Secondary Metabolites from Marine-Derived Bacteria with Antibiotic and Antibiofilm Activities against Drug-Resistant Pathogens ^†

Abstract

The search for new antibiotics against drug-resistant microbes has been expanded to marine bacteria. Marine bacteria have been proven to be a prolific source of a myriad of novel compounds with potential biological activities. Therefore, this review highlights novel and bioactive compounds from marine bacteria reported during the period of January 2016 to December 2021. Published articles containing novel marine bacterial secondary metabolites that are active against drug-resistant pathogens were collected. Previously described compounds (prior to January 2016) are not included in this review. Unreported compounds during this period that exhibited activity against pathogenic microbes were discussed and compared in order to find the cue of the structure–bioactivity relationship. The results showed that Streptomyces are the most studied bacteria with undescribed bioactive compounds, followed by other genera in the Actinobacteria. We have categorized the structures of the compounds in the present review into four groups, based on their biosynthetic origins, as polyketide derivatives, amino acid derivatives, terpenoids, as well as compounds with mixed origin. These compounds were active against one or more drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), vancomycin-resistant Enterococci (VRE), multidrug-resistant Mycobacterium tuberculosis (MDR-TB), and amphotericin B-resistant Candida albicans. In addition, some of the compounds also showed activity against biofilm formation of the test bacteria. Some previously undescribed compounds, isolated from marine-derived bacteria during this period, could have a good potential as lead compounds for the development of drug candidates to overcome multidrug-resistant pathogens.

1. Introduction

One of the most significant global concerns regarding health issues is the emergence and rapid spread of drug-resistant pathogens that have acquired new resistance mechanisms, leading to antimicrobial resistance [1]. Currently, the pan- or multidrug-resistant (MDR) microbes are responsible for about 40–60% of the infection cases in many low- to middle-income countries, such as Indonesia, Brazil, and Russia [2]. It is predicted that antibiotic resistance in these countries will rise four to seven times faster than in other 37 member countries of the Organization for Economic Co-operation and Development (OECD) [2]. The immense burden caused by the drug-resistant microorganisms affects not only human health but also the economy. The report by OECD in 2018 showed that around 2.4 million individuals could die in Europe, North America, and Australia from 2015 to 2050 due to antimicrobial resistance (AMR). AMR would cost about 3.5 billion USD purchasing power parity (PPP) per year to the healthcare services of this group of countries [2].

The mechanisms through which bacteria develop resistance to antibiotics can be due to either their intrinsic resistance to antibiotics or their ability to develop/acquire antibiotic resistance genes. Intrinsic ability of bacteria to resist a particular antibiotic is a result of inherent structural or functional characteristics [3]. Acquired resistance can occur through vertical evolution (including mutation and selection) or horizontal evolution from genetic exchange with other bacteria (including transformation, conjugation, or transduction) [4]. Bacteria may also develop adaptive resistance to one or more antibiotics through induction by a specific signal or environmental cues. The resulting transient adaptive resistance is not vertically inherited but will be back to the original state upon removal of the signal or environmental cues [5].

There are quite a few antibiotics that are produced by bacteria, especially from actinomycetes (Streptomyces), such as streptomycin, tetracycline, chloramphenicol, erythromycin, viomycin, lincomycin, meropenem, and daptomycin. Nonetheless, antibiotics are also produced from other phyla of bacteria, such as gramicidin A from Bacillus, colistin from Paenibacillus, mupirocin from Pseudomonas, and aztreonam, a monobactam antibiotic, which was obtained by synthesis based on the structure of the compound produced by Chromobacterium violaceum [6]. Intriguingly, although the emergence of antibiotic-resistant pathogens has stimulated a renewed effort to search for new antibiotics with unique structures and different mechanisms of action from actinomycetes or related taxa, this endeavor often leads to the rediscovery of the already known antibiotics [7].

In recent years, the interest in finding novel antibiotic-producing bacteria has shifted from terrestrial strains to their marine counterparts due to the following reasons: (i) The marine environment is considered an underexplored source, (ii) high-stress environment makes the marine-derived bacteria produce metabolites different from their terrestrial counterparts, and (iii) the advancement of technologies for bioprospection that can allow the collection of samples from deep sea, thermal vent, or polar region [8,9,10]. Potential secondary metabolites from marine bacteria with strong antibiotic activity against drug-resistant pathogens have been reviewed by Schinke et al. [11], covering the compounds obtained from marine-derived bacteria during the period of 2010–2015.

The present review discusses the structures of undescribed secondary metabolites with antibiotic/antibiofilm activities as well as the producing organisms, reported from January 2016 to December 2021, highlighting the metabolites that are active against drug-resistant pathogenic bacteria, such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESAKPE).

2. Secondary Metabolites from Marine-Derived Bacteria Which Were Tested against Drug-Resistant Bacteria

In order to facilitate readers’ comprehension, secondary metabolites isolated from marine-derived bacteria, which were tested against drug-resistant pathogens during the period of January 2016 to December 2021, were classified according to their biosynthetic origins in the first level. Then, each group was divided into subgroups according to their chemical scaffolds.

2.1. Polyketides

2.1.1. Anthraquinones and Their Analogs

Three unreported angucycline-type aromatic polyketides, nocardiopsistins A-C (1–3) (Figure 1), were isolated from a deep-sea actinobacterium, Nocardiopsis sp. strain HB-J378. The production of these compounds was significantly increased with the addition of lanthanum chloride (LaCl₃) to the SPY medium (39.5 g of sea salt, 20 g of soluble starch, 10 g of glucose, 5 g of peptone, 5 g of yeast extract, 2 g of CaCO₃, 0.5 g of K₂HPO₄, and 0.5 g of MgSO₄·7H₂O per liter). Incubation of the bacterium in the SPY medium was done at 28 °C for 7 days. The chemical structures of 1–3 were established using interpretation of 1D and 2D NMR as well as high-resolution electrospray ionization mass spectrometry (HRESIMS) data. Compounds 1–3 were active against methicillin-resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentration (MIC) values of 12.5, 3.12, and 12.5 μg/mL, respectively. Interestingly, 2 exhibited the same MIC value as chloramphenicol, a positive control used in this study [12].

Two undescribed angucycline-type glycosides, namely stremycins A (4) and B (5) (Figure 1), were isolated from the culture extract of Streptomyces pratensis strain NA-ZhouS1, which was obtained from a marine sediment in Zhoushan, East China Sea [13]. The structures of both compounds were determined using high-resolution time-of-flight mass spectrometry (HR-TOF-MS), 1D, and 2D NMR techniques. Compounds 4 and 5 have the same glycone moiety, comprising β-olivose-(4→1)-β-olivomycose-(3→1)-4-O-carbamoyl-β-amicetose, but slightly differ only in the tetracyclic benz[α]anthracene skeleton with the hydroxyl group on C-4 in 5. Both compounds were isolated from 25 L of a metal-stress medium culture (liquid gauze’s medium containing 20 g of soluble starch, 1 g of KNO₃, 0.5 g of K₂HPO₄, 0.5 g of MgSO₄·7H₂O, 0.01 g of FeSO₄·7H₂O, 35 g of sea salt per liter at pH 7.4 and supplemented with 100 µM of NiCl₂·6H₂O) and incubated at 28 °C for 10 days. Nickel was selected due to its capacity to activate cryptic gene clusters, which induce the production of bioactive metabolites that were not detected in the medium without metal. Both compounds showed antibacterial activity with a MIC value of 16 µg/mL against P. aeruginosa, methicillin-resistant S. aureus (MRSA), K. pneumoniae, and E. coli. When tested against B. subtilis, both compounds showed MIC values of 8–16 µg/mL. Therefore, the variation in the aglycone part does not significantly alter antibacterial bioactivities.

The marine sediment-derived actinomycete, Streptomyces sp. 182SMLY, yielded polycyclic anthraquinone compounds, namely N-acetyl-N-demethylmayamycin (6) and streptoanthraquinone A (7) (Figure 1). Compounds 6 and 7 were isolated from the fermentation of the bacterium at 28 °C for 7 days in the Gause’s liquid medium (20 g/L starch, 1.0 g/L KNO₃, 0.5 g/L K₂HPO₄, 0.5 g/L MgSO₄·7H₂O, 0.5 g/L NaCl, and 0.01 g/L FeSO₄·7H₂O). The structure of 6 was established using 1D and 2D NMR, HRESIMS techniques. The relative configurations of the stereogenic carbons of the amino sugar moiety were determined by NOESY correlations, while their absolute configurations were established by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. The antibiotic activities of 6 and 7 were tested against methicillin-resistant S. aureus ATCC 43300 and E. coli ATCC 25922, however, 7 was not active. Compound 6 showed inhibition activity only against MRSA with a MIC value of 20.0 µM. The positive control, norfloxacin, exhibited MIC values of 62.6 µM against both E. coli and MRSA [14].

Two unreported resistoflavin derivatives, chlororesistoflavins A (8) and B (9) (Figure 1), were obtained from the culture extract of Streptomyces sp. strain EG32, isolated from a sediment collected from the northern coast of Egypt (Mediterranean Sea) and cultured in the Waksman liquid medium (20.0 g glucose, 5.0 g peptone, 5.0 g beef extract, 3.0 g yeast extract, 3.0 g CaCO₃, 5.0 g NaCl, 50% seawater, 50% DI H₂O, 10 L in total) at 27 °C for 7 days. The structures of both compounds were established by extensive 1D and 2D NMR and HR-ESI-TOF-MS spectral analysis. Intriguingly, even though 8, 9, and resistoflavin, all possessing only one stereogenic carbon (C-11b), displayed the same sign of optical rotation (levorotatory) and shared the same biosynthetic origin, the ECD spectrum of 8 exhibited a strong Cotton effect near the n-π* transition of the carbonyl at its C-10, which is antipodal to the ECD spectra of resistoflavin and 9. The authors hypothesized that a chlorine atom imposes severe allylic-1,3 strain in 8, distorting the ring from the ideal conformation found in 9 and resistoflavin, thus altering the first-sphere contributions to the Cotton effect. By using molecular mechanics (MM) and density functional theory (DFT) calculations of both 8 and 9, they have found that 8 contained a cyclohexenone ring that has a severely distorted conformation from that of 9 and resistoflavin by steric influence of the bulky, electron-rich chlorine atom on C-11 and the carbonyl group at C-10. The conformational ring bending of the cyclohexenone in 8, when compared to 9, is interpreted as forcing the disposition of the heavy chlorine substituent from a negative-contributing quadrant to a positive-contributing quadrant. Therefore, the authors concluded that the absolute configuration of C-11b in 8 is the same as that of C-11b in 9 and resistoflavin, i.e., R configuration.

Compound 8 showed comparable inhibitory activity to resistoflavin (MIC = 0.25 µg/mL) but eight times stronger than 9 (MIC = 2.0 µg/mL) against MRSA. The positive control, ciprofloxacin, showed a MIC value of 0.2 µg/mL against MRSA. Interestingly, the position of the chlorine substituent on the resistoflavin scaffold was found to affect the activity of its analogs. For instance, when the chlorine atom is on C-11, as in 8, its activity is comparable to that of the parent compound, i.e., resistoflavin. However, when the chlorine atom is on C-4, as in 9, its activity is reduced when compared to resistoflavin [15].

The liquid culture of Nonomuraea sp. strain MM565M-173N2, isolated from a deep-sea sediment collected in Japan trench at a depth of 329 m off the Sanriku coast, furnished sealutomicins A-D (10–13) (Figure 1). Compounds 10–13 were obtained from 220 L of the fermented production medium containing 0.75% glycerin, 0.75% cotton seed meal, 0.25% L-glutamate, 0.15% NaCl, and 1.8% Daigo’s Artificial Seawater SP (pH value 7.4 before sterilization). The fermentation was performed at 27 °C for 7 days on a rotary shaker (180 rpm). The structures of 10–13 were established by extensive analysis of their 1D and 2D NMR and HRESIMS spectra. In the case of 10, the nuclear Overhauser effect (NOE) data revealed the relative configurations as 16S*, 17R*, 24S*, 25S*. Since its circular dichroism (CD) spectrum is nearly identical to that of a related compound, dynemicin A, its absolute stereochemistry was established as 16S, 17R, 24S, 25S. However, the absolute configuration of C-28 of the side chain could not be determined. The relative configurations of the stereogenic carbons of 11–13 were established by NOE data, whereas their absolute configurations were determined by comparison of their CD spectra with that of 10. Since 11–13 were products from the Bergman cyclization of 10, which retain the stereochemistry at C-16, C-24, and C-25, the absolute structures of 11–13 were determined.

Compound 10 displayed strong antimicrobial activities against susceptible and MDR Gram-negative bacteria, including New Delhi metallo-beta-lactamase (NDM) and K. pneumoniae carbapenemase (KPC)-producing strains, with MIC values of 0.05–0.1 μg/mL for E. coli, and 0.1–0.4 μg/mL for K. pneumoniae. Moreover, 10 showed potent antimicrobial activities against susceptible and MDR Gram-positive bacteria, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococci (VRE) strains, with MIC values of 0.00625–0.0125 μg/mL for S. aureus, and 0.025–0.1 μg/mL for Enterococcus faecalis/faecium. Compounds 11–13 exhibited moderate to strong antibacterial activity against Gram-positive bacteria, with MIC values of 0.2–1.6 μg/mL against susceptible and MDR S. aureus, and 0.8 to more than 6.4 μg/mL for susceptible and E. faecalis/faecium VRE. However, they did not show activity against Gram-negative bacteria. Meropenem, a positive control, showed MIC values ranging from 0.1–6.4 µg/mL. The positive controls, meropenem, showed MIC values ranging from 0.0125 μg/mL (toward E. coli K-12) to 6.4 μg/mL (toward S. aureus MRSA.), while colistin displayed MIC values ranging from 0.4 μg/mL (toward E. coli K-12) to 6.4 μg/mL (toward E. coli MCR) [16].

Antibacterial activity-guided isolation of a marine sediment-derived bacterium, Streptomyces sp. MBTI36, yielded an undescribed glycosylated 6,8,9-trihydroxy-3,4-dihydroanthracen-1 (2H)-one derivative, chromomycin A₉ (14) (Figure 1). Analysis of the 16S rRNA of the bacterium showed 99.9% sequence similarity to Streptomyces microflavus NBRC13062. Compound 14 was isolated from the fermentation of the bacterium at 28 °C for 14 days without shaking in a total of 20 L of the liquid GTYB medium (each liter of the medium contains 10 g of glucose, 2 g of tryptone, 1 g of yeast extract, and 1 g of beef extract in 1 L of artificial seawater). The structure of 14 was established based on 1D and 2D NMR and HRESIMS spectral analysis as well as a comparison of its ¹H and ¹³C NMR data with the previously reported chromomycins Ap, A₂, and A₃.

Compound 14 displayed potent antibacterial activities against all methicillin-sensitive S. aureus (MSSA) and MRSA strains, showing significant broad-spectrum antibiotic effects on MRSA strains with MIC values of 0.06–0.25 µg/mL, which are more potent than some of the major classes of antibiotics, such as daptomycin (MIC > 32 µg/mL), vancomycin (MIC = 0.5–2 µg/mL), platensimycin (MIC = 4–8 µg/mL), linezolid (MIC = 1–2 µg/mL), and ciprofloxacin (MIC = 0.13 > 32 µg/mL) [17].

S. aureus ATCC43300 was selected to evaluate its resistance development against ciprofloxacin and 14. A steady increase in MIC was observed for ciprofloxacin during the passage experiment in S. aureus ATCC43300, with a 128-fold change (MIC = 32 µg/mL) when compared with the initial MIC value (MIC = 0.25 µg/mL). On the contrary, a 2-fold increase in the MIC (from 0.13 to 0.25 µg/mL) was observed for S. aureus ATCC43300 after 21 passages. Therefore, during the passage experiment, MIC values for 14 did not increase more than 4-fold from its initial MIC value, confirming that there was no resistance development during the 21 passages [17].

2.1.2. Naphthoquinones

The undescribed napyradiomycins, i.e., napyradiomycin B7a (15), napyradiomycin B7b (16), and napyradiomycin D1 (17) (Figure 2), were isolated from the EtOAc extract of the culture of Streptomyces sp. strain CA-271078 (which showed 99.34% similarity with Streptomyces aculeolatus NBRC 14824(T)), fermented at 28 °C in 3 L of the R358 medium for 6 days.

The structures of 15, 16, and 17 were established by the interpretation of HR-ESI-TOF-MS, 1D, and 2D NMR spectra. The relative stereochemistry of both compounds was determined based on NOESY and ROESY correlations of the key protons as well as the values of coupling constants of some protons. The absolute configurations of the stereogenic carbons in 15 and 16 were assumed to be the same as those of all the previously reported napyradiomycins in the B series on the basis of their common biosynthetic origin as well as the same signs of their specific rotations. The structure of napyradiomycins can be recognized by the semi-naphthoquinone chromophore, a prenyl unit attached to C-4a, which is cyclized to form a tetrahydropyran ring in most cases, and a monoterpenoid subunit attached to C-10a. Compounds 15 and 17 displayed moderate inhibitory activity against MRSA, with MIC values of 48 and 12–24 µg/mL, respectively. In addition, both compounds also inhibited the growth of Mycobacterium tuberculosis H37Ra, with MIC values of 12–24 and 24–48 µg/mL, respectively, while 16 exhibited a MIC value > 64 µg/mL against both MRSA and M. tuberculosis. However, the assay did not include any positive control in the experimental design. The results suggested that the absolute configurations at C-3 of 15 and 16 have a significant influence on the antibacterial activity [18].

Bioassay-guided fractionation of the EtOAc extracts of solid agar cultures of Micromonospora sp. RJA4480, isolated from a marine sediment obtained at −85 m in Barkley Sound, British Columbia, that exhibited potent in vitro inhibition of MRSA and E. coli, led to the identification of four undescribed macrolides containing a naphthoquinone core, 3-amino-27-demethoxy-27-hydroxyrifamycin S (18), 3-aminorifamycin S (19), sporalactam A (20), and sporalactam B (21) (Figure 2) as the antibacterial componentes. Although no 3-amino ansa macrolide has been reported as a natural product, 19 has been previously obtained by semisynthesis [19].

The structure of 18 was established by interpretation of HR-ESI-TOF-MS, 1D, and 2D NMR spectral data. Curiously, the proton and carbon signals in the ¹H and ¹³C NMR spectra of 18, recorded in deuterated dimethyl sulfoxide (DMSO-d6), were doubled. The single-crystal X-ray diffraction analysis of 18 did not only determine its absolute structure as 12S, 20S, 21S, 22R, 23R, 24R, 25S, 26S, 27S but also revealed that 18 existed in two conformations, thus causing doubled signals in the ¹H and ¹³C NMR spectra. The structures of 20 and 21 were established by HRESIMS data and extensive analysis of their 1D and 2D NMR spectra. Interestingly, unlike the ¹H and ¹³C NMR spectra of 18, the signals in the ¹H and ¹³C NMR spectra of 20 and 21 are not doubled. The absolute configurations of the stereogenic carbons in 20 and 21 were assigned as 12S, 16S, 19R, 20S, 21S, 22R, 23R, 24R, 25S, 26S, 27S, based on the absolute stereochemistry of 18 and of tolypomycinone, a degradation product of tolypomycin Y which is biogenetically related to 20 and 21. In vitro antimicrobial assay showed that 18–21 inhibited the growth of MRSA with MIC₉₀ values of 0.0009, 0.0008, 7.0, and 1.8 µM, respectively. The reference compounds, 27-demethoxy-27-hydroxyrifamycin S (22) and rifamycin S (23) displayed MIC₉₀ values of 0.03 and 0.07 µM, respectively. Compounds 18–21 also inhibited the growth of M. tuberculosis in vitro, with MIC₉₀ values of 0.0009, 0.0008, 0.8, and 0.06 µM, respectively (whereas 22 and 23, displayed MIC₉₀ values of 0.04 and 0.006 µM, respectively), and of M. tuberculosis growing intracellularly in macrophage cells, with MIC₉₀ value ranging from 0.4 to 30 µM (whereas 22 and 23 displayed MIC₉₀ values of 3–10 and 0.07 µM, respectively). When comparing MIC₉₀ values of 18 and 19 with those of the reference compounds, 22 and 23, that lack only the amino substituent on C-3, it was found that the 3-amino substituent significantly enhances the potency of these antibiotics against MRSA, E. coli, and M. tuberculosis [20].

The previously reported 7,8-dideoxygriseorhodin C (24) (Figure 2) was obtained from the EtOAc extract of Streptomyces sp. strain 1425S.R.1a.1, isolated from a body tissue homogenate of a gastropod mollusk, Truncatella guerinii, collected in Cebu, Philippines, and was cultured in R2A broth (0.2% yeast extract, 1% malt extract, 0.2% glucose, and supplemented with 2% NaCl). Although the ¹³C NMR spectrum of 24 was similar to that of 7,8-dideoxygriseorhodin C in the previous report, in this work, the authors have unambiguously assigned all the ¹H and ¹³C NMR chemical shift values of 24 by 1D and 2D NMR spectral analysis. Since the absolute configuration at C-6 and C-6a of 7,8-dideoxygriseorhodin C had not been established in the previous report, Miller et al. [21] have attempted to determine the absolute configurations of these stereogenic carbons in 24 by comparison of the calculated and experimental ECD spectra. However, this method could only determine the absolute configuration of C-6 as 6S with certainty, while the absolute configuration of C-6a was ambiguous. Finally, a combination of the 3D modeling and the correlations observed in the ROESY spectrum have allowed the authors to establish the absolute configuration of C-6a as 6aS. Compound 24 inhibited the growth of S. aureus ATCC^® 43300™ MRSA, which is resistant to oxacillin and methicillin, with a MIC value of 0.08–0.12 µg/mL. The positive control, oxacillin, showed MIC values of 1.59–6.24 μg/mL. Treatment of ATCC^® 43300™ MRSA strain with a combination of 24 and oxacillin at 1xMIC showed the reduction of the individual MICs (MIC of 24 = 0.01–0.02 µg/mL; MIC of oxacillin = 0.02–0.298 µg/mL). Moreover, the combination index (CI) for the combination of 24 and oxacillin at 1xMIC was 0.12–0.24, indicating a synergistic effect between 24 and oxacillin [21].

Mersaquinone (25) (Figure 2), an unreported tetracene derivative, was isolated from the extract of a marine-derived Streptomyces sp. EG1, obtained from a sediment sample that was collected from the North Coast of the Mediterranean Sea, Egypt, and cultured in the Waksman medium at 28 °C for 7 days. The structure of 25 was established based on an extensive analysis of HRESIMS, IR, 1D, and 2D NMR spectra. Compound 25 inhibited the growth of the methicillin-resistant S. aureus (MRSA) strain TCH1516, with a MIC value of 3.36 µg/mL. The positive control, ciprofloxacin, showed a MIC value of 0.93 µM [22].

2.1.3. Macrolides

An undescribed anthracimycin congener, anthracimycin B (26) (Figure 3), was obtained together with the previously reported anthracimycin (27), from the EtOAc extract of the solid culture (R5A agar) of a marine-derived Streptomyces cyaneofuscatus M-169, which was isolated from a gorgonian coral (Order Gorgonacea) collected at 1500 m depth in the Avilés submarine Canyon, Cantabrian Sea [23]. The structure of 26 was determined by ESI-TOF-MS and 1D and 2D NMR spectral analysis as well as by comparison of its NMR data with those of 27 [24]. Since the optical rotation of 26 has the same sign (levorotatory) and similar magnitude of rotation to those of 27 whose absolute structure was established by X-ray diffraction by Jang et al. [24], the authors concluded that absolute configurations of the stereogenic carbons in 26 were the same as those of 27. Compounds 26 and 27 were tested against four Gram-positive MSSA and MRSA, vancomycin-sensitive E. Faecium, and vancomycin-sensitive E. Faecalis, two Gram-negative (E. Coli and K. Pneumoniae), and M. tuberculosis. Compound 26 displayed potent antibacterial activity with MIC values of 0.33–0.65 µM against S. aureus MRSA, 10.5–20.9 µM against S. aureus MSSA, 0.33–0.65 µM against E. faecium VANS and 0.65–1.26 µM against E. faecalis VANS while 27 not only exhibited more potent activities against these bacterial strains than 26 but also against M. tuberculosis [23].

Two unreported macrocyclic polyketides containing dodecahydropyrano-trioxacyclooctadecine dione (28) and trioxo-octadecahydro-1H-benzo[o]tetraoxacyclopentacosine carboxylate (29) (Figure 3) were isolated from the EtOAc extract of the agar nutrient culture of a heterotrophic Gamma-proteobacterium Shewanella algae MTCC 12715, associated with an intertidal red alga, Hypnea valentiae, which was collected from the Gulf of Mannar region in the southeast coast of India. The structures of 28 and 29 were established based on extensive analysis of HRMS, 1D, and 2D NMR spectra. The relative configurations of the stereogenic carbons of the macrocyclic rings were established based on NOESY correlations and supported with molecular mechanistic studies by MM2 force-field calculations by Chem3D Pro (ver-12.0).

Compounds 28 and 29 displayed potential antibacterial activity against methicillin-resistant S. aureus MRSA (≥20 mm) and vancomycin-resistant E. faecalis VREfs (≥25 mm, 30 μg on disc), whereas the standard antibiotic disc of streptomycin (30 μg) displayed a smaller clearance zone around the disc. In the same manner, 28 and 29 exhibited greater activity (MIC of 3–5 μg/mL) against drug-resistant pathogenic bacteria causing nosocomial infections than chloramphenical (≥6.25 μg/mL) in the microdilution method. The structural attributions and the correlations with bioactivity through various physico-chemical parameters of 28 and 29, adopted from ChemDraw Ultra ver-12.0 /ACD Chemsketch ver-8.0 databases, revealed that the optimum logarithmic value of the octanol-water coefficient (log Pow), in conjunction with greater electronic parameters and less steric bulkiness of 28 and 29, could be fundamental for bioactivity. Moreover, in silico docking studies of 28 and 29 exhibited significant inhibition at the allosteric site of the penicillin-binding protein (PBP2a) in S. aureus, which supports their in vitro activity profile [25].

Two undescribed bafilomycins, 21,22-en-bafilomycin D (30) and 21,22-en-9-hydroxybafilomycin D (31) (Figure 3) were obtained from a methanol extract of a GYM solid culture of Streptomyces sp. HZP-2216E, isolated from a fresh seaweed, Ulva pertusa, which was collected from the Turtle Islet in the South China Sea close to Shanwei City (Guangdong), China. The structure of the compounds was established by extensive analysis of HRMS and 1D and 2D NMR spectra [26]. The geometries of the double bonds were established from the NOE information, while the relative configurations of the stereogenic carbons were determined by NOE information as well as a close similarity of the carbon chemical shift values in the ¹³C NMR spectra of the macrocyclic portion of 30 and bafilomycin D (32) [27]. On the other hand, the hydroxyl group on C-23 was deduced as β-oriented since 30 could be prepared from bafilomycin D (32) via double bond migration in DMSO solution. In the case of 31, the α-orientation of the hydroxyl group on C-9 was based on a large coupling constant (J = 10.3 Hz) between H-8 and H-9. Compounds 30 and 31 showed growth inhibitory activity against MRSA with MIC values of 12.5 µg/mL for both compounds, while gentamicin, a positive control, showed inhibition with a MIC value of 0.36 µg/mL [26]. Compound 32 was also isolated, together with the undescribed 23-O-butyrylbafilomycin D (33), from the same bacterium cultured in a 2216E solid medium. Compound 33 exhibited inhibitory activity against MRSA with a MIC value of 7.4 µM, while norfloxacin, a positive control, showed inhibition with a MIC value of 31.3 µM [27].

The complete genome sequence of Streptomyces sp. IMB7-145, isolated from a marine sediment sample at a depth of 40 m in Heishijiao Bay, Dalian, China, revealed the presence of the cluster (named npm), which spans a 132.4 kbp DNA region and contains 31 individual open reading frames (ORFs), in which nine large genes (npmA−npmI) encode a 20-module type I polyketide synthase (PKS). Detailed bioinformatics analysis revealed that the predicted domain and module architecture were in good agreement with the assembly of the polyketide skeleton of niphimycin. The strain was then cultured in the M7 medium (composed of 5 g of glucose, 3 g of peptone, 3 g of beef extract, 2.5 g of starch, 2.5 g of CaCO₃, 0.001 g of FeSO₄, 0.001 g of MnCl₂, 0.001 g of ZnSO₄, 0.001 g of CuSO₄, and 0.001 g of CoCl₂ in 1 L of H₂O) at 28 °C for 5 days on a rotary shaker at 200 rpm, after which the supernatant and the mycelia were separated. The combined organic extracts of the supernatant and mycelia were fractionated by column chromatography of silica gel and Sephadex LH-20 and purified by a reversed-phase high-performance liquid chromatography (HPLC) to give four niphimycin congeners, niphimycins C−E (34–36), and 17-O-methylniphimycin (37) as well as 38 (Figure 3). The planar structures of 34–36 were established by HRESIMS, 1D, and 2D NMR spectral analysis. By using J-based configurational analysis, the relative configurations of C-2/C-3, C-6/C-7, and C-28/C-29 were established as anti, anti, and anti, respectively. On the other hand, the relative configurations of C-7/C-9/C-10/C-11 were elucidated as syn/anti/syn by application of Kishi’s universal NMR database, while the relative configurations of C-17−C-21 of the pyran ring were determined by ¹H-¹H coupling constants and ROESY correlations. Additionally, the full absolute configurations were predicted from the ketoreductase (KR) domain analysis of the npm gene cluster. It is worth mentioning that the authors have observed that during the isolation process, the previously described 38 [28], also isolated from the extract, could transform to 37 in the MeOH solution in the presence of a trace of acetic acid, suggesting that 37 was an artifact arising from ketalization of 38 with MeOH during the isolation process [29].

Compounds 34–37 were tested against representative members of ESKAPE pathogens. Compounds 34 and 38, with only one malonyl group, showed good antimicrobial activity against methicillin-resistant S. epidermidis (MRSE) and S. aureus (MRSA), with MIC values of 4–32 µg/mL, but modest activity against vancomycin-resistant E. faecalis and E. faecium (VRE), with a MIC value of 64 µg/mL. The similar potency of 34 and 38 suggested that the position of the malonyl substituent is not crucial for antibacterial activity. On the contrary, antibacterial activity of 35 and 36, which contain two malonyl groups, was two-fold less than that of 34 and 38, suggesting that more malonyl groups in the compounds cause a reduction of antibacterial activity. Moreover, 37 was as active as 34 and 38, indicating that antibacterial activity was not significantly affected by the ketalization of the hemiketal ring [29].

Interestingly, 34 and 38 showed inhibitory activity against M. tuberculosis strain FJ05120 (resistant to isoniazid and rifampicin) and FJ05195 (resistant to isonizaid, rifampicin, streptomycin, and ethambutol) with MIC values of 16 and 32 µg/mL, respectively [28].

The extract of the marine actinomycete, Streptomyces althioticus MSM3, isolated from samples of the seaweed Ulva sp., which was collected in the Cantabrian Sea (Northeast Atlantic Ocean) and cultured in R5A liquid medium at 28 °C at 250 rpm for 6 days, furnished an undescribed desertomycin G (39) (Figure 3), a glycosylated macrolide containing a primary amine group. The planar structure of 39 was established by extensive analysis of HRMS, 1D, and 2D NMR spectra, as well as by comparison of its NMR data with those of the previously described desertomycin A [29]. Compound 39 exhibited strong inhibitory activity against various Gram-positive multidrug-resistant clinical pathogens, such as M. tuberculosis, S. Aureus, S. Pneumoniae, S. Pyogenes, Clostridium perfringens, C. Urealyticum, E. Faecalis, E. Faecium, and moderate antibiotic activity against relevant Gram-negative clinical pathogens, such as Bacteroides fragilis, Haemophilus influenzae, and Neisseria meningitidis [30].

2.1.4. Spirotetronates

Spirotetronates are a class of compounds consisting of tetronic acid spiro linked with cyclohexane or cyclohexene moiety and can be divided into two sub-classes: Sub-class I, which consists of spirotetronates connected with a macrocycle, and sub-class II, which contains spirotetronates and macrocycle with an integration of decalin and sometimes oligosaccharide chains [31]. The biosynthesis of spirotetronates of both sub-classes starts with the attachment of acetyl and/or propanoyl CoA to the acyl carrier protein (ACP) to form a chain elongation in type I PKS (PKS I) (Figure 4). In sub-class I, the elongation of the chain is stopped and condensed by a glyceryl-ACP unit after reaching the appropriate length to form tetronic acid (i) then undergoes acetylation-elimination to form a butanolide (ii). Intramolecular Diels-Alder (IMDA) reaction connects butanolide with a diene portion. The biosynthesis of sub-class II is quite similar to that of sub-class I, but the elongation of the chain in sub-class II incorporates decalin. In addition, glycosylation adds sugar moiety to the structure.

The acetone extract of a culture of the actinomycete, Micromonospora sp. CA-214671, isolated from a marine cave sediment that was collected from the Canary Islands, Spain, and cultured in N4 medium (soluble starch 15 g/L, fish peptone 8 g/L, bacterial peptone 5 g/L, glycerol 6 g/L, KBr 0.2 g/L, CaCO₃ 2 g/L, and sea salt 10 g/L, and also 3% XAD-16 resin at pH 7.2–7.4) at 28 °C for 7 days, was found to exhibit activity against methicillin-resistant S. aureus (MRSA), M. tuberculosis H37Ra and M. bovis. Bioassay-guided fractionation of this extract led to the isolation of two unreported phocoenamicins B (40) and C (41), and the previously reported phocoenamicin (42) (Figure 5). The planar structures of 40 and 41 were established based on an extensive analysis of 1D and 2D NMR and also HRESIMS spectra, as well as by comparison of their NMR data with those of phocoenamicin (42). The relative configurations of their stereogenic carbons were established using NOESY correlations, however, their absolute configurations were not determined. Compounds 40 and 41 exhibited significant activity against MRSA S. aureus MB5393, with MIC values of 8–16 and 32–64 µg/mL, respectively, but negligible activity against vancomycin-resistant E. faecium (VRE) MB5571 (MIC > 128 µg/mL). Vancomycin, the positive control, showed stronger activity with a MIC value of 2–4 µg/mL [32].

Seven undescribed spirotetronate glycosides, microsporanates A-F (43–48), and tetrocarcon P (49) (Figure 5) were isolated from the acetone extract of the mycelium and of the culture broth absorbed on the XAD-16 resin of Micromonospora harpali SCSIO GJ089, isolated from a sediment sample obtained from the northern South China Sea, and cultured in N4 medium (soluble starch 15 g/L, fish peptone 8 g/L, bacterial peptone 5 g/L, glycerol 6 g/L, KBr 0.2 g/L, CaCO3 2 g/L, and sea salt 10 g/L, at pH 7.2–7.4) with 3% XAD-16 resin at 28 °C for 7 days. The structures of the compounds were determined based on extensive analysis of 1D and 2D NMR and also HRESIMS spectra. Compounds 43 and 44 showed moderate inhibitory activity against methicillin-resistant S. aureus shhs-A1 (MRSA, a clinical isolate) with a MIC value of 32 µg/mL, while the rest of the spirotetronate glycosides 45–49 showed MIC values of >128 µg/mL. Vancomycin, kanamycin, and ampicillin inhibited the same MRSA strain, with MIC values of 2.0, 64, and 1.0 µg/mL, respectively. The results indicated that the type of sugar moieties on C-9 was important for their bioactivities. Nitro sugars showed superior anti-MRSA compared to their amino sugar counterparts. The addition of sugar and C-17, as well as a change of C-23 α,β-unsaturated carbonyl moiety with a formyl group (CHO), have no effect on the anti-MRSA activity [33].

Abyssomicins are a family of antimicrobial compounds which are generally composed of C19 spirotetronates and often contain a four- or five-membered ring system within their architectures. A majority of abyssomicin analogs exist as monomers, and the only known dimer was abyssomicin J. In order to reanalyze the HPLC dataset of the metabolic profile of Streptomyces koyangensis SCSIO 5802, isolated from a sediment collected in the Northern South China Sea, Huang et al. [34] performed a large-scale (60 L) fermentation of the strain SCSIO 5802. Extraction and HPLC separation resulted in the isolation of monomeric abyssomicin congeners with undescribed frameworks (neoabyssomicins) and two undescribed abyssomicin dimers, named neoabyssomicins F (50) and G (51) (Figure 6). The structures of both compounds were established by HRMS, 1D, and 2D NMR spectral analysis. Key HMBC correlations indicated that the two monomers were connected via a C-9−S−C-9′ thioether bond to form a dimer. In the case of 50, X-ray crystallographic analysis confirmed the structure and determined the absolute configurations for all stereogenic carbons. Both 50 and 51 showed growth inhibitory activity against three clinical isolates of MRSA (MRSA-shhs A1, MRSA-699, and MRSA-1862) with the same MIC value of 16 μg/mL. Ampicillin (MIC values of 64, 4, 0.125 μg/mL) and vancomycin (MIC values of 0.5, 0.125, and 0.125 μg/mL) were used as positive controls [34].

2.2. Amino Acid Derivatives

A number of secondary metabolites produced by bacteria are originated from amino acids. These metabolites can be classified into several structural groups as follows.

2.2.1. Simple Amino Acid Derivatives

p-Terphenyls originate from a condensation of two precursors, which are derived from L-Phe/L-Tyr and L-Trp, which is catalyzed by a tridomain nonribosomal peptide synthetase to yield polyporic acid (PPA). Subsequent reduction and dehydration of PPA give terphenyl triol, a p-terphenyl scaffold [35].

By using the MS/MS-based Global Natural Products Social (GNPS) molecular networking analysis, two antibacterial p-terphenyl derivatives, nocarterphenyls D (52) and E (53) (Figure 6) were isolated from the EtOAc extract of a liquid culture (1% soluble starch, 1% glucose, 0.5% peptone, 0.2% yeast extract powder, 1% glycerinum, and 0.25% corn flour, pH = 7.0, in seawater) of the marine-derived actinomycete, Nocardiopsis sp. HDN154086, isolated from a sediment sample that was collected from the South China Sea. The structure of 52 was elucidated by 1D and 2D NMR and HRMS spectral analysis and was confirmed by single X-ray diffraction analysis using MoKα radiation. The structure of 52 consists of a p-terphenyl scaffold with the central benzene ring fused with a 2,2′-bithiazole moiety. On the other hand, the structure of 53 consists of a p-terphenylquinone with a methyl proprionate linked to a quinone ring through a sulfur atom [36].

Compound 52 displayed antibacterial activity against Proteus sp., B. cereus, M. phlei, B. subtilis, Vibrio parahemolyticus, and E. coli, with MIC values ranging from 1.5 to 6.2 μM, but inactive against MRSA (MIC > 50 µM). On the contrary, 53 showed activity against MRSA, with a MIC value of 6.2 μM, which is 8-fold more active than the positive control, ciprofloxacin (MIC = 50 µM), and also against Proteus sp. and B. subtilis, with MIC values of 12 μM but inactive against M. phlei, B. cereus, V. parahemolyticus, and E. coli [36].

2.2.2. Linear Peptides

Linear peptides can be formed by a direct condensation of two or more amino acid residues to form amide linkages or can be modified to form pyrazinone or diketopiperazine ring.

An undescribed lipopeptide, nesfactin (54) (Figure 7), was obtained from the culture extract of an actinomycete, Nesterenkonia sp. MSA31, isolated from the marine sponge Fasciospongia cavernosa, which was collected from the southwest coast of India [37]. The structure of 54 was elucidated by interpretation of the fragmentation patterns from the liquid chromatography-mass spectrometry (LC-MS/MS). Compound 54 was found to inhibit virulence phenotypes, including the production of hemolysin, protease, lipase, phospholipase, esterase, elastase, rhamnolipid, alginate, and pyocyanin, as well as motility of P. aeruginosa strain FSPA02 which is resistant to cefipime, ceftazidime, aztreonam, gentamycin, ampicillin, kanamycin, vancomycin, tetracycline, and streptomycin. By using high-performance thin layer chromatography (HP-TLC) and the reporter assay using CV026.G, it was found that 54 also inhibited the quorum sensing molecule, N-acyl-homoserine lactones (AHL), extracted from the culture supernatants of P. aeruginosa. Additionally, 54 also inhibited a biofilm formation in P. aeruginosa, as observed in a test tube, microtiter plate as well as confocal image analysis. Molecular docking studies showed that the LasR protein had binding energy—4.5 kcal/mol. It has two hydrogen-bonding interactions (Ala59 and Lys34 with binding distance of 2.8 and 3.5 Ằ) and one hydrophobic interaction (Ala58:4.01381) against 54 [38].

2.2.3. Cyclic Peptides

Two unreported cyclic lipotetrapeptides, named bacilotetrins A (55) and B (56) (Figure 7), were obtained from the culture extract of B. subtilis 109GGC020, isolated from a sediment sample collected from the Gageocho reef, Republic of Korea. The structures of 55 and 56 were elucidated by extensive analysis of HRESIMS, 1D, and 2D NMR spectra. The absolute configurations of the stereogenic carbons of the amino acids were established by hydrolysis of the lipotetrapeptides, followed by derivatization with Marfey’s reagent and chiral HPLC analysis. The absolute configuration of C-3 of 3-hydroxy fatty acids in both compounds was established as R by direct comparison of their specific rotation values with those of the previously reported 3-hydroxy fatty acids [39].

Compounds 55 and 56 were tested against several clinically isolated MRSA strains, i.e., ATCC25923, XU212, SA1199B, RN4220, and EMRSA15, by broth dilution method. Compound 55 showed antibacterial activity against ATCC25923, XU212, SA1199B, RN4220, with MIC values of 8, 16, 8, and 32 µg/mL, respectively), whereas 56 was active against ATCC25923, XU212, and SA1199B, with MIC values of 16, 16, and 32 µg/mL, respectively The positive control, norfloxacin, showed MIC values of 16, 16, 64, 2, and 4 µg/mL, against ATCC25923, XU212, SA1199B, RN4220, and EMRSA15, respectively [39].

The liquid culture extract of Streptomyces sp. IMB094, isolated from a marine sediment at a depth of 40 m in Heishijiao Bay, Dalian, China, yielded two unreported actinomycin analogs containing a tetracyclic 5H-oxazolo[4,5-b]phenoxazine chromophore, named neo-actinomycins A (57) and B (58) (Figure 8). The structures of the compounds were established by extensive analysis of HRESIMS, 1D, and 2D NMR spectra. The absolute configurations of amino acids were determined using advanced Marfey’s method after acid hydrolysis of 57 and 58. The presence of the phenoxazine ring system was proposed to originate from condensation between the previously reported actinomycin D (59) with α-ketoglutarate and pyruvate, respectively, as depicted in Figure 9. The hypothesis of the biosynthesis of 57 and 58 was supported by adding α-ketoglutaric acid and pyruvic acid (1 mg/mL) after the cultivation of Streptomyces sp. IMB094, followed by the detection of a 12-fold increase in the production of 57 in α-ketoglutaric acid-supplemented cultures compared to the unsupplemented control, while the yield of both 57 and 58 increased about 6-fold, 24 h after pyruvic acid was added into the cultures.

Compounds 57 and 58 were evaluated for their antibacterial activities against a series of Gram-positive and Gram-negative drug-resistant pathogenic bacteria. Compound 57 displayed moderate antibacterial activities against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococci (VRE) strains, with MIC values of 16–64 μg/mL, 64–256-fold less active than actinomycin D (59) which was used as a positive control. On the contrary, 58 did not show any antibacterial activity (MIC >128 μg/mL) [40].

Four unreported D-type actinomycin analogs, actinomycins D₁-D₄ (60–63) (Figure 8) were obtained from the culture extract of Streptomyces sp. LHW52447, which was isolated from a specimen of a marine sponge, Phyllospongia foliascens, collected from the Xisha Islands in the South China Sea. The structures of the compounds were elucidated by extensive analysis of HRESIMS, 1D, and 2D NMR spectra. The absolute configurations of the amino acid constituents were established by Marfey’s method. Compounds 60 and 61 represent naturally occurring actinomycins with the oxazole-bearing phenoxazinone chromophore. Compounds 60–63 were evaluated for their antibacterial activity against three strains of pathogenic methicillin-resistant S. aureus (MRSA), viz. P172, P175, and ATCC 33591. Compounds 60 and 61 (with MIC values of 0.125–0.25 µg/mL) displayed higher anti-MRSA activity than 62 and 63 (with MIC values of 0.25–1.0 µg/mL), suggesting that the anti-MRSA activity of this type of compounds might be potentiated by the presence of an oxazole moiety. The positive control, chloramphenicol, showed MIC values of 0.5, 0.5, and 1.0 µg/mL against P172, P175, and ATCC 33591, respectively [41].

2.2.4. Alkaloids

Four undescribed indole alkaloids, streptoindoles A-D (64–67) (Figure 10), were isolated from the EtOAc extract of Streptomyces sp. ZZ1118, isolated from a gut sample of a marine shrimp (Penaeus sp.) in the Zhoushan archipelago, Zhejiang, China, and cultured on the rice medium. Compounds 64 and 65 are enantiomeric bis-indole alkaloids and were separated by HPLC using a chiral column (ChiralCel OJ-RH). The planar structures of 64–67 were elucidated by extensive analysis of 1D and 2D NMR as well as HRESIMS data. While the absolute configurations of the stereogenic carbons of 64 and 65 were established by comparison of the calculated and experimental ECD spectra, the absolute configuration of the only chiral carbon (C-15) in 66 was determined by comparison of the experimental and calculated optical rotations. Moreover, the structure of 67 was also confirmed by X-ray analysis [42].

Compound 65 exhibited three times stronger antibacterial activity against MRSA (MIC value of 7 µg/mL) than 64 and 67 (MIC values of 25 µg/mL), while 66 showed no activity at a concentration of 50 µg/mL. The positive control, vancomycin, showed a MIC value of 0.75 µg/mL against MRSA [42].

Two undescribed chlorinated bis-indole alkaloids, dienomycin (68) and 6-OMe-7′,7″-dichorochromopyrrolic acid (69) (Figure 10), were obtained from the butanone and acetone extracts of a supernatant and a mycelia cake of Streptomyces sp. SCSIO 11791, isolated from a sediment sample collected from the South China Sea at a depth of 1765 m and cultured in the ISP-4 medium (containing 0.5% soluble starch, 0.05% yeast extract, 0.1% peptone, 0.1% K₂HPO₄, 0.1% MgSO₄·7H₂O, 0.2% (NH₄)2SO₄, 0.4% NaCl, 3% crude sea salt, 0.2% CaCO₃, pH 7.4). The structures of 68 and 69 were determined by analysis of HRESIMS, 1D, and 2D NMR spectral data. Compounds 68 and 69 were evaluated for their antibacterial activity against a panel of MRSA, isolated from human patients (MRSA 991, MRSA 1862, MRSA 669 A, and MRSA A2) and pig (MRSA GDQ6P012P and MRSA GDE4P037P). Compound 68 (MIC values = 2 µg/mL) showed stronger anti-MRSA activity than 69 (MIC values = 32–128 µg/mL) against all the MRSA strains but less potent than vancomycin, the positive control, against MRSA 1862, MRSA 669 A, MRSA A2, and MRSA GDQ6P012P (MIC values = 1 µg/mL). Interestingly, 68 showed stronger activity than vancomycin against MRSA GDE4P037P (isolated from pig), with a MIC value of 1 μg/mL compared to 8 μg/mL for vancomycin [43].

The undescribed indolizinium alkaloid, streptopertusacin A (70) (Figure 10), was obtained from the GYM solid culture extract of Streptomyces sp. HZP-2216E, isolated from a fresh seaweed, Ulva pertusa, which was collected at the Turtle islet in the South China Sea. The planar structure of 70 was established by extensive analysis of HRESIMS, 1D, and 2D NMR spectral data. The relative configurations of the stereogenic carbons in 70 were established through analysis of proton coupling constants and NOE data. The absolute configuration of the stereogenic carbon of its amino acid moiety (C-20) was determined by Marfey’s method after acid hydrolysis of 70, while the absolute configurations of C-1, C-2, and C-3 were established by comparison of the experimental and calculated ECD spectra. Compound 70 showed moderate inhibitory activity against MRSA with a MIC value of 40 µg/mL. The positive control, gentamicin, showed a MIC value of 0.36 µg/mL [26].

By using LC-MS-principal component analysis (PCA)-based metabolomics and molecular networking approaches, two antibacterial pyrrole-derived alkaloids, phallusialides A (71) and B (72) (Figure 11), were obtained from the acetone extract of a bacterium Micromonospora sp. WMMA-2495, isolated from a tunicate, Phallusia nigra, which was collected in the Florida Keys, and cultured in the artificial medium ASW-A (20 g soluble starch, 10 g glucose, 5 g peptone, 5 g yeast extract, 5 g CaCO₃ per liter of artificial seawater) supplemented with NaCl and KBr. The structures of the compounds were elucidated by extensive analysis of HRESIMS, 1D, and 2D NMR data. The relative configurations of the stereogenic carbons of 71 were established by a combination of NOE studies, coupling constant analyses, extensive molecular modeling, DFT calculations, and ROESY correlations. Since a comparison of the experimental and calculated ECD spectra allowed the determination of the absolute configuration of only C-4′, the absolute structure of 71 was based on the established relative configurations of the stereogenic carbons relative to C-4′ whose absolute configuration was unambiguously determined. The absolute configuration of the stereogenic carbons in 72 was established by comparison of the Cotton effects observed in its ECD spectrum with those in 71. Compounds 71 and 72 displayed antibacterial activity against MRSA (ATCC #33591), with a MIC value of 32 µg/mL (vancomycin, the positive control, showed MIC = 0.25 µg/mL). Comparing the structural feature of 71 and 72 with the co-isolated analogs that did not exhibit antibacterial activity revealed that halogenation at C-4 of the pyrrole moiety is crucial to antibacterial activity of 71 and 72, and the extra sugar moieties in the co-isolated analogs could modulate antibacterial activity [44].

2.3. Terpenoid Derivatives

Two undescribed halimane-type diterpenoids, micromonohalimanes A (73) and B (74) (Figure 11), were isolated from the culture extract of Micromonospora sp. WMMC-218, which was obtained from the ascidian, Symplegma brakenhielmi, collected in Florida at Stanblum State Park, USA, and cultured in ASW-medium. The structure of 74 was established by HRESIMS, 1D, and 2D NMR spectral analysis. The relative configuration of 74 was established by ROESY, 1D-double-pulsed-field-gradient-spin-echo (DPFGSE)-NOE, and DFT studies. The DFT studies of 74 revealed that stereochemical configurations of 74 were identical to those found in micromonohalimane A (73), which was co-isolated and whose absolute structure was determined by X-ray analysis. Compound 74 displayed antibacterial activity against MRSA (ATCC #33591), with a MIC value of 40 µg/mL, while 73 showed a MIC value greater than 200 µg/mL. Vancomycin was used as a positive control and showed a MIC value of 1 µg/mL. Moreover, 74 was found to be a bacteriostatic agent since the MRSA was able to grow in the LB plate in the presence of 74 [45].

Three unreported anti-MRSA diterpenoids, viz. 18-acetyl-cyclooctatin (75), 5,18-dedihydroxy-cyclooctatin (76), and 5-dehydroxy-cyclooctatin (77) (Figure 11) were isolated from the culture of Streptomyces sp. ZZ820, obtained from a soil sample, which was collected from a sea coastal in the East China Sea close to Zhoushan Archipelago, Zhejiang, China, and cultured in the SGYC liquid medium. The planar structures of 75–77 were elucidated by interpretation of HRESIMS, 1D, and 2D NMR spectra. The absolute configuration of C-5 in 75 was determined by Mosher’s method, while the absolute structures of 75 and 76 were established by comparison of their calculated and experimental ECD spectra. Since the structure of 77 differs from that of 76 in that Me-18 in 76 was replaced by a hydroxymethyl group in 77, and both compounds had positive optical rotation values and similar ECD curves, it was assumed that 77 had the same absolute structure as 76. Compounds 75–77 showed moderate anti-MRSA activity with MIC values of 27.45, 24.11, and 29.39 µg/mL, respectively. The positive control, gentamicin, showed a MIC value of 0.91 µg/mL [46].

2.4. Miscellaneous

An undescribed glycerol 1-hydroxy-2,5-dimethyl benzoate (78) (Figure 12) was isolated from the culture extract of Verrucosispora sp. strain MS100047, obtained from a deep-sea sediment sample collected at the depth 2733 m in the South China Sea, and cultured in the VER01 liquid medium. The structure of 78 was determined by analysis of HRESIMS and 1D and 2D NMR spectra. However, the absolute configuration of C-9 was not determined. Compound 78 showed selective activity against MRSA with a MIC value of 12.5 µg/mL [47].

An unreported 2,2′-pyridine containing methyl sulfonyl and carboxaldehyde oxime moieties, named maipomycin A (79) (Figure 12), was obtained from a rare actinomycete, Kibdelosporangium phytohabitans XY-R10, isolated from the root sediments of a mangrove plant, Kandelia candel (L.) Druce, collected from Mai Po Inner Deep Bay Ramsar Site, Hong Kong, China, and cultured in SGTPY medium (17 g sea salts, 5 g starch, 5 g glucose, 1 g tryptone, 1 g peptone, and 1 g yeast extract dissolved in 1 L of distilled water). The structure of 79 was established by analysis of HRESIMS, 1D, and 2D NMR spectra and confirmed by a single-crystal X-ray analysis. Compound 79 did not show antibacterial activity against a panel of Gram-positive and Gram-negative bacteria, except for a weak antibacterial activity against the reference strains and clinical isolate of Actinobacter baumannii (MIC = 128 mg/mL). On the contrary, 79 showed an effective inhibitory activity against the Gram-negative bacteria biofilm formation in a dose-dependent manner from 2 to 64 mg/mL. The percentage of biofilm biomass at a minimum biofilm inhibitory concentration (MBIC) was reduced by 84.3% for A. baumannii, and 82.6% for P. aeruginosa compared to the control. Confocal microscopy analysis showed an increase in the roughness coefficient and surface-to-biovolume ratio of the biofilm, suggesting the heterogeneity and incompleteness of biofilm development caused by 79. Interestingly, although 79 exhibited weak antibacterial activity, it could efficiently potentiate colistin against A. Baumannii. A combination of colistin with 79 resulted in the reduction of the MIC of colistin 4–8 folds. Moreover, 79 also showed a synergistic effect with colistin against A. Baumannii but only an additive effect for anti-biofilm activity [48].

The extract of Streptomyces sp. ZZ741, isolated from a mud sample in Jintang Island, Zhoushan, China, and cultured on a rice medium, furnished ten undescribed glutarimide antibiotics, streptoglutarimides A−J (80–89) (Figure 12). The planar structures of 80–89 were elucidated based on their HRESIMS data and extensive analysis of their 1D and 2D NMR spectra. The absolute configurations of C-11 and C-13 in 80 were determined as 11R, 13R by X-ray diffraction analysis and confirmed by comparison of their experimental and calculated ECD spectra. The absolute configurations of C-11 and C-13 in 81 were determined as 11R, 13S by comparison of their experimental and calculated ECD spectra. Therefore, 80 and 81 are diastereomers. Compound 82 has only one stereogenic carbon (C-13), and its absolute configuration was established as 13S, also by comparison of their experimental and calculated ECD spectra. The absolute configurations of the stereogenic carbons in 83–85 were determined by comparison their experimental and calculated ECD spectra as well as by ¹³C chemical shift calculations. The absolute configurations of the stereogenic carbons in 86–88 were determined by Mosher’s method and by comparison of their experimental and calculated ECD spectra, while the absolute configurations of the stereogenic carbons in 89 were established by NOE correlations and comparison of their experimental and calculated ECD spectra.

Compounds 80–89 displayed antimicrobial activities against MRSA, with MIC values ranging from 9–11 µg/mL, while the positive controls, vancomycin, and gentamycin, exhibited MIC values of 0.2 and 0.5 µg/mL, respectively [49].

The unreported N-isoprenoid bromo-phenazinone, marinocyanin A (90) (Figure 13), was isolated from the culture extracts of the strains CNS-284 (isolated from a sediment sample collected at a depth of 34 m in Palau) and CNY-960 (isolated from a sediment sample collected in the Solomon Islands), while the undescribed marinocyanins B-F (91–95) (Figure 13) were isolated from the culture extract of the strain CNS-284. Both strains were identified as members of MAR4 clade within the Streptomycetaceae. The planar structures of 90–95 were elucidated by HRESIMS, 1D, and 2D NMR spectral analysis. The structure of 90 was confirmed by X-ray crystallographic analysis. The absolute configurations of the stereogenic carbons in 93 and 95 were not determined due to the limited amount of the compounds and their stability [50].

Compound 90 strongly inhibited the growth of amphotericin-resistant Candida albicans, with a MIC value of 0.95 µM. Compounds 93–95 exhibited weaker inhibitory activity against C. albicans, with a MIC value of 14.67 µM. Compounds 91 and 92 showed MIC values of 5.79 and 3.90 µM, respectively. The MIC value of the positive control, amphotericin B, was 0.084 µM. Compound 90 exhibited a MIC value of 2.37 µM against S. aureus, while 91–95 only showed MIC values in the range of 30.71–36.62 µM. The structure–activity relationship indicated that the terpenoid ring system was important for the activity. Modification in the ring system significantly reduced the potencies of the marinocyanins [50].

Bioactivity-guided isolation of the culture extract of P. aeruginosa strain 1682U.R.0a.27, isolated from the gill of a giant shipworm, Kuphus polythalamius, which was collected in Sultan Kudarat, Mindanao, Philippines, and fermented in R2A medium (0.005% yeast extract, 0.005% protease peptone, 0.005% casamino acids, 0.005% dextrose, 0.005% soluble starch, 0.003% sodium pyruvate, 0.003% K₂HPO₄, 0.0005% MgSO₄, and 2% NaCl) yielded pyoluteorin analogs, mindapyrroles B (96) and C (97) (Figure 14). The structures of 96 and 97 were established by interpretation of HRMS, 1D, and 2D NMR spectra. Compounds 96 (MIC = 4 µg/mL) displayed more potent antibacterial activity than 97 (MIC = 8 µg/mL) against MRSA strain ATCC 43300 (oxacillin, the positive control, showed a MIC value of 8 µg/mL). Compound 96 also inhibited Gram-negative bacteria, such as P. aeruginosa and E. faecium, with MIC values of 8 µg/mL. It is interesting to observe that the marinopyrroles, which are coupled by C-C bonds, displayed weaker antibacterial activity than those with the C-N bonds. Within the marinopyrroles, the addition of the hydroxyphenylthiazole moiety led to an increase in antimicrobial activity [51].

3. Conclusions

Marine-derived bacteria have been acknowledged as an important source of compounds with unique structures and potential bioactivities. Moreover, the biomass of marine bacteria can be scaled up to an unlimited level in the laboratory for secondary metabolite production. The structures of antimicrobial compounds derived from marine bacteria reported from January 2016 to December 2021 consisted mostly of polyketides, amino acid derivatives, terpenoid derivatives, and also derivatives of mixed origins (Figure 15). As can be seen in

Table 1. Summary of the compounds with antimicrobial properties against drug-resistant pathogens and their sources.

No.	Bacterial Strain	Source	Compounds and Their Activities	Ref.
1.	Bacillus subtilis 109GGC020	Sediment sample from the Gageocho reef, Republic of Korea	55 and 56 active against MRSA	[39]
2.	Kibdelosporangium phytohabitans XY-R10	Root sediments of a mangrove plant, Kandelia candel (L.) Druce, from Mai Po Inner Deep Bay Ramsar Site	79 active against A. baumanii as antibiofilm	[48]
3.	MAR4 clade within the Streptomycetaceae strains CNS-284 and CNY-960	Sediment sample from Solomon Islands	90–95 active against amphotericin B-resistant C. albicans	[50]
4.	Micromonospora harpali SCSIO GJ089	Sediment from Northern South China Sea	43 and 44 active against MRSA	[33]
5.	Micromonospora sp. CA-214671	from the Canary Island	40 and 41 active against MRSA	[32]
6.	Micromonospora sp. RJA4480	Marine sediment from Barkley Sound, British Columbia	18–21 active against MRSA	[20]
7.	Micromonospora sp. WMMA-2495	Tunicate, Phallusia nigra, from Florida Keys	71 and 72 active against MRSA	[44]
8.	Micromonospora sp. WMMC-218	Ascidian, Symplegma brakenhielmi, Florida at Stanblum State Park, USA	74 active against MRSA	[45]
9.	Nesterenkonia sp. MSA31	Marine sponge, Fasciospongia cavernosa, from southwest coast of India	54 against MDR-P.aeruginosa	[38]
10.	Nocardiopsis sp. HDN154086	Marine sediment from South China Sea	52 and 53 active against MRSA, Proteus sp. and B. subtilis	[36]
11.	Nocardiopsis sp. strain HB-J378	Deep-sea sediment	1–3 active against MRSA	[12]
12.	Nonomuraea sp. strain MM565M-173N2	Deep-sea sediment from Japan trench	10–13 active against MDR-E. coli and MDR-K. pneumoniae, MRSA, and VRE	[16]
13.	Pseudomonas aeruginosa strain 1682U.R.0a.27	Gill tissue homogenate of the giant shipworm, Kuphus polythalamius, from Sultan Kudarat, Mindanao, Philippines	96 and 97 active against MRSA. 96 also active against P. aeruginosa and E. faecium	[51]
14.	Shewanella algae MTCC 12715	Red algae, Hypnea valentiae, from the Gulf of Mannar, India	28 and 29 active against MRSA and VRE	[25]
15.	Streptomyces althioticus MSM3	Seaweed, Ulva sp., from the Cantabrian Sea	39 active against M. tuberculosis, S. aureus, S. pneumoniae, S. pyogenes, Clostridium perfringens, C. urealyticum, E. faecalis, E. Faecium, B. fragilis, H. influenzae and N. meningitidis	[30]
16.	Streptomyces cyaneofuscatus M-169	Gorgonian coral from Avilés submarine Canyon, Cantabrian Sea	26 active against MRSA, MSSA, E. faecium, E. faecalis	[23]
17.	Streptomyces koyangensis SCSIO 5802	Marine sediment from Northern South China Sea	50 and 51 active against MRSA	[34]
18.	Streptomyces pratensis strain NA-ZhouS1	Marine sediment in Zhoushan, East China Sea	4 and 5 active against B. stubtilis, E. coli, K. pneumoniae, MRSA, P. aeruginosa	[13]
19.	Streptomyces sp. 1425S.R.1a.1	Mollusk, Truncatella guerinii, from Cebu, Philippines	24 active against MRSA	[21]
20.	Streptomyces sp. 182SMLY	Marine sediment	6 active against MRSA	[14]
21.	Streptomyces sp. CA-271078	-	15 and 17 active against MRSA and M. tuberculosis	[18]
22.	Streptomyces sp. EG1	Marine sediment	25 active against MRSA	[22]
23.	Streptomyces sp. EG32	Marine sediment from North Coast of the Mediterranean Sea, Egypt	8 and 9 active against MRSA	[15]
24.	Streptomyces sp. HZP-2216E	Seaweed Ulva pertusa from Turtle Islet Guangdong, China	33 active against MRSA	[27]
25.	Streptomyces sp. HZP-2216E	Seaweed, Ulva pertusa, from Turtle Islet Guangdong, China	30 and 31 active against MRSA	[26]
26.	Streptomyces sp. HZP-2216E	Seaweed, Ulva pertusa, from Turtle Islet Guangdong, China	70 active against MRSA	[26]
27.	Streptomyces sp. IMB094	Marine sediment Heishijiao Bay, Dalian, China	57 active against MRSA and VRE	[40]
28.	Streptomyces sp. IMB7-145	Marine sediment at -40 m in Heishijiao Bay, Dalian, China	34–37 active against MRSA, MRSE, and VRE. 34 also active against MDR-TB	[28]
29.	Streptomyces sp. LHW52447	Marine sponge, Phyllospongia foliascens, in Xisha Islands in the South China Sea	60–63 active against MRSA	[41]
30.	Streptomyces sp. MBTI36	Marine sediment	14 active against MRSA	[17]
31.	Streptomyces sp. SCSIO 11791	Deep-sea sediment from South China Sea	68 and 69 active against MRSA	[43]
32.	Streptomyces sp. ZZ1118	Marine shrimp (Penaeus sp.) in Zhoushan archipelago, Zhejiang, China	64, 65, and 67 active against MRSA	[42]
33.	Streptomyces sp. ZZ741	Sea mud in Jintang Island Zhoushan, China	80–89 active against MRSA	[49]
34.	Streptomyces sp. ZZ820	Coastal soil in Zhoushan Archipelago (Zhejiang, China)	75–77 active against MRSA	[46]
35.	Verrucosispora sp. strain MS100047	Deep-sea sediment from South China Sea	78 active against MRSA	[47]

, Streptomyces species constitute a major source of bioactive compounds. However, other bacterial taxa, such as Nonomuraea sp., Micromonospora sp. Shewanella sp., Bacillus sp., are also found to produce potential compounds with interesting activity against drug-resistant pathogens. Thus, marine-derived bacteria are an excellent source for further exploration to search for novel antimicrobial and antibiofilm compounds against multi-drug resistant pathogens.