Diversity and antimicrobial activity of marine nudibranch associated bacteria against tropical human skin pathogens

Introduction

The number of cases of skin disease that continue to appear shows the urgency of treatment for this problem. Hay (2020) estimates that skin diseases affect nearly a billion people globally each year. However, they are still a neglected topic by public health authorities even though most casualties from skin diseases are avoidable (Alfonso et al., 2017; Bongomin et al., 2017). One of the most commonly used treatment methods for skin diseases is antibiotic products. Microbial skin infections such as mycoses, including dermatophytosis, candida, and Malassezia, dominate in hot climates such as Indonesia. Cutibacterium acnes, Staphylococcus aureus, and Candida albicans are causative agents of skin pathogens. Furthermore, these pathogenic microbes also have a role in cases of infection associated with biofilms, which are often chronic, nosocomial, and persistent bacterial infections that exhibit resistance towards broad-spectrum antibiotics.

These pathogenic bacteria live abundantly, are widely distributed, and live permanently on human skin. The bacteria in the skin can move to other people or medical devices via contact during insertion of a catheter or other medical devices onto the human body.

Although skin diseases rarely develop into deadly diseases, they are complicated to treat and pose a serious burden to the public health system. Skin infections are common in Indonesia, especially in rural areas where bacteria can spread through the bloodstream and infect distant organs. Karimkhani et al. (2017) reported that skin diseases, including dermatitis, acne vulgaris, urticaria, psoriasis, fungal skin diseases, scabies, decubitus ulcer and alopecia areata, contributed 1.79% to the global burden of disease. Hence, due to the high number of skin infections and the limited choices in antibiotics to treat these infections, the exploration of new drugs is urgently needed.

Marine nudibranchs are a highly diverse group of gastropod molluscs that are soft-bodied and shell-less and are found worldwide in various ecosystems, including coral reefs (Thompson and Brown, 1984; Hoeksema, 2007). Scientists are interested in their ecology, natural product chemistry, color pattern evolution and natural history because of their beauty and morphological diversity (Korshunova et al., 2020, 2021). However, with a total of approximately 10,000 species, only about 4% have been chemically analyzed, therefore, a high number of compounds remain to be discovered (Avila and Angulo-Preckler, 2020). Having colorful bodies and lacking the physical protection of a shell, the ability of nudibranch species to defend themselves against predators has drawn attention to the chemical defense of its secondary metabolites. Data on nudibranch species with antipredator compounds, such as Doriopsilla albopunctata, D. areolata, D. janaina, and D. pharpa, have been reported (Fontana et al., 2003; Long and Hay, 2006; Gaspar et al., 2008). Several previous studies showed that secondary metabolites from nudibranchs also have antiviral, antibacterial, anticancer, and anti-inflammatory bioactivity (Zhukova, 2014; Avila and Angulo-Preckler, 2020; Hertzer et al., 2020). Besides, some active compounds from nudibranchs that are still in the preclinical and clinical pipeline include the anticancer active compound jorumycin, isolated from the skin and the mucus of the Pacific nudibranch Jorunna funebris (Fontana et al., 2003), bursatellanins isolated from Bursatella leachii (Mollo et al., 2005), and aplyronines and dolastatins isolated from the Dolabella auricularia (Mudianta et al., 2015; White et al., 2016).

It is well known that the development of drugs from nudibranch derivatives is not easy since synthesizing complex compounds in large quantities is not profitable because it requires a large supply of raw materials, which is a threat to its sustainability. So, it is rare to find research compounds of nudibranch-derived drugs up to the stage of clinical trials for further marketing (Chen et al., 2018). Microorganisms as raw materials, on the other hand, are feasible due to their sustainable and economical supply and their ability to provide bioactive compounds (Rajasabapathy et al., 2020). Symbiotic associations between marine invertebrates from different phyla, such as the arthropods (van As and van As, 2019), bivalvia (Zannella et al., 2017), or porifera (Anteneh et al., 2021), are known to host such symbiotic associations. Böhringer et al. (2017) found antibacterial activity in 71% (35 of 49 isolates) of bacteria associated with the nudibranch genus Chromodoris sp., Phyllidiella sp., Hexabranchus sp., and Doriprismatica sp. Some bioactive compounds may be produced by their bacterial symbionts (Blockley et al., 2017; Riyanti et al., 2020). Current studies show that nudibranch-associated bacteria may act as a chemical defense for nudibranchs via antimicrobial production (Kristiana et al., 2019; Avila et al., 2020).

The production of several biologically active secondary metabolites from microorganisms needs to be activated by specific gene clusters. Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are examples of gene cluster forms (Adamek et al., 2018). These gene clusters produce antibiotic compounds such as tetracycline and erythromycin (El Samak et al., 2018; Verma et al., 2011). Therefore, the detection of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes in the association of nudibranchs with bacteria could further explain their essential role in chemical defense in the nudibranchs. The purification of these nudibranch-associated bacterial genes has been conducted (Riyanti et al., 2009; Böhringer et al., 2017; Kristiana et al., 2020; Abdelrahman et al., 2021). The most recent study by Abdelrahman et al. (2021) reported that nudibranch-associated bacteria have antibacterial activity and possess natural product gene clusters. In the present study, nudibranch-associated bacteria with potential bioactivity found in the Jepara coastal waters of the Java Sea were molecularly identified to determine their diversity, antimicrobial activity, and the presence of PKS and NRPS gene fragments.

Methods

Sampling site collection

Nudibranch samples (J. funebris, G. rubropapulosa and G. atromarginata) were collected by scuba diving at depths of 5 to 8 m in Jepara coastal waters, i.e., Awur Bay (110°38’53.14" E, 6°36’36.82" S), Kartini Coast (110°38’15.12" E, 6°35’30.13" S), Panjang Island (110°37’24.01" E, 6°34’23,43" S) and Bandengan Coast (110°38’55.32" E, 6°32’59.28" S) in July 2020 (Figures 1 and 2). Three individual samples were taken for each nudibranch species. To prevent contamination with air before being brought to the surface, the specimens were placed in plastic bags. Then, the samples were stored separately in a plastic bag containing natural seawater until they were processed. Each sample was then placed in an ice cool-box and brought to the Marine Science Laboratory at Diponegoro University (Semarang, Indonesia) for bacterial isolation.

Figure 1. Sampling site location (Source: Google Earth).

Figure 2. Images of nudibranch species documented at Jepara coastal waters.

A. Jorunna funebris; B. Gymnodoris rubropapulosa; C. Glossodoris atromarginata.

Results

Antimicrobial screening of nudibranch-associated bacteria

Out of 115 isolates, 20.87% (n = 24) showed antimicrobial activity against skin pathogens (Table 2 and Figure 3; Sabdono et al., 2022a). Among 24 active isolates, 12 isolates could inhibit the growth of more than one pathogen, whereas the remaining isolates (n = 12) inhibited only one pathogen tested. Most of the active isolates (57.7%) were capable of inhibiting the growth of M. furfur, followed by 53.8, 34.6, and 19.2% of the bacterial pathogens C. acne, C. albicans and S. aureus, respectively.

Table 2. Pathogenic assay of nudibranch-associated bacteria against tropical skin pathogens.

No.	Nudibranch	Total isolate	Active isolate	Strains	Causative agents of skin diseases
					S.aureus	C.acne	C.albicans	M.furfur
1.	J. funebris	36	5 (13.9 %)	JF.2.5	-	-	-	+
2.				JF.2.6	-	-	+	+
3.				JF.2.7	-	-	+	-
4.				JF.2.8	+	-	-	-
5.				JF 4.5	+	+	-	-
6.	G. rubropapulosa	31	4 (12.9 %)	GR.1.4	-	+	-	-
7.				GR.1.8	-	+	-	+
8.				GR.1.11	-	-	-	+
9.				GR.1.12	-	-	-	+
10.	G. atromarginata	48	15 (31.3 %)	GA4.12	+	+	-	-
11.				GA9.3	-	-	-	+
12.				GA9.5	-	-	-	+
13.				GA.9.8	-	-	+	+
14.				GA.9.10	+	-	-	+
15.				GA2.2	-	+	-	+
16.				GA2.4	-	+	-	-
17.				GA2.6	+	+	+	+
18.				GA2.8	-	+	+	+
19.				GA2.10	-	+	-	-
20.				GA2.17	-	-	+	+
21.				GA2.22	-	+	-	-
22.				GA2.25	-	+	+	+
23.				GA2.26	-	+	+	+
24.				GA2.27	-	+	-	+
	Total	115	24 (20.87%)		4 (16.7%)	13 (54.2%)	8 (33.3%)	15 (62.5%)

Figure 3. Pathogenic assay of nudibranch-associated bacteria (Note: A: C. acne; B: S. aureus; C: M. furfur; D: C. albicans).

16S rDNA gene sequencing and phylogenetic analysis

The 16S rRNA gene sequencing analyses showed that these 24 isolates could be assigned to 11 different genera within the three phyla: Proteobacteria (genera Salinicola, Thalassospira, Halomonas, Paracoccus, Pseudovibrio, Pseudoalteromonas, Pseudomonas), Firmicutes (genus Bacillus), and Actinobacteria (genera Brevibacterium, Gordonia, Dietzia). A total of 11 of the 24 isolates (46%) were members of the genus Bacillus. The remaining 13 of the 24 isolates (54%) were members of the genera Salinicola (two isolates), Gordonia (two isolates), Streptomyces (two isolates), Thalassospira (one isolate), Halomonas (one isolate), Dietzia (one isolate), Brevibacterium (one isolate), Paracoccus (one isolate), Pseudovibrio (one isolate), Pseudoalteromonas (one isolate) and Pseudomonas (one isolate) (Table 3 and Figure 4). Identification of pairwise 16S rRNA gene sequence similarities was analyzed by using the NCBI-BLAST homology. The Phylogenetic Analysis Using Parsimony (PAUP) v.05 (Swofford, 1998) and CLUSTAL_X (Thompson et al., 1997) neighbor-joining algorithms were used to infer phylogenetic trees (Figure 5).

Table 3. Identification of nudibranch-associated bacteria by 16S rRNA gene.

No.	Strains	Closely related	% Identity	Accession no
1.	JF2.5	Salinicola zeshunii strain N4	86.28	NR_132717.1
2.	JF2.6	Gordonia hongkongensis strain HKU50	96.03	NR_152023.1
3.	JF2.7	Gordonia terrae strain CL6	96.24	JN862637
4.	JF2.8	Salinicola salarius strain M27	85.98	NR_042490.1
5.	JF4.5	Pseudovibrio denitrificans strain NBRC 100825	99.06	NR_113946
6.	GR1.4	Pseudomonas stutzeri strain CCUG 11256	98.92	NR_118798
7.	GR1.8	Bacillus altitudinis strain UAC-24	99.45	JX475123
8.	GR1.11	Bacillus altitudinis strain 41KF2b	97.94	NR_042337.1
9.	GR1.12	Streptomyces ambofaciens strain NBRC 12836	98.85	NR_041079.1
10.	GA4.12	Streptomyces ferralitis strain SFOp68	97.16	NR_029087
11.	GA9.3	Paracoccus beibuensis strain JLT1284	98.66	NR_116400.1
12.	GA9.5	Dietzia maris strain AUCM A-593	98.99	NR_037025.1
13.	GA9.8	Bacillus aerius strain 24K	97.51	NR_118439.1
14.	GA9.10	Halomonas meridiana strain DSM 5425	98.95	NR_042066
15.	GA2.2	Brevibacterium avium strain XJB-YJ9-1	96.07	KM186614
16.	GA2.4	Thalassospira povalilytica strain Zumi 95	99.35	NR_125450
17.	GA2.6	Bacillus altitudinis strain IARI-JR-46	99.93	KF054998
18.	GA2.8	Bacillus altitudinis strain XjGEB-71	98.14	JQ320096
19.	GA2.10	Bacillus stratosphericus strain NIBSM_OsR1	99.52	KY911276
20.	GA2.17	Bacillus safensis strain EGI287	100	MN704552
21.	GA2.22	Bacillus aerius strain UB02	99.72	MK696417
22.	GA2.25	Bacillus altitudinis strain JYM50	99.65	MN511801
23.	GA2.26	Bacillus altitudinis strain BM-Y1	96.01	FJ426274
24.	GA2.27	Bacillus altitudinis strain L9	99.72	MN134574

Figure 4. Generic composition of the 24 bacteria associated with nudibranchs of the Java Sea that have antimicrobial activity.

Figure 5. Phylogenetic tree of bacteria isolated from three nudibranchs of the Java Sea that have antimicrobial activities.

Micrococcus luteus strain DSM 20030 was used as the outgroup.

Screening of PKS and NRPS genes

The PKS and NRPS genes in nudibranch-associated bacteria were detected. However, none of the 24 antimicrobial bacterial strains had PKS-I, one possessed PKS-II, and five possessed NRPS genes (Table 4 and Figure 6; Sabdono et al., 2022b).

Table 4. Detection of PKS-I, PKS-II, and NRPS genes of antimicrobial strains.

No	Isolate	PKS-I	PKS-II	NRPS
1.	JF2.5	-	-	+
2.	JF2.6	-	-	+
3.	JF2.7	-	-	+
4.	JF2.8	-	-	-
5.	JF4.5	-	-	-
6.	GR1.4	-	-	+
7.	GR1.8	-	-	+
8.	GR1.11	-	-	+
9.	GR1.12	-	-	-
10.	GA4.12	-	+	-
11.	GA9.3	-	-	-
12.	GA9.5	-	-	-
13.	GA9.8	-	-	-
14.	GA9.10	-	-	-
15.	GA2.2	-	-	-
16.	GA2.4	-	-	-
17.	GA2.6	-	-	-
18.	GA2.8	-	-	-
19.	GA2.10	-	-	-
20.	GA2.17	-	-	-
21.	GA2.22	-	-	-
22.	GA2.25	-	-	-
23.	GA2.26	-	-	-
24.	GA2.27	-	-	-

Figure 6. Detection of PKS-I, PKS-II, and NRPS genes on antimicrobial isolates.

PKS-I: type I polyketide synthase; PKS-II: type II polyketide synthase; NRPS: non-ribosomal peptide synthetase.

Distribution of nudibranch-associated bacteria with antimicrobial potency

The distribution of antimicrobial isolates is demonstrated in Figure 7. Overall, in the present study, 24 antimicrobial strains were obtained (five bacterial isolates from J. funebris, four isolates from G. rubropapulosa and 15 isolates from G. atromarginata). The nudibranch G. atromarginata harbored not only the majority of bacterial isolates but also the highest number of bacterial genera (seven of the 11 genera), followed by G. rubropapulosa (three genera) and J. funebris (three genera). Among the 11 genera of bacterial isolates, Bacillus was the major genus isolated from the nudibranchs G. atromarginata and G. rubropapulosa. The Bray-Curtis analysis was used to compare antibacterial communities in the three nudibranch species. The result showed a 100% dissimilarity of bacterial communities between J. funebris vs. G. rubropapulosa and J. funebris vs. D. atromarginata, while the dissimilarity between G. rubropapulosa vs. D. atromarginata was 68.4% (Table 5). These results showed that the antimicrobial communities in different nudibranch species varied significantly.

Figure 7. The number and genera of bacteria isolated from nudibranchs with antimicrobial activities.

Table 5. The dissimilarity of antimicrobial strains associated with different nudibranchs (%).

Nudibranch	J. funebris	G. rubropapulosa	D. atromarginata
J. funebris	-	100	100
G. rubropapulosa	100	-	68.4
D. atromarginata	100	68.4	-

Discussion

Marine nudibranchs such as J. funebris, G. rubropapulosa, and D. atromarginata are common in Jepara coastal waters, particularly in Awur Bay, Kartini Coast, Panjang Island, and the Bandengan coast of the North Java Sea (Sabdono et al., 2021). The natural products of these species exhibit variability in their bioactivity that has potential applications in the pharmaceutical and medical fields. He et al. (2014) highlighted the antitumor and antimicrobial activity of renieramycin-type bistetrahydroisoquinolinequinone alkaloids, which were extracted from the skin of the nudibranch J. funebris. Furthermore, Li et al. (2019) reported the discovery of a novel spongian type diterpene anticancer and antibiotic produced by the nudibranch G. atromarginata from Weizhou Island in the South China Sea. Recently, some studies have shown that several species of nudibranchs have the potential to produce biological activities. Reddy et al. (2015) reported the antimicrobial potential of the nudibranchs Phyllidia varicosa, Plakobranchus ocellatus, Phyllidiella rosans, and Halgerda stricklandi against bacterial and fungal pathogens collected from the South Andaman Islands, India. Avila and Angulo-Preckler (2020) demonstrated that more than hundreds of marine nudibranchs possess ecological and pharmacological activities. The ecological activities consist of predator avoidance, toxicity, antimicrobials, and antifouling, while the pharmacological activities include cytotoxicity and antitumoral activity, antibiotic, antiparasitic, antiviral, and anti-inflammatory activity; and activity against neurodegenerative diseases.

Recent discoveries have shown that the actual producers of some of the bioactive compounds are marine invertebrates with microbial symbionts (Raimundo et al., 2018). This implies the considerable potential for finding nudibranch bacterial symbionts for treating newly developing diseases in humans. The diversity and functional roles of nudibranch-associated bacterial communities have been characterized and determined (Kristiana et al., 2020; Böhringer et al., 2017; Riyanti et al., 2009). In this study, isolation and screening of bacteria with antimicrobial activity associated with the three nudibranchs revealed that 24 out of 115 isolates demonstrated antimicrobial activity against skin pathogens (Table 2 and Figure 3). Analyses of 16S rRNA sequencing discovered that 24 isolates were closely related to 11 genera, including Bacillus as a dominant genus, followed by Streptomyces, Gordonia, Salinicola, Thalassospira, Halomonas, Dietzia, Brevibacterium, Paracoccus, Pseudomonas, and Pseudomonas (Table 3 and Figure 4). Members of the genus Bacillus are known as the largest source of bioactive natural products, which exhibit a wide range of antibiotic activities (Stoica et al., 2019). Bacilli genera have produced hundreds of antibiotics (Awais et al., 2010).

Our phylogenetic analysis (Figure 5) shows that these bacterial genera belong to the phyla Firmicutes, Proteobacteria, and Actinobacteria. Previous studies on nudibranch-associated bacteria support the results of our study. Riyanti et al. (2009) reported that Streptomyces sp. isolated from nudibranch Jorunna sp. and Chromodoris sp. displayed antimicrobial activity against multi-drug resistant (MDR) bacterial strains. Böhringer et al. (2017) discovered that the diversity of nudibranch-associated bacteria with antimicrobial activity was dominated by the genus Pseudoalteromonas and Vibrio. Besides, Kristiana et al. (2019) found that antibacterial strains Virgibacillus marismortui, V. dokdonensis, Bacillus kochii, Vibrio algynoliticus and Pseudoalteromonas piscicida were revealed from the nudibranch genera Chromodoris and Phyllidiopsis. It was also reported that nudibranch-associated bacteria, Pseudomonas rubra, and Virgibacillus salarius, produce anti-MRSA, cytotoxicity, and anti-herpes simplex virus-1 bioactive compounds (Kristiana et al., 2020).

The discovery of PKS and NRPS genes in nudibranch bacteria could explain their pivotal functions in the chemical defense process of the nudibranch. This study showed that six isolates (23%) were positive for one PKS-II or NRPS (Table 4 and Figure 6), including Bacillus, Gordonia, Salinicola, Pseudomonas, and Streptomyces genera. From these two genes, several new polyketide and peptide compounds were discovered in marine microbes with different biological activities and ecological functions (Baker et al., 2009). Stoica et al. (2019) reported that low molecular weight polypeptides are produced by Bacillus species via ribosomal or non-ribosomal mechanisms. However, very little is known about the distribution of these biosynthetic systems in other nudibranch-associated bacteria. So far, only one study has demonstrated that most of the nudibranch-associated bacteria with biological activities contain PKS and NRPS genes (Abdelrahman et al., 2021), but it is still not understood if these isolates produce compounds that contribute to nudibranch defenses. The result of our study was only positive for one class of biosynthetic genes; nevertheless, it suggested that these bacterial isolates confine genes for biosynthesis.

Figure 7 and Table 5 show that in this study, the number and genera of bacterial strains of the three nudibranch species were significantly different. Each nudibranch species had different numbers and types of bacterial groups. G. atromarginata nudibranch harbored not only the most abundant bacterial isolate but also the highest number of bacterial genera. The understanding of how the diversity of culturable bacteria varies among nudibranch species is limited. To the best of our knowledge, no previous research has investigated the diversity of antimicrobial activity in microbes associated with nudibranchs.

In conclusion, this study revealed the significant potential of nudibranch-associated bacteria from the North Java Sea as a source of anti-skin pathogens. However, further studies need to be conducted on the diversity of the culturable bacteria associated with the same nudibranch species from different geographic regions. Furthermore, there is still minimal knowledge about how the diversity of cultured bacteria differs in nudibranch species. Hence, it is also important to conduct research on the diversity of bacteria that can be cultured associated with the same nudibranch species at different locations.

Data availability