Biological and Chemical Diversity of Bacteria Associated with a Marine Flatworm

The aim of this research is to explore the biological and chemical diversity of bacteria associated with a marine flatworm Paraplanocera sp., and to discover the bioactive metabolites from culturable strains. A total of 141 strains of bacteria including 45 strains of actinomycetes and 96 strains of other bacteria were isolated, identified and fermented on a small scale. Bioactive screening (antibacterial and cytotoxic activities) and chemical screening (ultra-performance liquid chromatography–mass spectrometry (UPLC-MS)) yielded several target bacterial strains. Among these strains, the ethyl acetate (EA) crude extract of Streptomyces sp. XY-FW47 fermentation broth showed strong antibacterial activity against methicillin-resistant Staphylococcus aureus ATCC43300 (MRSA ATCC43300) and potent cytotoxic effects on HeLa cells. The UPLC-MS spectral analysis of the crude extract indicated that the strain XY-FW47 could produce a series of geldanamycins (GMs). One new geldanamycin (GM) analog, 4,5-dihydro-17-O-demethylgeldanamycin (1), and three known GMs (2–4) were obtained. All of these compounds were tested for antibacterial, cytotoxic, and antifungal activities, yet only GM (3) showed potent cytotoxic (HeLa cells, EC50 = 1.12 μg/mL) and antifungal (Setosphaeria turcica MIC = 2.40 μg/mL) activities. Their structure–activity relationship (SAR) was also preliminarily discussed in this study.


Introduction
Flatworms are invertebrates that belong to Platyhelminthes. About 4500 species of flatworms have been reported, of which over 1000 species are exclusively marine flatworms primarily belonging to Turbellaria polyclad [1]. Marine flatworms are 3-20 cm in length but some are quite small. Most marine flatworms are free-living organisms and they are found to hide under rocks or inside empty shells in the

Sample Identification
A marine flatworm ( Figure 2), collected at depth between 1 and 3 m in the intertidal zone of Yung Shue O, Hong Kong (114°21′ E, 22°24′ N) in January 2014, was originally identified as Stylochus sp. by a simple morphological comparison, but was later identified to be Paraplanocera sp. based on its 18S rRNA gene sequence (1260 bp). Maximum likelihood tree ( Figure 3) shows the phylogenetic position of the Paraplanocera sp. which is affiliated to the superfamily of Stylochoidea and forms the closest genetic distance with Paraplanocera oligoglena. The GenBank accession number for the 18S rRNA gene sequence of Paraplanocera sp. is MF319765.

Sample Identification
A marine flatworm ( Figure 2), collected at depth between 1 and 3 m in the intertidal zone of Yung Shue O, Hong Kong (114°21′ E, 22°24′ N) in January 2014, was originally identified as Stylochus sp. by a simple morphological comparison, but was later identified to be Paraplanocera sp. based on its 18S rRNA gene sequence (1260 bp). Maximum likelihood tree ( Figure 3) shows the phylogenetic position of the Paraplanocera sp. which is affiliated to the superfamily of Stylochoidea and forms the closest genetic distance with Paraplanocera oligoglena. The GenBank accession number for the 18S rRNA gene sequence of Paraplanocera sp. is MF319765.

Isolation and Taxonomy of Bacteria from the Marine Flatworm Paraplanocera sp.
A total of 141 strains of bacteria associated with the marine flatworm Paraplanocera sp. were isolated and identified, including 37 species of actinobacteria belonging to nine genera and 64 species of non-actinobacteria affiliating to 27 genera (the information of the isolates are detailed in Table 1). The 16S rRNA genes of all the cultured isolates have high similarity (98.5-100%) with their reference strains except the three novel species (marked in bold in Table 1). There were 45 strains of actinomycetes listed as follows, Streptomyces (14 strains of 13 species), Micromonospora (12 strains of eight species), Mycobacterium (nine strains of eight species), Tsukamurella (two strains of two species), Microbacterium (two strains of two species), Micrococcus (two strains of one species), Pseudonocardia (two strains of one species), Brevibacterium and Arthrobacter. There were 96 strains of non-actinomycetes, including Bacillus (33 strains of 22 species), Vibrio (11 strains of six species), Halobacillus (six strains of five species), Microbulbifer (four strains of three species), Ruegeria (11 strains of two species), Pseudovibrio (five strains of three species), Fictibacillus (two strains of two species), Pseudoalteromonas (two strains of one species), Photobacterium, Joostella (two strains of one species), Flammeovirga (two strains of one species), Arcobacter, Staphylococcus, Aquimarina, Tenacibaculum, Roseovarius, Cupriavidus, Oceanobacillus, Deinococcus, Pseudomonas, Paenibacillus, Stenotrophomonas, Paracoccus, Psychrobacillus, Alcaligenes (two strains of two species), Roseivirga and Methylobacterium. These results showed a great diversity of culturable bacteria associated with marine flatworm Paraplanocera sp.
Three novel bacterial strains were discovered from this study. Two strains, UST20140214-052 and UST20140214-015B, had been characterized to be new species of the genus Pseudovibrio and published elsewhere [15,16]. In these years, there is a growing interest in Pseudovibrio species as more and more isolates have been identified from sponges, corals, and sea squirts and, among them, some strains have genomic interactions with host marine invertebrates and gene clusters for producing secondary metabolites to protect the host from pathogens [27]. Another new species strain, XY-FW106, was characterized to be Deinococcus planocerae [17]. Interestingly, XY-FW106 showed resistance against ultraviolet irradiation [28], which might provide some protective function for the survival of the host flatworm, which lived in the shallow waters.

Isolation and Taxonomy of Bacteria from the Marine Flatworm Paraplanocera sp.
A total of 141 strains of bacteria associated with the marine flatworm Paraplanocera sp. were isolated and identified, including 37 species of actinobacteria belonging to nine genera and 64 species of non-actinobacteria affiliating to 27 genera (the information of the isolates are detailed in Table 1). The 16S rRNA genes of all the cultured isolates have high similarity (98.5-100%) with their reference strains except the three novel species (marked in bold in Table 1). There were 45 strains of actinomycetes listed as follows, Streptomyces (14 strains of 13 species), Micromonospora (12 strains of eight species), Mycobacterium (nine strains of eight species), Tsukamurella (two strains of two species), Microbacterium (two strains of two species), Micrococcus (two strains of one species), Pseudonocardia (two strains of one species), Brevibacterium and Arthrobacter. There were 96 strains of non-actinomycetes, including Bacillus (33 strains of 22 species), Vibrio (11 strains of six species), Halobacillus (six strains of five species), Microbulbifer (four strains of three species), Ruegeria (11 strains of two species), Pseudovibrio (five strains of three species), Fictibacillus (two strains of two species), Pseudoalteromonas (two strains of one species), Photobacterium, Joostella (two strains of one species), Flammeovirga (two strains of one species), Arcobacter, Staphylococcus, Aquimarina, Tenacibaculum, Roseovarius, Cupriavidus, Oceanobacillus, Deinococcus, Pseudomonas, Paenibacillus, Stenotrophomonas, Paracoccus, Psychrobacillus, Alcaligenes (two strains of two species), Roseivirga and Methylobacterium. These results showed a great diversity of culturable bacteria associated with marine flatworm Paraplanocera sp.
Three novel bacterial strains were discovered from this study. Two strains, UST20140214-052 and UST20140214-015B, had been characterized to be new species of the genus Pseudovibrio and published elsewhere [15,16]. In these years, there is a growing interest in Pseudovibrio species as more and more isolates have been identified from sponges, corals, and sea squirts and, among them, some strains have genomic interactions with host marine invertebrates and gene clusters for producing secondary metabolites to protect the host from pathogens [27]. Another new species strain, XY-FW106, was characterized to be Deinococcus planocerae [17]. Interestingly, XY-FW106 showed resistance against ultraviolet irradiation [28], which might provide some protective function for the survival of the host flatworm, which lived in the shallow waters. Table 1. The diversity of the culturable bacteria derived from the marine flatworm Paraplanocera sp. A total of 141 strains of bacteria, including 37 species of actinobacteria and 64 species of non-actinobacteria, were identified by comparison 16S rDNA sequences of the isolates with their reference strains in the GeneBank of NCBI. All the known isolates have high identity percentage values with 98.5-100% except the three new species (Pseudovibrio hongkongenesis UST20140214-015B [15], Pseudovibrio stylochi UST20140214-052 [16], and Deinococcus planocerae XY-FW106 [17]).

Species Isolate ID Accession Number of the Most Similar Strain Species Isolate ID Accession Number of the Most Similar Strain
Actinobacteria

The Bioactive and UPLC-MS Chemical Screening of the Isolated Bacteria
All 141 bacterial isolates were fermented on a small scale and the EA crude extracts of their bacterial fermentation broth were evaluated for anti-MRSA (strain ATCC43300) and cytotoxic (HeLa cells) activities. In total, eight extracts showed anti-MRSA activity and seven showed cytotoxic activity. Among them, extracts of strains XY-FW47 and XY-FW120 showed both potent antibacterial and cytotoxic activities. Four strains of Streptomyces showed the strongest anti-MRSA activity. In addition, one Arthrobacter soli strain and one Bacillus siamensis strain showed moderate activity against MRSA, and the latter strain also showed weak effects on HeLa cells (Tables 2 and 3). Two strains of Streptomyces revealed high activity against HeLa cells, while two other Streptomyces strains and one Paracoccus strain showed moderate activity against HeLa cells (Table 3). These results suggested that the bacteria associated with the marine flatworms might be a rich source of bioactive compounds. In this study, the strains of XY-FW47 and XY-FW120 were selected as the target strains for further chemical characterization due to their strongest bioactivities.  Further chemical analyses on the two strains of Streptomyces (XY-FW120 and XY-FW47) were proceeded by UPLC-MS on a reversed phase C18 column with a gradient solution ACN/H 2 O (5-95%, 25 min). As shown in Figure 4, XY-FW120 produced a main metabolite with a characteristic ultraviolet (UV) absorption of 430-440 nm and a high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) [M + H] + of 1255.6924. Combined database searching (Dictionary of natural products, SciFinder, AntiBase, MarinLit, etc.) with extensive literature searching suggested this metabolite was very likely to be actinomycin D (see Figure 4 and Figure S9) which had already been described by Meienhofer and Atherton [29]. As actinomycin D is a well-known antibiotic with antitumor activity, this also explains why the extract of XY-FW120 showed strong activity in both bioassays. It was also reported that the same species Streptomyces parvulus DAUFPE 3124 produced only actinomycin D [30]. However, the strain XY-FW120 produced not only a high level of actinomycin D, but also a few known actinomycin D analogs with trace amounts (data not shown). As the UV absorption patterns as well as the high resolution mass data of these actinomycin compounds were the same as those described in the literature, there is a very high chance that these compounds are exactly the same as those reported. However, there might be one case that our consumption could be wrong: enantiomers are also possible, although the odds are low. Based on these analyses, the metabolites of strain XY-FW120 are almost fully understood, thus we did not proceed for further investigation on this strain to characterize its metabolites. bioassays. It was also reported that the same species Streptomyces parvulus DAUFPE 3124 produced only actinomycin D [30]. However, the strain XY-FW120 produced not only a high level of actinomycin D, but also a few known actinomycin D analogs with trace amounts (data not shown).
As the UV absorption patterns as well as the high resolution mass data of these actinomycin compounds were the same as those described in the literature, there is a very high chance that these compounds are exactly the same as those reported. However, there might be one case that our consumption could be wrong: enantiomers are also possible, although the odds are low. Based on these analyses, the metabolites of strain XY-FW120 are almost fully understood, thus we did not proceed for further investigation on this strain to characterize its metabolites.  [19,32,33]. GM, previously isolated from Streptomyces hygroscopicus, is a benzoquinone antibiotic containing the typical structures of benzoquinone and dienamide. This class of compounds showed various bioactivities, including anticancer, anti-protozoa, antimalarial and antifungal activities [34,35]. Based on extensive investigation of the literatures and the analyses of UPLC-MS data, it is concluded that these compounds with HR-ESI-MS [M − H] − 547.2805 (retention time at 8.0 min), 533.2992 and 547.2636 (retention time at 12.0 min) are very likely novel GMs produced by the strain XY-FW47 (see Figure S10). As new analogs may provide more selectivity or low toxicity, it is worth isolating these potential new GMs. Consequently, this bacterial strain was chosen to be the target strain for further study. The 16S rRNA gene sequence of strain XY-FW47 was deposited in GenBank with the accession number MF664376.    [19,32,33]. GM, previously isolated from Streptomyces hygroscopicus, is a benzoquinone antibiotic containing the typical structures of benzoquinone and dienamide. This class of compounds showed various bioactivities, including anticancer, anti-protozoa, antimalarial and antifungal activities [34,35]. Based on extensive investigation of the literatures and the analyses of UPLC-MS data, it is concluded that these compounds with HR-ESI-MS [M − H] − 547.2805 (retention time at 8.0 min), 533.2992 and 547.2636 (retention time at 12.0 min) are very likely novel GMs produced by the strain XY-FW47 (see Figure S10). As new analogs may provide more selectivity or low toxicity, it is worth isolating these potential new GMs. Consequently, this bacterial strain was chosen to be the target strain for further study. The 16S rRNA gene sequence of strain XY-FW47 was deposited in GenBank with the accession number MF664376.
literatures and the analyses of UPLC-MS data, it is concluded that these compounds with HR-ESI-MS [M − H] − 547.2805 (retention time at 8.0 min), 533.2992 and 547.2636 (retention time at 12.0 min) are very likely novel GMs produced by the strain XY-FW47 (see Figure S10). As new analogs may provide more selectivity or low toxicity, it is worth isolating these potential new GMs. Consequently, this bacterial strain was chosen to be the target strain for further study. The 16S rRNA gene sequence of strain XY-FW47 was deposited in GenBank with the accession number MF664376.

Structural Elucidation of the Isolated Compounds
In total, four compounds were isolated and identified from strain XY-FW47. Compound 1 was isolated as a yellow amorphous powder. The molecular formula of Compound 1 was established as C 28 (Table 4), which were assigned with the assistance of the distortionless enhancement by polarization transfer (DEPT) spectrum to six methyls, four methylenes, seven methines, and nine quaternary carbons. The signals of 1 H-NMR spectra also revealed six methyl groups (δ H 3.41, 3.35, 1.91, 1.68, 0.98, and 0.96) and four methines. The gross structure of Compound 1 and all of the 1 H and 13 C NMR data associated with the molecule were determined by 2D NMR studies, including 1  H-10/CH 3 -26 and H-14/CH 3 -28, together with the HMBC correlations from CH 3 -22 to C-1/C-2/C-3, from OCH 3 -23 to C-6, from H-7 to C-8/C-9/C-24, from CH 3 -25 to C-8/C-9 and from CH 3 -27 to C-12, indicated the existence of the ansa ring (see Figure 6), which was similar to 17-O-demethylgeldanamycin [32]. Comparing with the 1D and 2D NMR signals of 17-O-demethylgeldanamycin, the lost of the double bond at C-4 and C-5 in Compound 1, which was replaced by two methenes. The NOSY correlations of H-6/H-7/H-12, and H-10/H-11/H-14 indicated the absolute configuration of Compound 1 that was also consistent with 17-O-demethylgeldanamycin. Thus, the structure of Compound 1 was identified.

Bioactive Evaluation and Structure-Activity Relationship
The bioactivities (anti-MRSA, anti-HeLa Cell and antifungal) of Compounds 1-4 were evaluated. These compounds showed no activity against MRSA. Compound 3 GM exhibited potent activity against HeLa cells with EC 50 1.12 µg/mL. It also showed antifungal activity against the plant pathogen Setosphaeria turcica with MIC 2.40 µg/mL. Previous cytotoxic tests (FRE/erbB-2 tumors) of 4,5-dihydrogeldanamycin (4) revealed much weaker activity than GM (IC 50 = 230 and 70 nM, respectively) [38]. Compounds 17-O-demethylgeldanamycin (2) and 17-O-demethylgeldanamycin hydroquinone showed much more cytotoxicity towards normal P19-derived neurons than GM at 1 nM [39]. These data together with the present results of Compound 1-4 indicate that the double bond at C-4,5 and the methoxy group at C-17 position are essential for increasing anti-HeLa cells and anti-S. turcica activities of GMs. As none of the GMs were active against MRSA, the potent anti-MRSA activity of the crude XY-FW47 extract might be contributed by the trace GMs or other types of metabolites which we were not able to isolate. We will need to optimize fermentation conditions of XY-FW47 to obtain enough quantities of these compounds in the future studies.

Bacteria Isolation and Identification
A marine flatworm was collected at depth between 1 and 3 m intertidal zone of Yung Shue O, Hong Kong (114 • 21 E, 22 • 24 N) in January 2014, and identified as Paraplanocera sp. based on 18S rRNA gene sequencing (1260 bp). The bacterial isolation method was in accordance with the method described by Xu et al. [15] using modified BD Difco TM R2A agar (adding 17 g/L seasalt). The 16S rRNA gene sequence of the isolations were determined by PCR using universal primers 27F and 1492R, then the isolates were identified by the blast program in the NCBI database.

Bacteria Fermentation
Small scale fermentation of the cultured bacteria was carried out as follows: seed cultures of the strain were collected in 50 mL Falcon centrifuge tubes, with each containing 15 mL of SGTYP medium with sea salts (5.0 g soluble starch, 5.0 g glucose, 1.0 g tryptone, 1.0 g yeast extract, 1.0 g peptone, 17.0 g sea salts per litre, pH 7.6 ± 0.2). Then fresh inoculum was inoculated in 250 mL flasks with each containing 80 mL of SGTYP medium with sea salts (5.0 g soluble starch, 5.0 g glucose, 1.0 g tryptone, 1.0 g yeast extract, 1.0 g peptone, 17.0 g sea salts per litre, pH 7.6 ± 0.2). The flasks were incubated at 28 • C for 5 days before harvesting. The fermentation broth was extracted with ethyl acetate (EA) three times of the total volume (1:3 v/v). The EA crude extract of the bacterial fermentation broth was obtained and prepared as 50 mg/mL stock solution in DMSO for testing.

Bioactive and Chemical Screening
The pathogen MRSA ATCC43300 was incubated in LB broth (10 g tryptone, 5 g yeast extract and 10 g NaCl per liter) at 28 • C for 12 h and then diluted 5000 times with fresh LB broth. The tested samples (2 µL) were added to each well of 24-well plates with 1 mL of the diluted pathogen solution. The pathogen was then incubated at 28 • C and the optical density at 600 nm was measured 24 h after inoculation with vancomycin (50 µg/mL) as positive controls. The cytotoxic assays were performed using the method described by Li et al. [40]. HeLa cells were inoculated and incubated in 24-well plates for 12 h before adding the tested samples. After incubation for 48 h, the CCK method was used to assay the cell viability. Three biological replicates were carried out for each sample and each bioassay experiment was repeated three times.
The ESI-TOF and mass spectra of the isolates were acquired from a UPLC-TOF-MS system (ultra-performance liquid chromatography-time of fly-mass spectrometry) using a Bruker microTOF-q II (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer coupled to a Waters ACQUITY UPLC system (Waters, London, UK).

Extraction and Compounds Isolation
Large scale fermentation (50 L) and the extraction of XY-FW47 were obtained as the described method of small scale. The EA crude extract of XY-FW47 was separated by reverse phase C18 chromatography with water and methanol solvent mixtures of H 2 O-MeOH  Table 4.

Bioactive Assays
The tests of Compounds 1-4 against MRSA ATCC43300 were determined as previously described. The antifungal activities against three plant pathogens (Setosphaeria turcica, Bipolaris maydis and Altemaria solani) were conducted on BD Difco TM Potato Dextrose Agar at 28 • C for 24 h. A series of two-fold dilution of tested samples was made with either LB broth or Potato Dextrose Broth in 24-well plates. The antibacterial tests were checked as the method described in the Section 3.3. The antifungal tests were performed at 28 • C for 24 h, and inhibition of ≥95% of the growth was observed by stereo microscope.
The cytotoxic tests were measured by using the previous method. Compounds 1-4 were prepared as 50 mg/mL in DMSO, and a series of two-fold dilution was made with the assayed media. After 48 h of incubation, the cytotoxicities were assayed by the CCK method.

Conclusions
This study firstly explored biological and chemical diversity of bacteria associated with the marine flatworm Paraplanocera sp. A total of 141 strains of bacteria were isolated including 45 strains of actinomycetes and 96 strains of other bacteria. Among them, there were three novel strains, suggesting that a rich biodiversity of bacteria may be associated with marine flatworms. The isolation and identification results of these bacteria indicated biological diversity and novelty of bacteria derived from this marine flatworm. One new GM analog (1) and three known GMs (2-4) were obtained from Streptomyces sp. XY-FW47. GM (3) showed potent bioactivity against HeLa cells with EC 50 1.12 µg/mL and against plant fungal pathogen Setosphaeria turcica with MIC 2.40 µg/mL. Preliminary discussion of SAR suggested that the existence of C-17 methoxy group and C-4,5 double bond might increase the bioactivities of GMs. Our study has provided new insights into the bacteria associated with marine flatworms.