Seasonal Variation in Antimicrobial Activity of Crude Extracts of Psammaplysilla Sp. 1 From Phillip’s Reef, South Africa


 Marine invertebrates constitute a diverse group of marine organisms beneficial to humankind due to their therapeutic significance. The marine sponge species Psammaplysilla sp. 1 was collected from Philips Reef, South Africa, over a four-season period and assayed for antimicrobial potential. The physicochemical parameters of the collection site were also recorded. The sponge crude extracts' antimicrobial activity was evaluated using an agar well diffusion assay against five pathogens. Phytochemical screening was conducted to identify the presence of 7 critical phytochemical groups. During the four seasons, the temperature was 17.35 oC ± 2.06, with autumn recording the highest (20 oC) temperature. Antifungal activity was observed by Psammaplysilla sp. 1 (30 mm) against C. albicans, and this was higher than that shown by standard drugs ICZ-10µg (15 ± 0.1 mm), FLU-15µg (21 ± 0.2 mm), VCZ-5µg (17 ± 0.1 mm) respectively. Similar bioactivities were observed seasonally with Psammaplysilla sp. 1 (22 mm and 24 mm) during autumn and spring, respectively, against C. difficile, while only crude extracts collected in spring showed bioactivity against C. albicans. Psammaplysilla sp crude extracts showed broad-spectrum bioactivity against all test pathogens. DCM:ME crude extracts tested positive for the presence of 2/7 of the phytochemicals (terpenoids and flavonoids). GC-MS revealed several previously reported biologically active compounds such as Bicyclo[4.2.0]octa-1,3,5-trien-7-ol and Phenol, 2,6-dibromo has been found in plants. This study revealed that sponge bioactivity is dependent on the season and further validated the antimicrobial potential of South African marine sponges.


Background
Nature has been identi ed as a good source of vast medically signi cant compounds for many years [1].
In the last decades, problems with antibiotic-resistant microbes have emerged, with an increased evolution of complex diseases, especially amongst immunosuppressed individuals [2]. Therefore, there is a need to discover and develop novel and extremely potent antimicrobial agents as conjugates or alternatives to existing antimicrobial therapy that cannot be overemphasized. The marine environment provides a possible source of antimicrobial compounds due to its vast diversity, with its organisms representing approximately 80% of the world's biota [3]. Marine sponges belonging to the phylum Porifera [4] are signi cant animals that have been reported to be a source of unique natural products ranging from food, fragrances, pigments, enzymes, inhibitors, insecticides, and essential medicines such as antibacterial, anti-cancer, antiviral, and antifungal agents [6]. Approximately 10,000 pharmacologically bioactive compounds were successfully reported from marine invertebrates such as tunicates, sponges, soft corals, sea hares, nudibranchs, bryozoans, and sea slugs [7]. Bioactive metabolites obtained from the sponge genera Helicona, petrosia, and Discodema were potent anti-cancer and anti-in ammatory agents. To further support the antimicrobial potential observed from sponges in previous studies, Lakshimi et al. a Whatman No.2 lter paper. The ltrates were concentrated using a rotary vacuum evaporator (Lasec Steroglass, Strike 202) at 40 o C at 300 rpm. The resulting crude extract residues, approximately 1 ml each per 50 ml of evaporated solution was stored at 4 o C for further analysis.

Test organisms/growth
Commercial strains of bacteria, methicillin-resistant Staphylococcus aureus, ATCC 49476, and Pseudomonas aeruginosa ATCC 10145 were resuscitated on Brain Heart Infusion (BHI) agar (oxoid, UK) at 37 o C for 24 hours. Clostridium di cile ATCC 9689 was resuscitated on BHI supplemented with 5 mg ml − 1 of yeast extract and incubated at 37 o C for 48 hours microaerophlically [11]. Fungal species of Candida albicans ATCC 10231 and Aspergillus fumigatus (ATCC 204305) were cultured on Potato dextrose agar (PDA) and incubated at 30 o C for three to ve days [15].

Antimicrobial assays
The crude extracts' antibacterial activity was evaluated using the standard agar well diffusion assay on Muller Hinton Agar (MHA) with slight modi cations (Selvin and Lipton, 2004). Inoculums of the test bacteria, methicillin-resistant S. aureus ATCC 49476, C. di cile ATCC 9689, and P aeruginosa ATCC 10145, were prepared by transferring colonies from an overnight culture into 0.9% normal saline and turbidity adjusted to 0.5 McFarland standards (the equivalent of one to two × 108 CFU ml − 1 ). Two hundred and fty microliters of each adjusted bacteria suspension were introduced into 500 ml of MHA (oxoid, UK), which was cooled to about 40 o C post sterilizations. The mixture was poured into Petri dishes and allowed to solidify. Wells of six mm diameter were punched into the MHA plates containing test bacteria using a sterile cork borer. Crude extract concentrations of 80% (v:v) were prepared. Experiments were done in duplicates for all crude extracts obtained using ethyl acetate and dichloromethane: methanol (1:1) (v:v).
Twenty microliters of each of the extracts were dispensed into different wells, and a negative control well was lled with the corresponding solvents. Antibiotic disks were also included as positive controls in the assays: vancomycin, VN (30 µg), clindamycin, CD (10 µg), trimethoprim. TM (5 µg), rifampicin, RP (5 µg) and amoxicillin, AMX (25 µg) was tested against MRSA, imipenem, IMP (10 µg), cipro oxacin, CIP (5 µg), and meropenem, MEM (10 µg) against P. aeruginosa and metronidazole, MNZ (5 µg), tetracycline, TE (30 µg) and clarithromycin, CLR (15 µg) were tested against C. di cile. S. aureus and P. aeruginosa inoculated plates were incubated at 37 o C for 24 hours while those of C. di cile were incubated at 37 o C for 48 hours microaerophilically. The resulting diameters of the zones of inhibition were measured in millimeters. The antifungal property of different extracts against the test fungi, C. albicans (ATCC 10231) and A. fumigatus (ATCC 204305), were determined as previously reported by [16]. However, the media used was PDA. Negative controls were wells lled with corresponding solvents while the antifungal agents, uconazole, FLU (15 µg), Itraconazole ICZ (10 µg), and Voriconazole, VCZ (5 µg) were included as positive controls. Incubation was done at 37°C for three to ve days [17]. All assays were done in triplicates, with a total dry weight of 24.5 mg being added into 1 ml of the parent extraction solvent to create a 24 mg/ml stock solution. The quantity 24 mg/ml was selected as it was the average mass of the crude extract powder, which was obtained following rotary evaporation.
Phytochemical screening of bioactive crude extracts Crude extracts that exhibited antimicrobial activity against the test bacteria and fungi were tested for phytochemical compounds such as avonoids, tannins, terpenoids, phenolic compounds, and saponins, and alkaloids.

Detection of Flavonoids
Flavonoids were detected using the Juglone test method as described by Sofowara [18]. Diluted ammonia solution was added to crude ltrate followed by 1 ml of concentrated sulphuric acid, and 2 ml of potassium hydroxide solution was added and allowed to mix. 1 ml of aqueous ltrate of the sample was added before being observed for visible color changes.

Detection of Tannins
Detection of tannins was carried out as reported by Iyengar [19]. A few drops of 0.1% ferric chloride were added to 0.5 ml of crude ltrate and observed for brownish green or a blue-black coloration which indicated the presence of tannins.

Detection of Terpenoids
The Salkowski test was used to detect terpenoids, as reported by Kiran et al. [20]. To a volume of 5 ml of crude ltrate, 2 ml of chloroform was added, followed by 3 ml of concentrated sulphuric acid being added. A reddish-brown coloration of the interface indicated the presence of terpenoids.

Detection of Phenolic compounds
The method reported by Mace [21] was followed. A volume of 2 ml of the crude ltrate and alcohol was mixed with a few drops of neutral ferric chloride (pH 7) solution. A dark green color indicated the presence of a phenolic group.

Detection of Saponins
Saponins were detected using the method described by Venkatesh et al. [22]. In this test, 0.5 ml of latex was dissolved in 5 ml of distilled water in a test tube. The solution was shaken vigorously and observed for a stable, persistent froth with a honeycomb structure.

Detection of Anthraquinones
The crude ltrate (2.5 ml) was shaken with 5 ml of benzene and 2.5 ml of 10% ammonia solution. A pink, red, or violet color indicated the presence of anthraquinones, as reported by Mujeeb et al. [23].

Detection of Alkaloids
Samples containing alkaloids were identi ed using the method described by Evans [24]. A volume of 2 ml of 1 M hydrochloric acid was added to 1 ml of the crude ltrate. A few drops of Mayer's reagent (Mercuric chloride 1.36 g, potassium iodide 5.0 g dissolved separately, and the volume made up to 100 ml with distilled water) were added to the test tube. A white or creamy precipitate indicated a positive result for Alkaloids.
Gas chromatography -Mass spectrum analysis GC-MS (Shimadzu UV-1800) with NIST library was used to identify the compounds present in the extract. A volume of 3µl microliters of the EA extract was injected into the GC-MS using a microsyringe. The signals obtained because of the compound elution from the Gas chromatography into the detector represented as peaks. The intensity of the signals and retention time were measured. The retention indices and mass spectra patterns obtained from the detected compounds were compared with those already documented in the NIST library for identity assignment [25].

Statistical analysis
Means of zones of inhibition obtained from the bioactivity assays were derived and the standard deviations calculated using Microsoft Excel. A two-way Analysis of Variance (ANOVA) was carried out to determine if there were any statistical differences between the mean zones of inhibition of the ethyl acetate and dichloromethane: methanol crude extracts of Psammaplysilla sp collected during the four seasons (Winter, Summer, Autumn, and Spring) against the pathogens of interest. The GraphPad Prism version 7.04 (Prime 7 for Windows) Microsoft Software was used for this analysis. The signi cance value was set at p < 0.05.  (Table 1).  Taxonomic identi cation of marine species

Results And Discussion
The collected specimen was identi ed as Psammaplysilla sp. 1 based on morphological appearance. Analyzed specimens were deposited at the Department of Environmental Affairs Oceans and Research facility in Cape Town, South Africa.
Antimicrobial susceptibility-based evaluations against P. aeruginosa For activity against P. aeruginosa (Fig. 3), no bioactivity was obtained during winter and summer. The highest inhibition zones were recorded with DCM:ME spring crude extracts of Psammaplysilla sp. 1 (26 mm) as seen in Fig. 3, followed by crude extracts obtained from the autumn collection (20 ± 03 mm) while ethyl acetate crude extracts recorded lower inhibition zones (16 ± 0.2 mm). Signi cant differences in the mean zones of inhibition (p = 0.0127) were observed against P. aeruginosa for collections from all four seasons. The antibiotic Cipro oxacin (30 ± 0.3 mm) performed slightly better than DCM:ME crude extract collected in spring (26 mm).
Antimicrobial susceptibility-based evaluations against C. di cile Slightly similar bioactivities were observed seasonally with Psammaplysilla sp. 1 (22 mm and 24 mm) during autumn and spring, respectively, against C. di cile (Fig. 4). A p-value of 0.1342 was obtained for samples collected during all four seasons against C. di cile. Thus, illustrating no statistically signi cant differences in the bioactivity pro les of specimens collected during autumn and spring. These results could also further imply that both EA and DCM:ME crude extracts collected in spring and autumn are similar in terms of e cacy.
Antimicrobial susceptibility-based evaluations against A. fumigatus Only Psammaplysilla sp 1 EA crude extracts collected during autumn and spring showed marked activity against A. fumigatus. Inhibition zones of 31 ± 0.1 mm and 16 mm in autumn and spring, respectively (Fig. 5). Crude extracts of DCM:ME collected in all four seasons showed no activity against A. fumigatus. EA crude extracts obtained in autumn showed more signi cant bioactivity than all the standard antifungal drugs ICZ (16 ± 0.8 mm), FLU, 15 µg (24 ± 2.1 mm), and VCZ, 5 µg (21 ± 0.2 mm), as seen in Table 1. Statistically signi cant differences were observed in EA and DCM:ME bioactivity potentials of crude extracts collected in spring and autumn against A. fumigatus (p = 0.0164). This result proves that seasons may play a role in sponges producing bioactive metabolites.
Antimicrobial susceptibility-based evaluations against C. albicans Only crude extracts collected in spring showed bioactivity against C. albicans, as seen in Fig. 6. EA and DCM:ME crude extracts of Psammaplysilla sp recorded zones of inhibition of 30 mm and 16 mm, respectively. Psammaplysilla sp EA crude extracts bioactivity was higher than those recorded for the standard antifungal agents; ICZ (15 ± 0.1), FLU, 15 µg (21 ± 0.2), and VCZ, 5 µg (17 ± 0.3). Statistically signi cant differences (p = 0.041) were observed in the mean zones of inhibition of crude extracts tested against C. albicans.
Phytochemical screening crude extracts Psammaplysilla sp. 1 that was tested for antimicrobial properties was also tested for the presence of seven (7) commonly reported phytochemicals. These phytochemical classes were also selected due to their antimicrobial properties. DCM:ME crude extracts tested positive for the presence of 2/7 of the phytochemicals (terpenoids and avonoids), while EA crude extracts tested positive for 1/7 of the phytochemicals (saponins), as seen in Table 3.

Discussion
The vast nature of the marine environment favors a great deal of ecological diversity. Such ecosystems are home to a great diversity of important marine species such as sponges that have been reported to produce structurally diverse metabolites. Biological diversity is due to many physical and chemical parameters such as pH, temperature, salinity, and turbidity (Hamed et al., 2015). Differences in these parameters exert a driving force on the adaptive survival strategies, which lead to the synthesis of new metabolites [1]. As of 2014, approximately 30% of all marine natural products were isolated from marine sponges [51]; because of this, the primary interest in discovering new antimicrobials is tied to marine sponge research. Based on the results obtained from this study, turbidity recorded during spring could have a role in the antimicrobial activity observed against all test pathogens, as seen in Figs. 2, 3, and 5. According to the South African Water Quality Guidelines and WHO, a turbidity reading > one NTU indicates contaminated seawater [26]. It can therefore be concluded, as reported by Zasloff et al. [27], that the contamination could cause increased microbial pathogen interaction with the sponge species and, as a result, yielded high synthesis of diverse secondary metabolites, particularly during spring with high bioactivities. The same could not be accounted for in winter as no bioactivity was observed during that season. However, such observation requires further validation as no studies have looked at the correlation between environmental parameters such as pH, temperature, turbidity, and salinity and the bioactivity potential of marine sponge.
Our crude extracts were tested for the presence of seven (7) commonly reported phytochemicals. Terpenoids and avonoids were found in DCM:ME crude extracts, while EA crude extracts tested positive for the presence of saponins only. A study reported by Govinden-Soulange et al. [28] on two Mauritian sponge species Stylissa spp and Biemna tubulosa, EA crude extracts contained saponins. Both sponges showed bioactivity against S. aureus of which our collected sponge samples also showed bioactivity against MRSA. In another study by Fouad et al. [29], it was observed that saponins obtained from marine sponge crude extracts only showed bioactivity against C. albicans and not MRSA. Calabro et al. [30] further reported the same ndings as their marine sponge Poecillastra compressa from the Mediterranean Deep-Sea contained saponins that only showed antifungal properties when tested against A. fumigatus. In our study, both antibacterial and antifungal activity was observed in the ethyl acetate crude extract, which contained saponins. Therefore, our ndings contrast with those observed by the previous two studies. Our ndings were further in agreement with the ndings reported by Warad et al. [31] which the marine sponge Callyspongia diffusa crude extracts contained avonoids that showed bioactivity against C. albicans. Our Psammaplysilla sp. 1 crude extracts showed broad-spectrum bioactivity. These crude extracts were bioactive against both bacterial and fungal species, thus illustrating the e cacy or potential of the metabolites present within the sponge compared to the previously reported studies. Kim et al. [32] reported crude extracts found Psammaplysilla sp. 1 possessed bactericidal activity in Staphylococcus aureus strains (MRSA). Their study further reported the inactivity of some of the extracts against different Gram-negative bacteria strains such as Escherischia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Klebsiella oxaytoca, Enterobacter cloacae, and Citrobacter freundii. Our study observed different results as bioactivity was observed in Pseudomonas aeruginosa (DCM: ME-26 mm). These results could be due to our sample containing different bioactive metabolites within the same phytochemical class.
The bioactivity assays results obtained (Figs. 2-6) from this study revealed that marine sponge species can produce highly bioactive compounds effective against pathogenic microbes, as previously reported by Putra et al. [5]. Sponges have been noted to produce a broad array of marine natural products [33] and possess signi cant chemical diversity [7,34]. These ndings can be further supported by the results of the crude extracts of Psammaplysilla sp (35 ± 0.4 mm), which performed better than control antimicrobials tested against three bacterial species (MRSA, P. aeruginosa, and C. di cile) and two fungal species (A. fumigatus and C. albicans). In our study, it was observed that during the winter and summer seasons, no bioactivity was observed against the test microorganisms except for the spring and autumn seasons. This study is the rst to report complete seasonal evaluations of the antimicrobial potentials of Psammaplysilla species. No existing data was available on the seasonal bioactive pro les of Psammaplysilla. However, for other sponge's experimental data was available.
A study reported by Page et al. [35] revealed that wild M. hentscheli (sponges) exhibited a variation in the concentrations and e cacy of bioactive compounds (mycalamide, pateamine, and peloruside A) from different geographic regions and at different times of the year, thus illustrating that seasons do play a role in the different bioactivity pro les of marine sponges across various geographic locations. Devi et al. [36] from sponges Clathria sp and Axinella sp collected in South and East India during summer revealed no bioactivity for crude extracts of both species tested against P. aeruginosa and MRSA. The same observation was made with this study's crude extracts collected in summer as no bioactivity was recorded against MRSA and P. aeruginosa. In another study done by Cita et al. [3] on East Java, Indonesian sponges were collected during the rainy season. Their study revealed a different outcome as the same species (Axinella sp and Clathria sp) showed bioactivity against MRSA and P. aeruginosa as zones of inhibition of 15 ± 0.2 and 7 ± 0.21 were obtained, respectively. These ndings mean that different biogeographic locations' different seasons result in different marine sponges' bioactivity potentials being reported. In addition to this, our collected species were only bioactive during spring and autumn against P. aeruginosa, which are opposite seasons to those reported by Cita et al. [3] and Devi et al. [36].
Upon GC mass-spectrometric analysis, several bioactive compounds previously reported were found in the crude extracts. Khatua et al. [37] isolated 2-Heptacosane and Methyl 11,12-octadecadienoate from Trichosanthes dioica root extract, and it showed antibacterial activity against Proteus mirabilis and Bacillus subtilis. A study reported by Pangal et al. [38] revealed that tert-Butyl-p-benzoquinone has antibacterial properties against E. coli and S. aureus. Even though these two studies reported antibacterial activity against different bacterial species than those used in our study, this further con rms the broadspectrum antibacterial properties exhibited by Psammaplysilla sp 1. The compounds found in the sponge crude extract were like those reported to be found in plant extracts. Kumar and Pal [39] made a similar observation when they found that D-Phytosphingosine, a corresponding 4-hydroxy analog of Dsphinganine is a signi cant sphinganoid base found in both higher plants and many invertebrates.
Our study indicates that seasons may play a role in the variation of bioactivity potential of sponge species. These observations were also observed even for the same sponge species because of different geographical locations. Kanagasabhapathy et al. [40] reported that Psammaplysilla sp collected from the Gulf Mannar in India showed bioactivity against P. aeruginosa (11 mm). This bioactivity was much less than the bioactivity results obtained in or study. The highest bioactivity recorded was 26 mm from a DCM:ME crude extract collected during spring. Our crude extracts e cacy was slightly less than trimethoprim (5 µg) 28 ± 1 mm. These nding agree with the study done by McClintock and Gauthier [11], which reported that most orders of Demosponge show signi cant bioactivity even though bioactivity varied even amongst the same species located in different regions. Green et al. [41] further explained that the variations reported in different studies were due to the different methodologies used for extraction and geographical locations. Our study further agrees with Thompson et al. [42], which revealed that the season has a role in the bioactivity potentials of sponge species.
Therefore, it is inevitable that the season might have a direct role in the bioactivity potentials of sponge species. The biogeographical location further in uences the chemical diversity and presence of bioactive compounds. Secondly, both ethyl acetate and dichloromethane: methanol (1:1) have comparable bioactive metabolite extraction potentials from Psammaplysilla sp. 1 tissue, and therefore are both recommended extraction solvents for future studies. Nevertheless, more studies are needed to validate further these ndings concerning Psammaplysilla sp. 1 as this study is the rst to report seasonal bioactivity of this species. Our study also validated the antimicrobial potential of South African marine sponges against infectious pathogens, as previously reported by Zoraghi et al. [43] and Veale et al. [44].
Davies-Coleman et al. [52] reported that South Africa has enormous biodiversity of marine sponges. Many of them also produce a broad diversity of biologically active compounds that can also be used for different pharmaceutical applications, such as anti-cancer drugs [52,53]. This study in the future will investigate the anti-cancer and antiviral properties of Psammaplysilla sp. 1 based crude extracts and the possible application in drug delivery systems.

Conclusion
Our study revealed that selected marine invertebrates such as sponges synthesize biologically active compounds against bacterial and fungal pathogens. The study has also identi ed Phillip's Reef in Algoa Bay Port Elizabeth as a perfect environment worth exploring for the discovery of marine natural products for antimicrobial application, with spring and autumn being the best seasons for the collection of marine species for bioactive compounds isolation. This study's scienti c approach was different from previous studies done in South Africa as most look at the antimicrobial potential of microbial ora found on the sponges. While those that look at the sponge matrix seek to isolate bioactive compounds for anti-cancer, antioxidant, and anti-in ammatory properties, a positive correlation can be made between the bioactivity potentials of certain sponge species with the season. A new sponge species was successfully identi ed as Psammaplysilla sp. 1. Psammaplysilla sp. 1 is an excellent source of potential antibacterial agents for treating bacterial and fungal-related infections as this species showed broad-spectrum bioactivity. This study further recommends evaluating the bioactive crude extracts for the constituents present in the  Figure 4 Antibacterial activity of Ethyl acetate (EA) and Dichloromethane: Methanol (DCM:ME) crude extracts against C. di cile