AYajjmpDLunawN9mRtBUbWAMSNG9on1NRLA. baumanniiis one of most the virulent pathogens causing infections in the critical care units in Egypt (See et al., 2013) and worldwide A. baumannii com-monly causes nosocomial infections, predominantly the aspiration pneumonia, catheter-associated blood stream infection, catheter-associated urinary tract in-fections and soft tissue infections (Wong et al., 2017). The previous authors concluded that the community-acquired infections of Acinetobacter spp. have in-creased over time. Disease spread of Acinetobacter spp. is assisted by host immunity evasion, pathogen resistance to dehydration, and antimicrobial agents. Multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria showed high resistance to all available antibiotics, commonly used in the treating infections caused by A. baumannii, which significantly increase mortality (Kengkla et al., 2018). In 2017, the World Health Organization (WHO) reported that, A. baumannii is one of the most virulent pathogens, requiring urgent control, so seeking new antimicrobial drugs is very important. The important sources of natural bioactive compounds are microorganisms, which have enormous potential for the exploration of new compounds for the pharmaceutical applications (Demian 1999; Keller et al., 2005; Porras & Bayman, 2011; Qadri et al., 2013). During the fermentation, a wide range of secondary metabolites are produced by filamentous fungi including: antibiotics, antitumor compounds, antiviral, anti-parasitic agents, immune-suppressants, and the toxins (Ranadive et al., 2013; Nigam & Singh, 2014). Fungal bioactive compounds are therefore more beneficial for human compared to any other natural products (Gulwani et al., 2014). For example, secondary metabolites of the Chaetomium globosum act as anti-cancer agents (Moubasher & Hamed, 2015). Additionally, secondary metabolites of Aspergillus terreus act as anti-cancer, antibiotic and anti-inflammatory agents (Boruta & Bizukojc, 2017).

Microbial co-cultivation, which is known as mixed fermentation, involves the cultivation of two or more microorganisms in the same environment (Lami et al., 2019; Chasoya et al., 2021). This method induces the production of active metabolites by metabolite pre-cursor formed enzymes or by inducing epigenetic modifications in the producer strain to form these metabolites (Wakefield et al., 2017). Co-cultures of A. terreus/ C. globosum stimulated lovastatin production (Boruta et al., 2019; González et al., 2020). Whereas, production of enzymes was induced in A. terreus / A. niger co-culture (Rehman et al., 2014). It was con-cluded that many compounds such as butyro-lactones, terrain, orsellinic acid, and anthranilic acid were induced in A. terreus / Bacillus subtilis or Bacillus cereus co-cultures (Chen et al., 2015). These com-pounds were not detected in axenic fungal culture (González et al., 2020). This study aimed to induce fungal secondary metabolites using co-culture tech-niques and to detect their effect against A. baumannii.

Isolation and identification of bacteria

Isolation and identification of bacteria from clinical specimens (sputum, blood and wounds) were carried out in the microbiology laboratory of the Clinical Path-ology Department, Faculty of Medicine, Cairo Univer-sity, Egypt. Collection and handling of the clinical specimens were performed by experienced nurses. Bacterial identification was performed according to morphological characteristics, response to Gram stain, and biochemical reactions. Also, enzymatic reactions using VITK MS automated mass spectrometry micro-bial identification system (Biomerieux) that uses Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) technology were also used in the identification. Antibiotic susceptibility test of the clinical isolates was done according to Clinical Labo-ratory Standard Institute guidelines (Weinstein, 2020) in the same department.

Isolation of fungi

Fungi were isolated from soil and air, by using the methods of Moubasher et al. (2022) on Czapeks-Dox agar w/v (3% sucrose; 0.1% KH2PO4; 0.05% Mg-SO4.7H2O; 0.05% KCl; 0.001% FeSO4.7H2O 0.02%; NaNO3; 1.5% agar and 0.005 % Rose Bengal as bacteriostatic agent). Fungi collected from jam were isolated on the same medium containing 6% w/v sucrose instead of 3% w/v. The inoculated plates were incubated at 28°С for 5 days. 

Antibacterial activity of the isolated fungi

Fifty fungi species were isolated from different sources were tested to determine their inhibitory effect against the selected bacterium. One ml of 0.5 McFarland standard (MCF) (Gayathiri et al., 2018) of bacterial suspension was spreaded on plates containing Mueller Hinton agar w/v (0.2% beef extract; 1.75% casein hydrolysate; 0.15% starch; 1.7 %  agar), Czapeks-Dox agar, malt extract agar w/v (2% malt extract; 2% glu-cose; 0.1% peptone; 2%  agar) or sucrose peptone agar w/v (2%  sucrose; 0.5% peptone; 0.05%  KH2PO4;    0.025%  MgSO4.7H2O; 1.5%  agar,  pH was adjusted at 7.2 - 7.4 by adding known volume of 40% NaOH). Disc from fungal growth margin (1cm diameter) was transferred into a central bore of each plate. The Petri dishes were incubated at 38°С for 24-72 hrs. At the end of the incubation time, the most potent fungus against the multi-drug resistant bacterium was deter-mined. The potent fungus was kindly identified at Assiut University Mycological Center, Egypt (AUMC).

Induction of the secondary metabolites production using co-culture 

Five discs (1cm diameter each) were cut from the growth margin of the potent fungus and transferred into 4L Erlenmeyer flask containing 1L sucrose pep-tone broth (supplemented with 4 % sucrose). Flasks were incubated at 28°С for 3 days at standing and shaking (150 rpm) states. At the end of the incubation time, each flask was inoculated with 1ml of 0.5 McFarland standard (MCF) suspension of the selected bacterium. The co-cultured flasks were incubated at 38°С for 10 days at standing and shaking states. 

Separate flasks containing either fungus or bacterium isolates were used as controls. At the end of incubation period, filtration of the growth culture of each flask was done by using filter paper Whatmann No.1 to remove fungal mycelia. Then the filtrate centrifuged at 10,000 g for 10 min. at 4°С. Partitioning of the super-natant was carried out successively by chloroform, ethyl acetate and methanol. Drying of the extracts was performed using a rotary evaporator at 50 °С and then the extracts were weighed separately. One milligram of each extract was dissolved in 200 µl of its solvent to give a final concentration of 5µg/µl. Bacterial suscepti-bility to the different types of fungal extracts was carried out using filter paper disc diffusion method (Balouiri et al., 2016). Discs impregnated with 50 µl of each crude extract, which was allowed to dry. This disc was placed on the growth medium previously inoculated with the selected bacterium isolates to assess antibacterial effect of extracts. After incubation at 38°C for 24 hrs the diameter of inhibition zones around the discs was measured to detect the most efficient extract.

Separation, purification and identification of the most potent compound

The most efficient extract was fractionated by silica gel column chromatography. Silica gel 60 P (Carl Roth GmbH+ Co. KG – Schoemperlenstr- Germany) was used as stationary phase. Silica gel (100 gm) was sus-pended in a known volume of solvent mixture (75 ml chloroform + 25 ml ethyl acetate) to pack column with dimensions 10 cm diameter and 100 cm height. In the first column a mixture of chloroform (CHCl3): ethyl acetate (EtOAc) (2: 1 v/v) was the mobile phase with increasing gradient of ethyl acetate up to 100% (v/v) then used a mixture of  ethyl acetate-methanol, 1: 12 (v/v) with increasing gradient of polarity by increasing methanol (Moubasher & Hamed, 2015). The collected fractions were categorized into groups by using pre-coated thin layer chromatography (TLC) (Fluka 60778254 nm). Mixture of CHCl3: EtOAc (1:1 v: v) was used as developing system. Antibacterial activity of each group was tested. The most potent group was subjected to further purification using the second column of silica gel chromatography. Mixture of CHCl3-EtOAc, 2: 1 (v/v) was used as the developing system with increasing ethyl acetate gradient up to 100% (v/v) then used ethyl acetate-methanol mixture, 2: 1(v/v) with the increasing gradient of polarity by increasing methanol of polarity. Sub-fractions were collected from the second column were running on pre-coated TLC, with using CHCl3-EtOAc, 1:1 (v/v) as developing system. According to their polarities these sub-fractions were categorized into groups by using (Fluka 60778254 nm). Further purification of the most effective group, against the selected bacteria, was per-formed by using preparative TLC silica gel 60 P (Carl Roth GmbH+ Co. KG – Schoemperlenstr- Germany). Mixture of CHCl3-EtOAc, 6:1 (v/v) was used as deve-loping solvent. The observed fractions were scraped off from TLC surface and dissolved in 1 ml of EtOAc to determine their antibacterial effect against the 3 isolates of A. baumannii. Checking the purification of the fraction which showed highest antibacterial effect against the tested isolates, was done by using pre-coated TLC and CHCl3 as developing system. The purified compound which showed highest antibacterial effect was characterized by subjecting to IR, LC-MS and 1H NMR spectroscopic studies. LC-MS was done by Waters Acquity QSM column reverse phase C-18 using A, B, C, D solvents which were Water + 0.1% formic acid (A); methanol + 0.1% formic acid (B); methanol 90% + Water 10% (C), and acetonitrile (D), with running time 32 min and flow rate 45 min in gra-dient from 100% to 10%. Column average temperature was 34.2°С. 1HNMR was done by BRUKER 400 MHz using CDCl3 as a solvent, at Center for Drug Dis-covery Research and Development, Ein Shams Univer-sity. Finally, IR was done by JASCO 460 plus with wavelength (400-4000) nm and frequency (50-60) Hz at Micro Analytical Center, Cairo University.

Determination of minimum inhibitory concentration (MIC)

Different concentrations (192,160, 128, 96 µg.µl-1) of the most potent purified compound were prepared. The minimum inhibitory concentration for the purified compound was executed by the paper disc-diffusion method (Booq et al., 2021). One ml of 0.5 McFarland standard suspension of A. baumannii was spread on plates containing Mueller Hinton by using sterile cot-ton swap. Discs impregnated with the different concen-trations of the purified compound (50µl) were placed on the inoculated plates and incubated at 38 °С. Inhi-bition zone diameters were measured after incubation for 24 hrs.

Identification and antibiotics susceptibility test of the selected bacterium

The clinically isolated bacterium was identified as Acinetobacter baumannii. The isolates showed resis-tance to carbapenem group antibiotics (meropenem and imipenem).

Determination of the highly potent fungus against MDR A. baumannii isolates

It was found that only one fungal species, among 50 spp. showed inhibitory effect against the three isolates of A. baumannii (Fig. 1). The fungus isolated from jam was the potent species and identified as Aspergillus terreus (AUMC 14766).

Fig. 1:  A. terreus against A. baumannii

Fig. 2:  Dry weight (mg/l) of ethyl acetate (EtOAc), chloroform (CHCl3) and methanol (MeOH) extracts of co-and monocultures filtrates of A. terreus & A. baumannii that were grown under shaking and standing condition.

Susceptibility of A. baumannii to A. terreus and A. baumannii co-culture and monocultures secondary metabolites

The suitable medium for co-culture of A. terreus / A. baumannii was sucrose peptone broth with 4% suc-rose. Under high osmotic pressure, greater amounts of secondary metabolites were induced in co-culture of A. terreus / A. baumannii than in each monoculture of both organisms, particularly in ethyl acetate extract (Fig. 2). Also, the induction of secondary metabolites in standing condition was highly stimulated in compa-rison shaking state. Moreover, secondary metabolites production in axenic culture of A. terreus was more than in axenic culture of A. baumannii.

Determination of the highly potent extract against A. baumannii isolates

The most potent extract was ethyl acetate extract of A. terreus /A. baumannii co-culture against the three isolates of MDR A. baumannii (Table 1), followed by chloroform extract of co-culture. However, all extracts of both A. terreus and A. baumannii monocultures as well as methanol extract of co-culture were ineffective against the selected isolates. The effect of the co-cul-ture extracts on the 3 isolates was nearly similar (Table 1)

Table 1:  Antibacterial activity of co- culture, monocultures of A. terreus and A. baumannii extracts.

IZD* (inhibition zone diameter), (1) Co- culture of A. terreus/ A. baumannii, (2) monoculture of A. terreus, (3) monoculture of A. baumannii.

Fractionation of ethyl acetate extract 

Twenty eight fractions were collected from co-culture ethyl acetate extract by the first column. According to polarities, fractions were categorized into 10 groups by using pre-coated thin layer chromatography (TLC). Group (2) showed inhibitory effect against the three isolates of MDR A. baumannii with 20 mm inhibition zone diameters (Fig. 3). After further purification of this group by the second column, eleven sub-fractions were collected, and categorized into 7 groups, accor-ding to their polarities, on pre-coated TLC. Only groups (3) and (4) showed inhibitory effect against A. baumannii isolates. 

By using agar disc-diffusion method, group (3) showed higher antibacterial effect (23 mm inhibition zone diameter) more than group (4) which showed 19 mm inhibition zone diameter. Further purification of group (3) was executed by the preparative TLC. Only sub-fraction (2) out of (6) sub-fractions exhibited inhibition effect against the MDR isolates of A. baumannii. 

Structure elucidation and characterization of the purified compound

After complete purification of sub-fraction 2 by using pre-coated TLC its structure elucidation and character-ization were performed. LC-MS technique was carried out by Waters Acquity QSM column reverse phase C-18.

Fig. 3:  Bactericidal activity of groups No.1, 2, and 3 which were separated from first Column against A. baumannii and C was ethyl acetate disc as a control

The pure compound showed main base peak at [M+] = 359.1HNMR in CD3Cl CH2O (3.3 ppm), CH2N (2.1 ppm), C2H2O (1.4 ppm), 6 CH2 (1.3ppm), CH3 (1.1 ppm), CH2 (1.9 ppm), CH (1.98 ppm), CH (0.2 ppm). IR was done by JASCO I460 plus (Fig. 4). In which at (34440 cm-1) NH, (2956 cm-1) COOH,(2854 cm-1) CHO,(1739 cm-1) ester,(1462 cm-1) C=O,(1261 cm-1)  C-OH,(1097 cm-1) C-OH,(1029 cm-1) C-OH. Depen-ding on the elucidation of the purified antibacterial compound and by the help of the published data, the purified compound was identified as terreusinone A with molecular formula C20H26N2O4 and molecular weight 358. The chemical structure of the identified compound was illustrated as shown in Fig. 5.

Fig. 4: IR by JASCO I460 plus of purified compound.

Fig. 5: Chemical structure of terreusinone.

Determination of the minimum inhibitory con-centration (MIC) of terreusinone A

Different concentrations of terreusinone A (192,160, 128, 96 µg /µl) showed different effects against the isolates of A. baumannii. (Fig. 6). Clear zones appe-ared at two concentrations of terreusinone A (160 and 192 µg.µl-1), whereas the lower concentrations were ineffective. MIC of terreusinone A was 160 µg.µl-1.

Inappropriate antimicrobial treatment and intensive care unit (ICU) stay led to the emerging of antibiotic resistant bacteria more rapidly among pathogens (Provenzani et al., 2020; Vivi et al., 2022). The World Health Organization (WHO) reported that carbapenem resistant A. baumannii is one of the most important MDR bacteria associated with hospital acquired infections (Provenzani et al., 2020). Many factors are res-ponsible for emerging of bacterial carbapenem resis-tance such as carbapenemase enzymes, the combined activity of ESBL and efflux pumps, or porin mutations (Shahen et al., 2019; El-kholy et al., 2021). 

The same authors observed high prevalence rate of carbapenem resistant A. baumannii in Egypt. High prevalence rates of A. baumannii occurred also in Mediterranean area, Southern Europe, Middle East and North Africa. While, low prevalence was recorded in Oceania, Western Europe, the Nordic region, and part of the central Europe (Ma & McClean, 2021). A. baumannii has a tolerance system for environmental changes by the biofilm formation, which provides pro-tecttion from environmental stresses such as disinfect-tion regimes, desiccation and host immune responses (Upmanyu et al., 2022).

Fig. 6: Effect of different concentrations of terreusinone A on A. baumannii. Discs (1, 2) conc. 96 µg.µl-1, disc (3)  conc. 128 µg.µl-1, disc (4) conc. 160 µg.µl-1, disc (5) 192 µg.µl-1 disc, and (C) ethyl acetate as a control.

The high level of intrinsic and acquired mechanisms of multidrug resistant bacteria makes their treatment diffi-cult. Therefore, it is important to focus on bioactive compounds such as fungal secondary metabolites, to find a suitable and effective alternative treatment in-stead of carbapenem, the commonly used antibiotic in treating Acinetobacter infection (Tiwari et al., 2015). It was reported that A. terreus was the only species among 50 spp. which showed antimicrobial activity against A. baumannii in the current study. This finding is in harmony with Barakat and Gohar, (2012) that showed that A. terreus among 26 fungal isolates had highest antibacterial activity. It was found that incu-bation conditions can affect the growth of micro-organisms and their metabolites (Atalla et al., 2008; Mathan et al., 2013). The results of this experiment and other reports revealed that the growing of A. terreus was significantly affected by the type of growth medium. In the current study, the most suitable media for the growth of A. terreus and A. baumannii were malt extract agar (Zain et al., 2009) and Mueller Hinton agar (Aryal, 2022) respectively. However, for co-culture, sucrose peptone broth was the best. Tempe-rature is an important factor regulating microbial growth and activity (Mathan et al., 2013). From this experiment the selected fungus can grow at 28°С and 38°С, which is on line with the study of Atalla et al. (2008); Mathan et al. (2013) reported that A. terreus can grow in a wide range of temperature from 25°С to 45°С, but the optimum temperature is 27°С. Pieti-käinen et al., (2005) concluded that fungal growth rates had an optimum temperature ranged from 25 to 30°C. The activity of A. baumannii decreased at 25°С and increased by increasing temperature (Monem et al., 2020). In our study incubation temperature at 38°С was suitable for A. terreus / A. baumannii co-culture. Both growth and yield of secondary metabolites were higher at standing condition than shaking condition. This finding is in harmony with that of Atalla et al. (2008) who suggested that agitation may fragment mycelia to cells that need some time to repair this dam-age, which will lead to decrease in secondary meta-bolites yield. In this work A. terreus grew well in sucrose peptone medium with pH 7.2 -7.4 that was confirmed by Pang et al. (2020). In our study A. terreus was isolated from jam and grew under high osmotic stress. (Atalla et al., 2008; Mathan et al., 2013) concluded that Aspergillus spp. can grow at broad range of osmotic pressure. Under osmotic stress A. terreus can grow and produce important pharma-ceutical compounds with anti-alzahimers disease activity (Ghoran & Kijjoa, 2021), in addition to other secondary metabolites such as gluconic acid (Dowdell et al., 2010). There are 165 compounds produced by A. terreus that was reported in the Antibase 2014 data base of secondary metabolites (Boruta et al., 2015; Jawaid et al., 2019). It was found that some of these compounds have antimicrobial activity (Al-Fakih & Almaqtri, 2019). Many bioactive compounds having industrial and medical applications are induced by several biosynthetic gene clusters (BGCs) in micro-organisms. However, most BGCs are not expressed and remain inactive under normal in vitro conditions, which prevent metabolic potential from being exp-loited. So, it is important to apply new techniques to stimulate and induce the production of certain meta-bolites which activate these biosynthetic pathways. Co-culture is an important way to elicit cryptic com-pounds. Wakefield, (2017); Chasoy et al. (2021) con-cluded that co-culture assay is an efficient way to stimulate the metabolic potential of fungi for produc-tion of new therapeutic agents as well as increasing  their concentrations in  response to quorum sensing molecules, that could control emerging of health issues (Sharma & Jangid,  2017). 

In Boruta et al. (2019) reported that A. terreus / Chaetomium globosum co-culture elicited lovastatin like compound that is monacolin J acid to produce. Also, in the co-culture of A. terreus/ Paecilomyces lilacinus, production of new compound terreins was stimulated (Lei et al., 2020). Boruta et al. (2020) found that the production of lovastatine, butyrolactone I, geodin and asterric acid was induced in co-culture of A. terreus / Penicillium rubens and Mucor racemosus. From this study, crude ethyl acetate extract of A. terreus / MDR A. baumannii co-culture was the most active extract against MDR A. baumannii when com-pared with methanol and chloroform crude extracts. In A. terreus monoculture, ethyl acetate (Choudhary et al., 2004; Silva et al., 2017) and methanol (Moha-mmed et al., 2016) crude extracts had bactericidal and antitumor effects, while in this experiment chloroform, ethyl acetate and methanol extracts of monocultures had ineffective against MDR A. baumannii. This may be referred to inability of A. terreus and A. baumannii in monocultures to produce bioactive compounds in sufficient concentrations to control bacterial growth. From our observations, the pure compound exhibited lower toxicity effect than crude extracts against A. baumannii. This may be referred to the presence of other compounds in addition to terreusinone A in the crude extract, which aids in increasing antibacterial activity through synergism. The most potent com-pound was identified as terreusinone A, after subject-ting to structure elucidation and characterization. Also Lee et al. (2004) isolated this compound from A. terreus. Terreusinone A is a yellow pigment and it belongs to the dipyrrolobenzoquinone derivatives. Nguyen et al. (2013) concluded that dipyrroloquinone alkaloids are sources of new anti-tumor and derma-tological drugs. Terreusinone A acts as a potent UV-A protectant (Lee et al., 2004; Vala et al., 2019). It showed cytotoxic effect against cancer cells (Wei et al., 2015). However, according to our available know-ledge terreusinone A antibacterial activity has not yet been reported. 

A. terreus exerted antibacterial effect against A. baumannii. Ethyl acetate crude extract of A. terreus / A. baumannii co-culture was effective against A. baumannii. Terreusinone A was separated from EtOAc fraction, after subjecting to several purification steps, in addition to structure elucidation and characterization processes. This is the first report that indicates anti-bacterial activity of terreusinone A particularly against A. baumannii in vitro. Terreusinone A may act as a promising drug candidate for the treatment of A. baumannii infections after exposing to several clinical trials.

Ethical approval

This work was performed in line with the principle of the Declaration of Helsinki. Approval was granted by Ethics Committee of Kasr Al-Ainy Medical School 2020/22383.

We are grateful to all the Dear Professors for providing their information and support regarding this research. 

The authors declare that there is no conflict of interest