This study began with the isolation of fungi associated to Mexican wetlands along the Pacific coast (Guerrero and Oaxaca, Mexico). The outcome of this procedure was a collection of 250 axenic isolates. Then, each microorganism was fermented in a solid medium (PDA). After the incubation period, the organic extract for each fungal strain was prepared and evaluated against multidrug-resistant A. baumannii (Fig. S1). Through this process, the 28 most promising fungi (inhibition greater than 40%: preselected fungi) were selected for up-scaling in a solid medium (Cheerios® cereal), taxonomic identification, and subsequent antibacterial evaluation. This procedure selected strains IQ-503 (Aspergillus sp.), IQ-548 (Aspergillus sp.), and IQ-567 (Talaromyces sp.), from which nine active molecules were isolated (Fig. S1-S2).

Diversity of Mangrove Fungi

Mexico is ranked in the sixth position in mangrove abundance worldwide (Lacerda et al. 1993). Mangroves are highly biodiverse ecosystems that harbor a wide range of species, especially heterotrophic microorganisms such as fungi, with ecological, chemical, and economic importance (Sosa-Rodríguez et al. 2009). These microorganisms perform various functions in this environment, such as decomposition of wood or organic matter from sediments, fragmentation of leaves, and as endophytes in symbiotic processes with mangrove roots and leaves (Sosa-Rodríguez et al. 2009). A review by Devadatha et al. (2021) indicates that approximately 850 mangrove fungi have been identified by 2020, being Xylaria spp., Aspergillus spp., Penicillium spp., Trichoderma spp., and Fusarium spp. the most represented, in agreement with outcomes from this work. In this scenario, endophytic fungi isolated from Mexican mangroves were studied looking for molecules with promising biological activity.

The taxonomic identification of 24/28 preselected microorganisms was performed using molecular methods. For this purpose, the ITS region of the ribosomal DNA was amplified and sequenced. The data are available in GenBank. To show the evolutionary relationships among the organisms, the sequences were then aligned and placed in a phylogenetic tree constructed using the maximum likelihood method (Fig. 1). In this study, 24 nucleotide sequences from fungi associated with Mexican Pacific wetlands and 38 reference sequences were included in the analysis. All identified fungi belong to the orders Botryosphaeriales, Eurotiales, and Hypocreales of the phylum Ascomycota. Within the Eurotiales, the genera Talaromyces, Aspergillus, and Penicillium were the most abundant, while the genera Phyllosticta and Lasiodiplodia, Botryosphaeriales, were less copious.

Fig. 1

The phylogenetic tree of 24/28 preselected fungal microorganisms. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site

Prioritization of MicroorganismsActivity Screening

The 28 preselected isolates were cultured in solid medium (Cheerios®) to increase yields. The antibacterial potential of each extract was assessed against A. baumannii to confirm its activity. Extracts with growth inhibition greater than or equal to 40% at 250 µg/ml toward A. baumannii A564 were selected. Some of the most outstanding extracts were (Fig. S3) IQ-503 (Aspergillus sp., 54.3 ± 5), IQ-512 (Aspergillus sp., 75.9 ± 3%), IQ-547 (Fusarium sp., 35.0 ± 3%), IQ-548 (Aspergillus sp. 60.2 ± 2), IQ-567 (Talaromyces sp, 75.0 ± 5), IQ-568 (Penicillium sp., 41.9 ± 6), IQ-573 (Penicillium sp., 42.7 ± 7), and IQ-574 (Talaromyces sp., 40.3 ± 8). Aspergillus and Penicillium correspond to the two strains with the highest reported number of naturally occurring, structurally diverse products, with 2607 and 2192 products, respectively (NPAtlas, 2023).

Untargeted Metabolomic Studies

Simultaneously, the chemical composition information obtained by LC-MS/MS for the 28 extracts was organized into molecular networks using the GNPS platform with the Derreplicator+ and MolNetEnhancer algorithms. The molecular network was constructed with 6123 mass spectra grouped into 930 nodes and categorized into 13 groups of composite families (Fig. 2). The molecular families (clusters) illustrated in the molecular networks are based on the similarity of the mass spectra. The largest group was that of benzoic acids with 108 nodes, whereas the pyrones and pyrans group with 13 nodes was remarkable. In contrast, the smallest group comprised benzodiazepines with only two nodes. Furthermore, this analysis facilitated the dereplication of 32 metabolites (with mass error < 10 ppm and cosine similarity > 0.7, Table S2), including some mycotoxins such as citrinin, harmane, meleagrin, and sterigmatocystin (Fig. 2). It is worth noting that out of the 32 identified molecules, nine have been reported to possess hepatotoxic (Flajs and Peraica 2009), neurotoxic (Lešić et al. 2019), nephrotoxic (Hamed et al. 2021), and/or mutagenic activities (Gupta et al. 2018) (Table S1). This analysis was performed to exclude extracts containing previously reported cytotoxic compounds (15 out of 28: Table S2) thus prioritizing the isolation of chemical entities with promising biological activity toward A. baumannii. Based on the results of antibacterial activity (inhibition > 40%, Fig. S1) and dereplication (Table S1), three extracts were prioritized. The selected microorganisms included Aspergillus sp. IQ-503, Aspergillus sp. IQ-548, and Talaromyces sp. IQ-567, all endophytic microorganisms from the species Rhizophora mangle (red mangrove), collected in Tecomate Lagoon, Guerrero, Mexico.

Fig. 2

Untargeted metabolomic studies. A Molecular network generated using the MS2 data, obtained for 28 extracts from preselected fungi. B Selected examples of dereplicated compounds through the GNPS platform

Chemical Study

The bioassay-guided chemical study of the bioactive extract of Aspergillus sp. IQ-548 led to the isolation of seven molecules characterized as asperazine (1) (Varoglu et al. 1997; Loach et al. 2016), aurasperone B (2) (Siriwardane et al. 2015; Priestap 1986), aurasperone F (3) (Antonov et al. 2021), TMC-256A1 (4) (Sakurai et al. 2002), fonsecin B (5) (Priestap 1986), dianhydroaurasperone C (6) (Pilevneli et al. 2021), and aurasperone A (7) (Campos et al. 2005). In parallel, investigation of Aspergillus sp. IQ-503 and Talaromyces sp. IQ-567 allowed the isolation of pyrophen (8) (Zhang et al. 2010) and penicillide (9) (Suzuki et al. 1991), respectively. All isolated molecules were identified by matching their spectroscopic and spectrometric data with those reported in the literature (Supporting Information).

Asperazine (1) belongs to the diketopiperazine group and inhibits the growth of human leukemia, murine, and human colon cell lines. It was isolated from a marine A. niger (Varoglu et al. 1997). Compounds 2–7 (aurasperone F, TMC-256A1, fonsecin B, dianhydroaurasperone C, and aurasperone A) are benzophenones derived from Aspergillus spp., principally A. niger; however, this kind of molecules and other benzophenones (e.g., penibenzophenones, tenellones, asperphenin) have also been isolated from Penicillium spp., Diaporthe spp., and Phomopsis spp. (Ibrahim et al. 2023). Pyrophen (8) is a pyrone that was first isolated from A. niger from maize in Puerto Rico (Barnes et al. 1990) and later in the species A. brasiliensis (Perrone et al. 2007). Finally, penicillide (9) is a benzodioxocin previously isolated from Penicillium sp., Pestalotiopsis sp., and various Talaromyces species with relevant biological activities (see below) (Salituro et al. 1993; Tao et al. 2017).

After the structural characterization, compounds 1–9 were manually annotated into the constructed molecular networking (Fig. S4). Asperazine (1) falls within the indole derivatives group and can be observed within a two-node cluster. Compounds 2–7 are found in small clusters. Compounds 2 and 3 exhibit a high degree of similarity in their molecular structure, differing only in small changes at positions 2′, 3′,  and 8, specifically a dehydration and loss of methoxy group, respectively. Likewise, compounds 6 and 7 also formed a single cluster because these benzophenones have distinct substituents at C-8, hydroxy, and methoxy groups, respectively. However, although compounds 4 and 5 are related, both were found in different clusters. Compound 8 appears as a member of a cluster formed by 10 nodes, indicating that some other analogues of pyrophen are present in fungal extracts. Penicillide (9) was found only in a single node.

Antimicrobial Potential of Isolates

The antimicrobial potential of the isolated molecules was determined according to the CLSI guidelines (CLSI 2015). The results of this evaluation showed that compounds 1 (IC50 6.9 ± 0.7 µg/ml), 2 (IC50 9.9 ± 0.8 µg/ml), 3 (IC50 8.3 ± 0.9 µg/ml), 4 (IC50 7.4 ± 1 µg/ml), and 9 (40 ± 9% inhibition, at 100 µg/ml) inhibited the growth of multidrug-resistant A. baumannii A564 (Fig. 3A and Table S3) in a concentration-dependent manner, with IC50 values below 10 µg/ml for compounds 1–4. Compound 9 displayed an inhibition of 40 ± 9% at 100 µg/ml. In all cases, the inhibition of bacterial growth exceeded that of the positive control gentamicin (20 ± 1% at 100 µg/ml). These results highlight the potential of benzophenones 2–4 and asperazine (1) as suitable candidates for developing antibacterial agents against A. baumannii A564, an intrahospital isolate resistant to last-generation drugs, including ciprofloxacin, gentamicin, meropenem, imipenem, doripenem, cefepime, ceftriaxone, ceftazidime, piperacillin, and ampicillin. In addition, it shows moderate resistance to colistin, a last-generation antibiotic (García-Patiño et al. 2017; Raorane et al. 2019). Based on these findings, compounds 1–4 and 9 are proposed as possible references for developing antibiotics to help combat AMR, particularly against A. baumannii, as it has become one of the main causes of resistant infections worldwide.

Fig. 3

Antimicrobial potential of isolated metabolites. A Concentration response curves for compounds 1–4 vs A. baumannii A564. B Effect of compounds 1–9 on the activity of AbFtsZ. C + indicates positive control (berberine)

In 2018, Zulqarnain et al. (2020) reported the antifungal activity of asperazine (1) against Fusarium oxysporum (at a minimum inhibitory concentration of 60 µg/ml). Furthermore, a more recent study in 2021 showed that 1 inhibited the proliferation of cervical cancer cells (Abdou et al. 2021). To date, this is the first report of this molecule as a growth inhibitor of a multidrug-resistant A. baumannii. Compounds 2 and 3, aurasperones B and F, are benzophenones with antioxidant (Leutou et al. 2016; Zhang et al. 2007), antifungal (Zhang et al. 2007), anticancer (Antonov et al. 2021; Fang et al. 2016), and antimicrobial activities against B. subtilis and E. coli (Bouras et al. 2005; Song et al. 2004). However, there are no reports of these molecules as inhibitors of multidrug-resistant A. baumannii strains. Compound 4 belongs to the naphthopyrone group and has been reported as an antioxidant (Leutou et al. 2016); furthermore, it exhibits anticancer activity against human cervical (Sakurai et al. 2002), liver (Huang et al. 2011), brain (Huang et al. 2011), and mama (Huang et al. 2011) cells and showed inhibition of IgE antibody activity, leading to its proposal as an antiallergic agent (Sakurai et al. 2002); to date, this is the first report of compound 4 as a bacterial inhibitor. Penicillide (9) has been reported to be an anticholesterolemic (Zeng et al. 2022), anticancer (Tao et al. 2017), and antimicrobial agent against strains such as S. aureus, S. albus, Vibrio alginolyticus, and V. parahaemolyticus (Song et al. 2022). This is the first report of compounds 1, 2, 3, 4, and 9 as growth inhibitors of A. baumannii.

AbFtsZ1-412 as a Putative Mechanism of Action

To establish a possible mechanism of action for the isolated molecules with antimicrobial potential, the enzyme AbFtsZ1-412 was selected as a candidate. This selection was based on the fact that molecules with similar nuclei to compounds 2–7, such as mycopyranone and viriditoxin (binaphthopyranones), interact with FtsZ proteins of S. aureus and E. coli (Rivera-Chávez et al. 2019a, b, c), suggesting this protein as a putative molecular target. To test this hypothesis, AbFtsZ1-412 was cloned and expressed in a heterologous host (Supporting Information).

Enzymatic Assay

Once the enzyme was obtained, a photometric assay was developed in a 96-well plate format at 37 °C to screen pure molecules as protein ligands. For this assay AbFtsZ1-412 (4 μM) and GTP (50 μM) were used in a final volume of 100 μl at pH 7. Assay conditions were adapted from the literature with minor modifications (Quan and Robinson 2005, Baykov et al. 1988, Martín-García et al. 2012). Malachite green was used to monitor enzyme activity and inhibition at 630 nm. To validate the ability of the assay to detect molecules capable of altering the GTPase activity of AbFtsZ1-412, berberine was used as a positive control. This alkaloid has previously been reported as an inhibitor of the FtsZ enzyme in S. aureus (Domadia et al. 2008). In this study, berberine was found to inhibit AbFtsZ1-412 activity by no more than 50% at a concentration of 186 µg/ml (500 μM) (Fig. S6).

Compounds as AbFtsZ Binders

Compounds 1–9 were evaluated against AbFtsZ1-412 at 100 µg/ml. Compound 4 showed inhibition of AbFtsZ1-412 activity, while compounds 2–3 and 5–9 showed interaction with the enzyme and enhanced its activity (Fig. 3B, Table S3). In addition, compound 4 also inhibited bacterial growth of A. baumannii A564 strain, suggesting AbFtsZ as a possible molecular target. In 2008, Rai et al. (2008) found that curcumin activates the GTPase activity of the FtsZ enzyme in E. coli. Similarly, in 2012, Ma et al. (2013) reported a group of trisubstituted benzimidazoles that activate GTPase activity of FtsZ in M. tuberculosis. In addition, vitamin K increases the GTPase activity of Streptococcus pneumonia FtsZ enzyme (Pushpakaran et al. 2022). In these reports, the authors discussed that increasing the enzyme activity raises the concentration of GDP in the bacterial cell, leading to depolymerization of the Z-ring and subsequently inhibiting cell division, resulting in enlargement and finally death. None of the FtsZ binders (2–9) identified in this study has previously been reported as ligands of any FtsZ enzyme.

Compound 5, fonsecin B, has been previously reported (Carboué et al. 2019) to possess antioxidant capacity. Lee et al. (2010) demonstrated that fonsecin B has the ability to affect the expression of specific genes involved in the production of immunoglobulins in germinal cells. Compound 6, dianhydroaurasperone C, is also a naphthopyrone with activity against skin cancer. This compound increases the intracellular concentration of vinblastine in KB-8-5 cancer cells and prevents its efflux, potentially enhancing the efficacy of vinblastine in the treatment of drug-resistant cancer (Ikeda et al. 1990). Compound 7, aurasperone A, has been reported to have antibacterial activity against B. subtilis, E. coli, and P. fluorescence, and antifungal activity against Candida albicans and Trichophyton rubrum (Lu et al. 2014). No inhibitory activity against A. baumannii was detected in this study, but it acts as a ligand for the enzyme AbFtsZ1-412. Moreover, pyrophen (8) and penicillide (9) showed significant biological activity as AbFtsZ ligands.

In prior studies, pyrophen (8) was found to hinder the growth of Aeromonas hydrophila (Agrawal et al. 2020), Micrococcus luteus (Agrawal et al. 2020), and Listeria innocua (Agrawal et al. 2020). Additionally, penicillide (9) exhibited inhibition against the microorganisms S. aureus (Song et al. 2004), C. albicans (Song et al. 2004), and E. coli (Song et al. 2004). This is the initial report of these compounds (2–9) with activity against FtsZ of A. baumannii.

Based on these considerations, it is proposed that compounds 2–9 have the potential to be FtsZ-targetting antimicrobials. The bacterial growth inhibition observed in A. baumannii by compounds 2–4 and 9 supports this hypothesis. Despite being activators of the AbFtsZ protein, compounds 5–8 did not demonstrate bacterial growth inhibition against A. baumannii, suggesting the activation of varied resistance mechanisms in A. baumannii, including changes in membrane permeability that inhibit the entry of molecules, as well as effluence of xenobiotics mediated by efflux pumps (García-Patiño et al. 2017; Raorane et al. 2019; Kyriakidis et al. 2021). Based on a structural analysis of compounds 2 and 3 in comparison to 6 and 7, it can be inferred that having a hydroxy group at position C-2 in molecules 2 and 3 affects their antibacterial activity, probably due to the hybridization (sp3) of C-2 and in consequence, the geometry of itself. In contrast, compounds 6 and 7 lacking this moiety displayed no inhibition against multi-resistant A. baumannii. There is currently no other documented fungal inhibitor