**1. Introduction**

Global health care is continually facing new challenges from human pathologies including cancers, diseases of old age (e.g., Alzheimer's and Parkinson's), rare orphan diseases, emerging infectious diseases, and, increasingly, the threat of drug-resistant infectious diseases. Infectious diseases claim over 13 million lives worldwide each year, with over 700,000 deaths currently attributed to antibiotic-resistant infections [1]. Drug resistance threatens to overturn the gains in antibiotic discovery since penicillin. Thus, there is an overwhelming urgency to discover new antibiotics, especially to fight antibioticresistant strains of MRSA, *Pseudomonas aeruginosa*, and *Klebsiella pneumoniae*. New natural products are needed not only to address antibiotic resistance, but also the aforementioned healthcare challenges.

To date, bacteria have provided the majority of the antimicrobial agents so far discovered [2,3]. Historically, the main source of bacteria for isolation of antibiotics has been soils [4] and, more recently, marine sediments [5,6]. Spore-forming actinomycetes that are defined as "chemically diverse bacteria" due to their ubiquitous production of antibiotics are abundant in soils but are also found in the marine environment. It is uncertain if they originate in the ocean or are washed into the sea in large numbers [6,7]. The study of

**Citation:** Dzeha, T.; Hall, M.J.; Burgess, J.G. Micrococcin P1 and P2 from Epibiotic Bacteria Associated with Isolates of *Moorea producens* from Kenya. *Mar. Drugs* **2022**, *20*, 128. https://doi.org/10.3390/md20020128

Academic Editors: Max Crüsemann and Ipek Kurtboke

Received: 8 July 2021 Accepted: 14 January 2022 Published: 7 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

bacteria that live on the surfaces of other organisms or symbiotically within other organisms has also been fruitful [8,9]. There is evidence that some organisms such as seaweeds and cyanobacteria harbor interesting holobionts, which are capable of producing bioactive compounds [10]. Cyanobacteria are also well known as sources of a diverse range of bioactive compounds. On the other hand, the study of cyanobacteria-associated bacteria in the discovery of novel antibiotics is in its infancy.

Bacteria–cyanobacteria associations are pre-historic at 440 Ma years ago, according to fossil evidence [11]. Bacteria attach themselves to cyanobacteria for buoyancy and to obtain nutrients released during photosynthesis; there is often a symbiotic relationship between these prokaryotes. Cyanobacterial filaments can be surrounded by a tough polysaccharide sheath that can harbor a multitude of heterotrophic bacteria that are difficult to remove [12]. The dominating heterotrophic bacteria associated with these sheaths are Gram-negative species from the phyla Proteobacteria and Bacteroidetes [13].

The association of bacteria with a cyanobacterial host is quite diverse and encompasses all phyla including Firmicutes, Actinobacteria, and γ-proteobacteria [14]. These assemblages constitute highly specific symbiotic relationships. Four specific strains of highly colored bacteria producing a quinone antibiotic were associated with *Lyngbya majuscula* from Puerto Rico [15]. The isolation of two minor hydroxyquinone compounds, quinone alkaloid 1 and 2 from a Curacao strain of *L. majuscula*, led to the speculation that these compounds may be synthesized by associated bacteria [16]. In a later study, the filamentous non-heterocystous marine cyanobacteria *Lyngbya* sp. was shown to harbor a maximum of six isolates of cultivable bacteria [12]. A study of other species of the order Oscillatoriales, comprising *Oscillatoria pseudogeminata*, *O. subtilissima*, *O. amphibian*, *O. cortiana*; *Phormidium lucidum*, *Phormidium* sp., *P. valderianum*, *P. tenue*, *P. foveolarum*; *Lyngbya confervoides* BDU 140301, *Lyngbya* sp. BDU 91711, and *Lyngbya* sp. BDU 141561, led to the isolation of 46 cultivable bacterial isolates [12].

The filamentous marine cyanobacterium *Moorea producens* is known to grow symbiotically with a number of other microorganisms, some of which are difficult to isolate [17]. *M. producens* has also been linked with swimmers' itch arising from debromoaplysiatoxin (DAT) [18]. In common with other Oscillatoriales, a Kenyan isolate of the filamentous marine cyanobacteria *M. producens* has associated bacteria including those with the ability to resist UV radiation [19]. Taxonomically, Moorea has recently been renamed Moorena [20]. This cyanobacterium is also a source of the cyclodepsipeptides homodolastatin 16 and antanapeptin A [21] and the potent anticancer molecule, dolastatin 16 (this study). The quest for a sustainable supply of these compounds generated an interest in obtaining the metagenomic DNA (gDNA) of the cyanobacteria/bacterial community. The immense diversity of the *Oscillatoriales* suggests that morphological identification is unreliable [13]. By contrast, molecular identification is accurate and specific as it is based on the genomic content of the organism. Whereas the latter method works efficiently for axenic species, its use for the speciation of non-axenic cyanobacteria is inadequate due to intragenomic gene heterogeneity arising from the presence of bacteria and other microorganisms [13,22,23]. Currently, molecular identification of non-axenic cyanobacteria utilizes the multiple displacement amplification (MDA) method, which has a limited total genomic coverage [24,25].

Various approaches for obtaining axenic cultures of cyanobacteria from non-axenic strains are documented [26]. However, they do not elaborate on how to isolate gDNA from non-axenic strains. Bacteria, yeast, and viruses are easily killed on contact with Cu2+ ions by reactive hydroxyl ions and hydroxy radicals that damage the cell while denaturing DNA in a Fenton-type reaction [27].

$$\mathrm{Cu^{+} + H\_{2}O \to Cu^{2+} + OH^{-} + OH^{\bullet}}$$

The use of copper and its alloys in "contact" killing of microorganisms dates back to both the Sumerian and Akkadian civilizations and only lost its prominence in medicine at the advent of commercially available antibiotics in 1932 [28]. However, the contact killing of microbes by copper is still being applied in Agriculture to control bacterial and fungal diseases [27]. Thus, we report here a novel method for the CuSO4·5H2O assisted differential gDNA isolation of non-axenic filamentous marine cyanobacteria. It is based on the treatment of cyanobacterial biomass with CuSO4·5H2O prior to exhaustive differential isolation of bacterial gDNA from cyanobacterial biomass to provide a residual substrate for isolation of the cyanobacteria gDNA. CuSO4·5H2O provides the Cu2+ ions needed for the contact killing of bacteria on the cyanobacteria biomass during treatment. A Kenyan isolate of *M. producens* was used for these studies. In addition, this method was used to confirm the identity of *L. majuscula* CCAP 1446/4 from the Culture Collection, Oban, Scotland, and to delineate the Kenyan *M. producens* from other species elsewhere. Bacteria associated with *M. producens*, including those associated with the filaments of *M. producens*, are described. The current study also highlights the first isolation of the antibiotics micrococcin P1 (**1**) and micrococcin P2 (**2**) from organic extracts of *B. marisflavi* and establishes an IC50 value of micrococcin P1 (**1**) against *Staphylococcus aureus*.

### **2. Results and Discussion**
