg ml −1 were obtained for S. aureus, B. subtilis, and most streptococci and the growth of enterococci was inhibited at slightly higher concentrations (16-32 μg ml −1 ) (Ola et al. 2014).
In a study conducted by Ismaiel et al. (2016), emodin (23) was isolated from A. awamori (WAIR120; LC032125), which isolated from stored wheat grain sample obtained locally from a retail supermarket (Zagaig, Egypt). This compound showed antibacterial activity against E. faecalis (AHR7), with MIC of 125 μg ml −1 . Emodin was found to induce morphogenic effects including swelling and elongation of bacterial cell as shown by light microscopy. In addition, cellular effects were also resulted, in which emodin caused considerable changes in the nature of cell membrane and submicroscopic structure of bacterial cell as shown by transmission electron microscopy.
Ethyl acetate extracts of the fungi A. flavus, A. fumigatus, and A. niger, isolated from the soil of .62-500 μg ml −1 , respectively. Penicillin was used as a positive control and exhibited IZDs and MICs of 9.2-15.2 mm and 15.62-250 μg ml −1 , respectively. The results of gas chromatography-mass spectrometry indicated to the presence of many fungal metabolites, but β-thujaplicin (42) was at high yield (Monggoot et al. 2018).
Four compounds, namely isoversicolorin C (43), isosecosterigmatocystin (44), glulisine (45), and versicolorin C (46) were isolated from the mangrove-derived endophytic fungus A. nidulans (MA-143), obtained from leaves of the plant R. stylosa. Using the microplate assay, these compounds were tested for antibacterial activities against three human pathogens (E. coli, M. luteus, and Vibrio vulnificus) and four aquatic bacteria (Edwardsiella ictaluri, Vibrio alginolyticus, V. anguillarum, and V. parahaemolyticus). Compound 43 showed potent antibacterial activity against V. alginolyticus and E. ictaluri, with MIC values of 1 and 4 μg ml −1 , respectively, while compound 44 exhibited activity against E. coli, M. luteus, V. alginolyticus, V. parahaemolyticus, and E. ictaluri, with MIC values ranging from 1 to 8 μg ml −1 . Chloramphenicol was used as a positive control and exhibited MIC values ranging from 0.5 to 8 μg ml −1 (Yang et al. 2018). Twelve bisabolane sesquiterpenoid derivatives were isolated from the endophytic fungus A. versicolor, obtained from the leaves of Elaeocarpus decipiens (Elaeocarpaceae). The activity of these compounds were tested against the enterobacterial plant pathogen E. carotovora subsp. carotovora, and the results showed that all compounds displayed antibacterial activities, with MIC values ranging from 15.2 to 85.2 μg ml −1 (Guo et al. 2018).
Under specific culture conditions, the endophytic A. fumigatus (CBA2743), obtained from the plant Copaifera multijuga (Fabaceae), released metabolites into the fermentation broth. The fractions of the fermentation broth were obtained by different chromatographic techniques and tested against Mycobacterium smegmatis and M. tuberculosis. The results showed that F4 fraction had positive antimycobacterial activity, with MIC value of 256 μg ml −1 (Silva et al. 2018).

Antibacterial properties of crude extracts
In this section, we discuss the crude extracts having antibacterial activities from terrestrial Aspergillus spp. These extracts with their susceptible bacteria are listed in Table 1. In preparing enzyme concentrates from certain moulds, antibacterial activity was noted in extract from a number of species characterized by high proteolytic capacity. Thus, two strains of A. clavatus were able to synthesise and release bactericidal products, into the liquid media, which possessed antibacterial activities against S. aureus and other bacteria (Wiesner 1942). In a study conducted by Foster and Karow (1945), antibacterial substances present in culture filtrates of A. niger (YWA), A. nidulans, A. oryzae (TP), and A. flavipes are either identical with, or closely related to, authentic penicillin produced by Penicillium notatum. This was shown by solubility properties, thermostability, pH stability, antibacterial spectra against 18 different bacteria, destruction by the enzyme penicillinase, and (in one instance) by efficacy in protecting animals against a lethal bacterial infection. Wilkins and Harris (1945) observed that Aspergillus spp. produced bacteriostatic substances against Bacterium coli, S. aureus, and Pseudomonas pyocyanea. Brian and Hemming (1947) reported that the culture filtrates of different species of Aspergillus displayed antibacterial activity against S. aureus and Salmonella typhi. Irobi et al. (2000) worked on A. quadrilineatus isolated from moldy Acha, a common cereal grown among the plateau state of Nigeria. They found that the petroleum ether defatted crude extract of this fungus has remarkable antibacterial activity against S. aureus and B. Subtilis, with IZDs of 18 and 20 mm, respectively at pH 6.4.
Antibacterial activity of crude and partially purified ethyl acetate extracts, derived from two species of endophytic fungus Aspergillus, was tested. These fungi were isolated from mangrove fern Acrostichum aureum and mangrove angiosperm associate, Acanthus ilicifolius of Nethravathi Mangrove, on the southwest coast of India. The results showed different activities against a panel of bacteria including Gram-negative and Gram-positive bacteria, with IZDs ranging from 0 to 15.2 mm (Maria et al. 2005).
The antibacterial activity of the mycelia extracts and crude culture broth of the endophytic fungus Aspergillus sp., isolated from the leaf samples of Justicia adathoda (Acanthaceae), was evaluated by agar well diffusion method against some bacterial strains. Crude mycelial extract inhibited all the strains significantly, with a mean strongest IZD of 12 mm against E. coli. Crude culture broth inhibited the strains of P. aeruginosa and Klebsiella pneumoniae alone and had a mean stronger IZD of 13 and 15 mm, respectively, than crude mycelial extract (Prabavathy and Nachiyar 2012).
The crude ethyl acetate extract of A. fumigatus, isolated from the plant Garcinia spp. (Clusiaceae), was screened for antibacterial activity. It showed antibacterial activities against B. subtilis, E. coli, K. pneumoniae, Shigella flexneri, and S. aureus, with IZDs of 16, 18, 20, 11, and 16 mm, respectively (Ruma et al. 2013). Al-Shaibani et al. (2013) studied the inhibitory effect of the culture filtrate of A. niger, obtained from the inflamed eyes of patients against P. aeruginosa, S. aureus, S. epidermidis, and Bacillus sp., which isolated from patients of microbial keratitis, who attended to Ibn Al-Haetham Eye Teaching Hospital, Baghdad, Iraq. The results revealed that A. niger possessed inhibitory effect against P. aeruginosa, S. aureus, S. epidermidis, and Bacillus sp., with IZDs of 15, 25, 30, and 32 mm, respectively.
Aspergillus sp. was isolated from the roots and leaves of Triticum durum (Poaceae) collected from the Bordj Bou Arreridj region (Algeria). Antibacterial activity of the crude ethyl acetate extract of this fungus was evaluated using agar diffusion assay against 12 pathogenic bacteria. Except Enterobacter agglomerans, P. aeruginosa, and Citrobacter freundii, the results revealed inhibition activity against the tested bacteria, with IZDs of 11.3-21.7 mm, and the largest zone was 21.7 mm against E. coli (Sadrati et al. 2013).
Many endophytic fungi were isolated from the leaves and stems of the plants Suaeda maritima and S. monoica (Amaranthaceae). Among these fungi, A. terreus (SM-EF 3) and A. flavus (SM-EF 2) were isolated. The fungal culture of these fungi were extracted with ethyl acetate and used as crude extract for checking their antibacterial activities by well diffusion method. The extract of A. terreus showed antibacterial activity, with IZDs of 11. 6, 10.3, 10, 9.3, 9, 8.6, and 5.6 mm against S. typhi, S. aureus, V. cholera, E. coli, K. pneumoniae, S. paratyphi, and Klebsiella oxytoca, respectively. The extract of A. flavus showed antibacterial activity against S. paratyphi, P. mirabilis, and S. aureus with IZDs of 7, 5.3, and 5.6 mm, respectively (Kalyanasundaram et al. 2015).
The crude extract of the endophytic fungus A. niger, isolated from the leaves of Opuntia dillenii (Cactaceae), was investigated for its antibacterial potential against five bacterial species. The fungal extract showed activity against S. aureus, B. subtilis, and E. coli, with IZDs of 9, 15, and 7 mm, respectively (Ratnaweera et al. 2015).
The ability of A. fumigatus, isolated from Sudanese indigenous soil, to produce antibacterial compounds was tested by Hassan and Bakhiet (2017). The crude extract (5%) of the fungus exerted activity against Salmonella typhimurium, Listeria monocytogenes, and P. aeruginosa, with IZDs of 8, 19, and 25 mm, respectively.
Using agar well diffusion method, the mycelial extracts of the fungal endophytes, A. fumigatus and A. niger, isolated from different medicinal plants, were investigated for antibacterial activity against some bacterial species. A. fumigatus showed inhibition, with IZDs of 9, 9, 6, 13, 8, and 6.5 mm against Proteus mirabilis, S. aureus, K. pneumoniae, P. aeruginosa, B. subtilis, and P. fluorescense, respectively, while A. niger showed lowest inhibition, with IZDs ranging from 2 to 6 mm. Tetracycline was used as a positive control, with IZDs ranging from 13 to 21 mm (Akinyemi 2017). Mishra et al. (2017) observed that ethyl acetate extract of the endophytic fungus A. clavatonanicus (MJ31), isolated from roots of Mirabilis jalapa (Nyctaginaceae), displayed antibacterial potential against B. subtilis, M. luteus, and S. aureus, with MICs of 0.078, 0.156, and 0.312 mg ml −1 , respectively.
At different concentrations, the fungal extract of A. niger (MTCC-961), obtained from Microbial Type Culture Collection, Chandigarh, India, was tested for its antibacterial activity against eight bacterial species by agar well diffusion method. The results displayed antibacterial activity, with IZDs ranging from 11 to 18 mm, at a concentration of 100 μg ml −1 . It also observed that the activity was concentration dependent. Gentamicin was used as a positive control and exhibited IZDs ranging from 15 to 19 mm (Kalyani and Hemalatha 2017).
A. oryzae (DBM4336), obtained from the Department of Biochemistry and Microbiology (DBM), University of Chemistry and Technology (Prague, Czech Republic), was screened for its antibacterial activity. At different solvent extractions, the antibacterial properties of fungal extracts were screened against seven bacterial species by disc diffusion method. From the results of the study, it was observed that different solvents used for preparation of the mycelial extracts gave different degrees in inhibitory activity. For this, the ethanol extract was found to be active against all seven testing bacteria used, with IZDs ranging from 1 to 10 mm, while aqueous extract was found to be not active (Synytsya et al. 2017).
The extracts of different endophytic fungi, A. nomius, A. oryzae, A. niger, A. terrus, and A. nidulans, isolated from different tissues (leaf, stem, and root) of Calotropis procera (Apocynaceae), were evaluated for their antibacterial potential against nine bacterial strains. The tested extracts showed activity against all tested bacterial strains, with IZDs ranging from 10 to 17.3 mm and MICs ranging from 15.6 to 250 μg ml −1 (Rani et al. 2017).
Ten species of Aspergillus were isolated from soil samples obtained from two different Algerian regions namely: Laghouat and Teleghma. The ten species were identified as A. fumigatus, A. niveus, A. wentii, A. fumigatiaffinis, A. quadrilineatus, A. nidulans, A. terreus, A. flavus, A. sclerotiorum, and A. niger. Using disc diffusion method, the extracts of these fungi exerted activity against S. aureus (ATCC 25923), B. subtilis (ATCC 6633), and E. coli (ATCC 25922), with IZDs ranging from 7 to 31.7 mm, and the highest activity was by A. fumigatus against S. aureus, with IZD of 31.7 mm. The extracts of all species had no activity on P. aeroginosa (ATCC 9027), except the extracts of A. niveus and A. wentii, with IZDs 8 and 7 mm, respectively (Amina et al. 2017).

Conclusions
The rapid development in antibiotic resistance in bacteria has generated an increased demand for the development of novel therapies to treat bacterial infections. The present review focuses on antibacterial activity of Aspergillus spp. isolated from terrestrial environment from all over the world. Aspergillus spp. show antibacterial activity against pathogenic bacteria. According to literature, isolated compounds and extracts from Aspergillus spp. exhibit higher antibacterial activity against Gram-positive than Gram-negative bacteria. While several compounds exhibited higher IZDs or MICs than an antibiotic, some of them, such as the compound CJ-17,665, exhibited good activity against multi-drug resistant S. aureus, which is one of the most important current public health problem. Therefore, Aspergillus spp. may provide metabolites that may help treat infectious diseases that have increased resistance to current antibiotics, and could provide alternative medical treatment.