Antimicrobial metabolites from the Indonesian mangrove sediment-derived fungus Penicillium chrysogenum sp. ZZ1151

Abstract A total of 14 compounds including a novel tetrasubstituted benzene derivative peniprenylphenol A were isolated from a scaled-up culture of the Indonesian mangrove sediment-derived fungus Penicillium chrysogenum ZZ1151 in rice medium. Structures of the isolated compounds were determined based on their NMR spectroscopic analyses, HRESIMS data, optical rotation calculations and comparison with the reported data. New peniprenylphenol A (1) was found to have antimicrobial activities against human pathogenic methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli and Candida albicans with MIC values of 6, 13, and 13 µg/mL, respectively. The known compounds of penicimumide (2), preparaherquamide (5), uridine (6), thymine (7), 1,2-seco-trypacidin (8) communol G (9), clavatol (10), 4-hydroxybenzeneacetic acid methyl ester (11), 2,5-dihydroxyphenylacetic acid methyl ester (12), 2-hydroxyphenylacetic acid methyl ester (13) and 4-hydroxyphenylethanone (14) showed antimicrobial activity against at least one of the three tested pathogens with MIC values of 3–25 µg/mL. Graphical Abstract


Introduction
From antiquity infectious disease caused by wide array of pathogenic microorganisms possess prodigious threat to mankind ubiquitously. Despite of commendable advances in the field of medicine and treatment, the world continues to be confronted by threats of stubborn emerging and reemerging infectious disease. High population density in areas with poor public health systems, changes in land use pattern, globalization, climate change, and the increase of interaction between human and animal populations are among major factors said to responsible for increasing array of infectious disease (Bloom and Cadarette 2019). Moreover, ever increasing trend of antimicrobial resistance of pathogen is another looming specter. Although at present more than half a million deaths have been recorded annually due to antimicrobial resistant infectious disease worldwide, it has been estimated that without effective control interventions, could rise to 10 million per annum by 2050 (Monteiro et al. 2020). Presently, approximately 20% of global morality are associated with infectious diseases (Martens and Demain 2017) and multidrug resistant (MDR) pathogens are thought be responsible for more than 15% of the nosocomial infections (Ginting et al. 2019).
Discovery of novel antibiotic is racing against ever increasing microbial drug resistance. Therefore, this unmet necessity requires new antimicrobials for treating infections caused by multidrug or extensively drug resistant pathogens. It is worth to mention that although 60% of drug candidates developed targeted towards Grampositive bacteria including Staphylococcus aureus but nearly 75% of them are discontinued. Furthermore, developing new classes of antibiotics against Gram-negative bacteria are also considered as challenging (Lepore et al. 2019). Small molecule antimicrobial agents always hold therapeutic importance for their advantages over other large molecules like peptides. Often, they showed promising properties conducive to be used as oral drug with less side effects (Mantravadi et al. 2019). For last few decades, marine natural products from microbial origins were considered as significant sources of promising drug leads. Extreme physio-chemical conditions in the marine environment are responsible for the development of adaptation mechanisms, including the production of specific active biomolecules which their terrestrial counterparts are unable to do (Xie et al. 2018). In last couple of decades, 76% new natural products from marine-derived fungi showed antibacterial activity. In recent years, fungi have been serving as a top contributing source for the exponential increase on the number of marine natural products (Carroll et al. 2020). Marine members of genus Penicillium have been considered as prolific producer of bioactive metabolites.
Over past few years, many bioactive secondary metabolites with different range of activity against major drug resistant pathogenic microbes have been isolated from marine Penicillium fungal strains. For example, pyrrospirones C-F, H and I and penicipyrrodiether A from Penicillium sp. ZZ380 showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli with MIC values ranging from 2 to 5 mg/mL Song, Chen, Ge et al. 2018). Another seven membered lactone derivative penicillilactone A from Penicillium sp. LS54 was found to show activity against Vibrio harveyi . A marine mangrove-associated fungal strain Penicillium brocae MA-231 yielded two novel compound brocazine G and brocapyrrozin A, with strong potency against S. aureus (Meng et al. 2015(Meng et al. , 2017. Similarly, pinophilin G from marine fungi Penicillium pinophilum was found to exhibit moderate efficacy against Vibrio anguillarum with an MIC value of 25.0 mmol/L (Ma et al. 2017). Whereas a South China Sea sediment derived fugal strain Penicillium sp. SCSIO sof101 produced penicacid D with weak antibacterial activity against pathogenic E. coli with an MIC value of 64 mg/mL (Song et al. 2020). In another study, purpurides E and F from Penicillium minioluteum ZZ1657 exhibited considerable activity against drug MRSA, E. coli and Candida albicans (Ma et al. 2020). Penicilazaphilone C from Penicillium sclerotiorum M-22 showed extremely strong antibacterial activity against S. aureus, Pseudomonas aeruginosa, Klebsiella pneumonia and E. coli with MIC values ranging from 0.037 to 0.150 mM (Zhou et al. 2016).
With an aim to explore the bioactive potential of marine associated microbes of tropical region, sediment sample was collected from intertidal zone of mangrove swamp of Pangkep district of South Sulawesi province, Indonesia. Microbial investigation of collected sediment sample focused on actinomycetes and fungi, resulted in isolation of a marine derived fungus Penicillium chrysogenum ZZ1151. A large-scale culture of P. chrysogenum ZZ1151 in rice medium was carried out and ethyl acetate (EtOAc) extract of this culture showed antimicrobial activity against MRSA, E. coli and C. albicans. Chemical investigation of this bioactive extract resulted in the isolation of 14 compounds (Figure 1), including a novel compound, named peniprenylphenol A (1). In this article, isolation, structure elucidation, and antimicrobial activity evaluation of the isolated compounds have been reported.

Results and discussion
The Indonesian mangrove sediment derived fungus ZZ1151 (Figure S 1 , Supplementary materials) was identified as Penicillium chrysogenum ZZ1151 according to the result (Figure S 2 ) of ITS rDNA sequence analysis. The corresponding sequence alignment description list showed complete similarity with several Penicillium chrysogenum strains (Table S 1 ). An EtOAc extract prepared from the culture of the strain ZZ1151 in rice medium was subjected to repeated column chromatography, followed by HPLC purification to give compounds 1 À 14 (Figure 1).

Antimicrobial activity determination
The isolated compounds 1-14 was assessed for their antimicrobial activities against human pathogenic methicillin-resistant Staphylococcus aureus (MRSA), E. coli and C. albicans by the micro-broth dilution method as described in previous publication (Ye et al. 2016). Vancomycin, gentamicin and amphotericin B were used as positive controls for MRSA, E. coli and C. albicans microtiter plate, respectively. According to the antimicrobial assay results (Table S 17 ), the new peniprenylphenol A (1) was found to have antimicrobial activity against all three pathogens of MRSA, E. coli and C. albicans with MIC values of 6, 13, 13 mg/mL, respectively. The known compounds preparaherquamide (5), uridine (6), 1,2-seco-trypacidin (8) and 4-hydroxybenzeneacetic acid methyl ester (11) also exhibited antimicrobial activity against the three pathogens with MIC values in a range from 3 to 25 mg/mL. While thymine (7) and clavatol (10) showed antibacterial activity against both MRSA and E. coli with MIC values of 13-25 mg/mL and 2-hydroxyphenylacetic acid methyl ester (13) showed activity against both MRSA and C. albicans with MIC values of 13 and 7 mg/mL, respectively. In addition, penicimumide (2) exhibited antibacterial activity against E. coli (13 mg/mL), communol G (9) and 4-hydroxyphenylethanone (14) had activity against MRSA (MIC: 25 mg/mL) and 2,5-dihydroxyphenylacetic acid methyl ester (12) showed antifungal activity against C. albicans (MIC: 25 mg/mL).

Conclusions
The findings of the present study have reiterated genus Penicillium 0 s astounding production capability of antimicrobial agents. In this study, a novel phenol derivative with a prenyl-derived group as a side chain, named peniprenylphenol A, was isolated and characterized from a mangrove sediment fungus Penicillium chrysogenum ZZ1151 cultured in rice medium. The new peniprenylphenol A showed antimicrobial activities in inhibiting the growth of all three pathogens of MRSA, E. coli and C. albicans and 11 of the 13 known compounds exhibited antimicrobial activity against at least one of the three tested pathogens. The data from this study enriched the chemical and bioactive diversities of the natural products produced by the marine-derived Penicillium fungi.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was supported by the National Natural Science Foundation of China (No. 81773587), the National Key R&D Program of China (No. 2018YFC0311002) and the HPC Center of Zhejiang University (Zhoushan Campus).