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Article

Preliminary Findings on Antibacterial Activity of Selected Marine Invertebrates

by
Marina Brailo Šćepanović
1,
Jasna Maršić-Lučić
2,*,
Romana Beloša
3 and
Sanja Tomšić
1
1
Department of Applied Ecology, University of Dubrovnik, 20000 Dubrovnik, Croatia
2
Laboratory for Aquaculture, Institute of Oceanography and Fisheries, 21000 Split, Croatia
3
Independent Researcher, Od izvora 24, 20236 Nova Mokošica, Croatia
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(6), 3101; https://doi.org/10.3390/app15063101
Submission received: 2 January 2025 / Revised: 4 March 2025 / Accepted: 7 March 2025 / Published: 13 March 2025
(This article belongs to the Section Marine Science and Engineering)
Versions Notes

Abstract

:

Featured Application

Extracts of selected marine invertebrates show antibacterial activity and could be regarded as potential sources of novel antibiotic substances.

Abstract

Antibacterial resistance has become a major problem where new promising drugs are needed. The extracts obtained from marine invertebrates Mytilus galloprovincialis, Patella sp., Gibbula sp. and Arbacia lixula were tested against bacteria using the disc diffusion method. Citrobacter sp. from seawater and Paenibacillus sp., Bacillus sp. and Geobacillus sp. from soil were used as well as the reference bacterial strains Staphylococcus aureus NCTC 12981, S. aureus subsp. aureus Rosenbach ATCC 6538, Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Salmonella enterica subsp. enterica serotype Typhimurium NCTC 12023, Listeria monocytogenes ATCC 19111, Klebsiella aerogenes ATCC 13048 and Escherichia coli NCTC 12241. The most successful bacterial inhibitors, inhibiting 8 of 13 strains were extracts of M. galloprovincialis, Patella sp., Gibbula sp., Enteromorpha sp., C. sinuosa and U. lactuca, extract of A. lixula showed antibacterial activity against five bacteria, while extract of C. officinalis showed no antibacterial activity. These results indicate the potential of these marine organisms as a source of antibacterial compounds and may serve as a basis for further research and development of new antibacterial agents.

1. Introduction

Bacterial infections have always caused problems for humanity whilst the discovery of antibiotics revolutionized medicine, saving countless lives. Unfortunately, the unrestricted use of antibiotics and relentless microbial adaptability have led to an alarming increase in antimicrobial resistance. Antibiotic resistance has become the greatest threat to public health in the 21st century [1]. In addition, antibiotic-resistant infections can be difficult and sometimes impossible to treat, leading to high mortality [2].
Antibiotic resistance is responsible for nosocomial bacteria receiving more attention, with Methicillin-resistant Staphylococcus aureus being the most notorious. The most common infectious agents found in surgical wounds include S. aureus, coagulase-negative Staphylococcus, Enterococcus species and Escherichia coli [3], but the importance of Enterobacter has also been recognised; in addition, Enterobacter is a microorganism associated with cephalosporin resistance [4]. Additionally, special attention needs to be paid to foodborne diseases and illnesses caused by foodborne pathogenic bacteria that are increasing day by day and have become an important issue for various food industries [5]. Salmonellosis is one of the most common foodborne diseases in the world [6], while invasive listeriosis is life-threatening and is one of the major causes of foodborne diseases leading to hospitalisation in Western countries [7]. Other important foodborne pathogenic bacteria include Escherichia coli, Salmonella Typhimurium [8,9], Bacillus cereus, S. aureus [10] and Listeria monocytogenes [7,9,11].
Researchers are exploring alternative antimicrobial agents to develop new substitutes for existing antibiotics that can be used against multidrug-resistant strains [1]. The growing number of bacteria that are resistant to conventional antibiotics has become a major medical problem that has sparked great interest in the use of “natural” alternatives [11]. Novel natural compounds with antimicrobial activity can be obtained from underexplored habitats such as the oceans, as they represent the largest ecosystem on earth with a great diversity of organisms [2]. The marine environment is a wealthy source of plants, animals and microorganisms, which due to their adaptation to this unique habitat, produce a wide variety of primary and secondary metabolites that have demonstrated significant biological activities against, e.g., cancer and inflammation, as well as in analgesia, immunomodulation, allergy and antiviral assays [12].
Aquatic invertebrates, due to their high genetic richness, are a major source of marine natural products of social value, as they produce molecules (enzymes, biopolymers, bioactive compounds, secondary metabolites) that can find applications in various fields such as pharmaceutics, nutraceutics, cosmetics, antibiotics, antifouling products, biomaterials and more [13]. Mytimycins, cysteine-rich antimicrobial peptides that exhibit antifungal properties, have been isolated from mussels Mytilus sp. [14], while some gastropods are able to de novo biosynthesize defensive compounds or biotransform compounds obtained from their diet [15]. Sea urchins have also generated interest for their peculiar defence system which protects them against microbial infections and fouling [16]. Extracts isolated from celomoocites of the sea urchin Arbacia lixula could contribute to the protection of blood vessels against the development of early atherosclerosis and open up new perspectives for the development of drugs/supplements for the prevention and/or treatment of cardiovascular diseases [17].
The specific habitat where an organism is growing has an influence on the chemical nature of the marine primary and secondary metabolites. It is thus essential to study different strains of the same organism from various locations [12]. The biotechnological and biomedical potential of animals inhabiting the Adriatic Sea is still poorly understood. Apart from research on one coral [18] and several sponge species [19,20,21,22], information on bioactive compounds from invertebrates of the Adriatic Sea is hard to find. Keeping in mind that most of the research on marine natural products is concentrated on less than 1% of the invertebrate species [15] the aim of this study was to give attention to these particular organisms deepening the understanding of the biological processes and chemical interactions that take place in this specific environment. The goal of this research was to test common inhabitants of the littoral zone along the south-eastern Adriatic coast for the presence of metabolites with antibacterial activity.

2. Materials and Methods

2.1. Marine Organisms and Extract Preparation

The selected marine organisms were collected in the Gulf of Gruž, Dubrovnik, Croatia (42°39′47.4″ N; 18°04′39.0″ E). Four soft tissue samples of marine invertebrates were selected: Mediterranean mussel Mytilus galloprovincialis, limpet Patella sp., top snail Gibbula sp. and sea urchin Arbaca lixula.
The collected organisms were cleaned of epiphytes, washed with tap water and cut into small pieces. Then, the animal tissue was homogenised with ceramic beads in a Bead Ruptor Homogenizer (OMNI International, Kennesaw, GA 30144, USA). For extraction, 1 mL of a homogeneous mixture of animal samples was transferred to 2 mL microtubes. Extraction was performed in two ways, using one solvent method and with three solvents in combination. Single solvent extracts were obtained by adding 1 mL of 99.8% ethyl acetate (Gram-Mol, Zagreb, Croatia), or 99.7% methanol (VWR Chemicals, Leicestershire, UK) or a mixture of deionized water and methanol (50:50) to the microtubes. That was followed by shaking for 72 h at 1000 rpm at 35 °C in a thermomixer (uniTHERMIX 2 pro, LLG Labware, Meckenheim, Germany) and by 24 h of concentration by evaporation at room temperature. The procedure with combined solvents was performed by the addition of 1 mL of 99.8% ethyl acetate (Gram-Mol, Croatia) to tubes and the mixture was heated for 72 h at 1000 rpm at 35 °C in a thermomixer, followed by 24 h of concentration by evaporation at room temperature. Next, 1 mL of 99.7% methanol (VWR Chemicals, UK) was added to the same tubes and the samples were again placed in a thermomixer for 72 h at 1000 rpm and 35 °C and then concentrated by evaporation at room temperature for 24 h. Finally, 1 mL of a mixture of deionized water and methanol (50:50) was added to the tubes and the samples were again placed in a thermomixer for 72 h at 1000 rpm and 35 °C, followed by concentration by evaporation at room temperature for 24 h. The extracts thus obtained were analysed for their antibacterial potential.

2.2. Bacteria

The reference bacterial strains were obtained from the Public Health Institute of Dubrovnik-Neretva County, namely: Staphylococcus aureus NCTC 12981, Staphylococcus au-reus subsp. aureus Rosenbach ATCC 6538, Salmonella enterica subsp. enterica serovar En-teritidis ATCC 13076, Salmonella enterica subsp. enterica serotype Typhimurium NCTC 12023, Listeria monocytogenes ATCC 19111, Klebsiella aerogenes ATCC 13048 and Escherichia coli NCTC 12241.
In addition, bacteria were also isolated from environmental sources; a seawater sample and a sample of the coastal soil were taken at the marine organisms’ collection site. A tryptic glucose–yeast agar culture medium was used for bacterial growth. Mixed cultures were prepared from 1 mL seawater and 1 g soil suspended in 1 mL saline solution. After incubation of the agar plates at 37 °C for 48 h, pure bacterial cultures were isolated and incubated again in same conditions. Samples from pure cultures were subjected to species identification.

2.3. Disc Diffusion Method

Each bacterial sample was suspended in 1 mL saline and placed in a Petri dish containing autoclaved medium; the dish was mixed in a circular motion to ensure uniform bacterial growth and allowed to solidify. A 5 mm filter paper discs (sterilised for 24 h at 105 °C) were immersed in extracts (≈25 µL) and placed on a solid culture medium containing bacteria. The plates were incubated for 48 h at 37 °C and then the antimicrobial effect of the extracts was manifested as the inhibition of bacterial growth around the disc. Diameters of the inhibition zone (mm) were measured using a ruler starting from the edge of the filter disc.
First, a preliminary study was performed with extracts obtained using a single solvent method with solvents as controls. After obtaining these results the extracts prepared with three combined solvents were used for further analysis.

2.4. Sequencing of 16S DNA and Bacterial Identification

Platinum Direct PCR Universal Master Mix (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was used for the direct amplification of DNA from samples of pure bacterial cultures. A short lysis protocol suggested by the manufacturer was performed for each of the bacterial colonies. Each colony from the Petri dish was completely immersed in the lysis solution, incubated for 5 min at 22–24 °C and then incubated for 1 min at 98 °C. After incubation, the lysate was briefly centrifuged, and 1 μL of the supernatant from the lysate was used as a template to prepare the PCR reaction mixture for amplification of the 16S rDNA gene using the universal primer set OTU 27f (f5′-AGAGTTTGATCCTGGCTCAG-3′) and OTU1492r (r5′-GGTTACCTTGTTACGACTT-3′). For each sample, 20 μL of the reaction mix was used, which consisted of 10 μL 2× Platinum Direct PCR Universal Master Mix, 0.4 μL of each primer at a final concentration of 0.2 μM, 1 μL of the lysate supernatant and 8.2 μL of sterile nuclease-free water. Cycling conditions were followed according to the manufacturer’s instructions: 94 °C for 2 min, followed by 35 cycles of 94 °C for 15 s, 60 °C for 15 s and 68 °C for 20 s (Eppendorf, Mastercycler nexus GX2). The concentration and purity of the DNA obtained were determined by absorbance measurements using the NanoPhotometer (IMPLEN). The final PCR product was loaded into agarose gel. electrophoresed, excised and sent to Macrogene Europe (The Netherlands) for purification and sequencing. Each extraction replicate was sequenced as a single sample.
The 16SrRNA sequence was used for a similarity search using the SEQUENC_MATCH utility through Ribosomal Data Base Project II (https://bio.tools/rdp (accessed on 28 March 2024)) to determine the taxonomic identity for each sequence. Sequences that were ≥97% similar were grouped as a phylotype.

3. Results

3.1. Environmental Bacteria Identification

The list of bacterial strains from environmental sources determined by taxonomic affiliation using SEQUENC_MATCH is shown in Table 1. The bacteria derived from soil were classified as members of the genera Paenibacillus sp., Bacillus sp. and Geobacillus sp., while all seawater bacteria were identified as representatives of Citrobacter sp.

3.2. Antibacterial Activity of Marine Organisms

Methanol and water–methanol extracts did not show any antibacterial effect, and ethyl acetate extract showed an antibacterial effect against all bacterial strains; while only solvents used as controls produced similar results.
Diameters of the inhibition zone of the combined solvent extracts of selected marine organisms against environmental bacteria are shown in Table 2. Among these bacteria, all marine-derived strains of Citrobacter sp. were very sensitive, being inhibited by all four extracts, as was Bacillus licheniformis from the soil. The soil bacteria Geobacillus sp. was sensitive to two out of four extracts, while Paenibacillus cookii was not inhibited at all.
The results of the antibacterial activity of the combined solvent extracts of the selected marine organisms against the reference bacterial strains are shown in Table 3 as diameters of the inhibition zone. Among these bacteria, Listeria monocytogenes ATCC 19111 was the most sensitive, being inhibited by all four extracts. Next, both Salmonella enterica ATCC 13076 and S. enterica NCTC 12023 showed the same result, they were inhibited by three identical extracts. Interestingly, Staphylococcus aureus ATCC 6538 was inhibited by two of the four extracts, while S. aureus NCTC 12981 was not inhibited at all, like Klebsiella aerogenes ATCC 13048 and Escherichia coli NCTC 12241.

4. Discussion

This study provides the first comprehensive evaluation of antibacterial activity from extracts of marine invertebrates: limpets, snails, mussels and urchins, collected in the Adriatic Sea, revealing significant inhibitory effects on both environmental and reference bacterial strains. Furthermore, to our knowledge, this is the first study on the antibacterial activity of extracts from marine organisms against the members of soil-derived Paenibacillus sp., Bacillus sp. and Geobacillus sp. as well as of seawater Citrobacter sp. representatives (Table 1). The results show a very diverse sensitivity of the tested bacterial strains to combined solvent extracts from marine invertebrates, while those environmentally derived were more sensitive than the reference strains (Table 2 and Table 3). All three strains of seawater Citrobacter sp. were inhibited by all four extracts tested. The potent in vitro antibacterial activity could be due to the fact that all selected organisms have very limited mobility, which prevents their escape from predators, epiphytes or pathogens, so it is possible that they possess some chemical defence mechanisms; in fact, marine invertebrates, living within very difficult, competitive, and hostile surroundings represent a great reservoir for compounds with improved bactericidal activity [11]. It is important to point out that some members of Citrobacter sp. are multidrug-resistant [23] and are considered a growing threat to public health. They are frequently detected in neonatal illnesses, urinary tract infections and patients with severe underlying chronic diseases, including hypertension, diabetes, cancer and respiratory infections, or in immunocompromised patients [24]. The soil-associated Bacillus and Paenibacillus species are increasingly associated with various human diseases and are considered strains of increasing clinical relevance due to their virulence properties [25]. In addition, B. licheniformis was identified as a cause of recurrent sepsis [26], while Paenibacillus infections in infants are probably underestimated so effective treatments are urgently needed [27]. Unfortunately, the P. cookii tested in this study was not inhibited at all, but it is noteworthy that B. licheniformis was inhibited by all four extracts of marine invertebrates as members of Citrobacter sp. These results suggest that the selected organisms may be a promising source for further pharmacological research in the search of novel antibiotic substances. In this study, Geobacillus sp. was inhibited by three out of four extracts and interesting sensitivity results were found in the literature; these bacteria are generally antibiotic-sensitive but resistant to heavy metals [28].
The extract of M. galloprovincialis showed antibacterial activity against 8 of the 13 strains tested (Table 2 and Table 3). This is not surprising as their immune system is very effective and makes them particularly resistant to adverse conditions and pathogens [13]. When compared to the results of the present study, some differences can be found in the literature; for example, the protamine-like proteins of M. galloprovincialis exhibited bactericidal activity against E. coli (ATCC 8739), E. aerogenes (ATCC 13048), Proteus mirabilis (ATCC 7002), P. vulgaris (ATCC 12454), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (ATCC 19430), Enterobacter cloacae (ATCC 10699) and Klebsiella pneumoniae (ATCC 27736) as well as Enterococcus faecalis (ATCC 14428) and two strains of S. aureus (ATCC 13709, ATCC 6738) [11]. However, some authors indicated different results with respect to the use of different solvent extracts; the ethyl acetate extract showed the highest activity against E. coli and K. pneumoniae and no activity against S. aureus, while the glycerol–water extract showed inhibitory activity against S. aureus and E. coli but no effect against K. pneumoniae [29]. To our knowledge, this is the first record that L. monocytogenes and Salmonella enterica ATCC 13076 and S. enterica NCTC 12023 were inhibited by M. galloprovincialis. It is interesting to note that Salmonella enterica subsp. enterica serotype Typhimurium was found in big clams Callista chione but not in the mussel M. galloprovincialis when commercial mussels from the north coast of Morocco were analysed [6].
The extract from Patella sp. had an antibacterial effect and inhibited the growth of 8 of the 13 bacteria tested (Table 2 and Table 3), but had no effect on S. aureus ATCC 25923, K. aerogenes ATCC 13048 or E. coli NCTC 12241. Comparable results were found in the literature; extracts of P. rustica inhibited the growth of S. aureus, B. subtilis, S. typhi, E. feacalis, K. pneumoniae and P. aeruginosa, but had no effect on E. coli or Streptococcus pneumoniae [30]. It appears that this is the first evidence of inhibition of L. monocytogenes and Salmonella enterica ATCC 13076 and S. enterica NCTC 12023 by Patella sp. extract, making this organism a valuable source for potential studies on new antibiotic agents.
The extract from Gibbula sp. also showed inhibitory activity against 8 out of 13 bacteria (Table 2 and Table 3) and based on the available information, this is the first report of the inhibition of L. monocytogenes and Salmonella enterica ATCC 13076 as well as S. enterica NCTC 12023 by extracts from this snail. However, tetrodotoxin, a potent low-weight marine toxin, was found in gastropod species Gibbula umbilicalis [31], as well as some other biotoxins (paralytic shellfish toxin, okadaic acid and 13-Desmethyl Spirolide C) [32]. The results of the present study and those found in the literature could place this organism at the center of future research on new farmaceuticals from the marine environment.
Extract from Arbacia lixula showed an antibacterial effect on 5 out of 13 different bacterial strains (Table 2 and Table 3), but among the reference pathogens only on L. monocytogenes, in fact, this is the first notice of L. monocytogenes inhibition using an extract from this sea urchin. Information on its antibacterial effects is scarce; in a study of coelomic fluid and coelomocyte lysate antimicrobial activity on S. aureus, P. aeruginosa or Enterococcus sp., an inhibitory effect was not noticed [33]. However, it was found that A. lixula extracts demonstrate anticancer properties on triple-negative breast cancer cells [34], and that was a source of astaxanthin, which has particular bioactivity for the prevention of neurodegenerative diseases [35]; therefore, further research is highly recommended.
It is important to note that the results presented here are the outcome of the extraction procedure with combined solvents. Since only ethyl acetate extract showed an antibacterial effect, which may be attributed to the toxicity of the solvent itself, it steered the research into checking if combined solvent extracts (containing compounds with different polarities) would exhibit a different synergistic effect. During the procedure, only mixed solvents were not used as control (factoring in the potential toxicity of the solvents themselves), as it was concluded that the results would be the same for all bacteria/organisms. It is important to point out that the extracts of all four invertebrates inhibited the growth of L. monocytogenes, soil-origin B. licheniformis, and all three marine-origin members of Citrobacter sp., while three out of the four extracts showed an antibacterial effect against S. enterica ATCC 13076 and S. enterica NCTC 12023 and nearly all of these bacteria are pathogenic and multidrug-resistant; therefore, any substance that could reduce their activity should be welcomed. The marine invertebrates tested in this research are common inhabitants of the Adriatic Sea and their abundance and easy accessibility could be a fundament to their exploitation for medical purposes.

5. Conclusions

Extracts derived from M. galloprovincialis, Patella sp., and Gibbula sp., inhibited 8 of the 13 bacteria each, making it very difficult to determine the most effective one, but it should be noted that the highest number of wider inhibition zone diameters was recorded for the extract of Gibbula sp. Some new interactions between marine invertebrates and both the environmental and the reference bacteria were described for the first time in this research. Therefore, a more profound study of the antimicrobial effect of extracts from these marine invertebrates is suggested as well as the identification of the active compounds in order to elucidate the potential mechanism of action of the antibacterial effect shown in this study.

Supplementary Materials

The sequences of bacteria obtained from the environment can be downloaded from GenBank with the following accession numbers: PP549971, PP549972, PP549973, PP549974, PP549975 and PP549976. https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 28 March 2024).

Author Contributions

Conceptualization, M.B.Š. and S.T.; methodology, M.B.Š. and J.M.-L.; formal analysis, M.B.Š., J.M.-L. and R.B.; investigation, M.B.Š., J.M.-L. and R.B.; data curation R.B. and S.T.; writing—original draft preparation, R.B., J.M.-L. and M.B.Š.; writing—review and editing, S.T. and J.M.-L.; visualization, J.M.-L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data supporting the findings of the study can be obtained from the corresponding author upon reasonable request.

Acknowledgments

The authors thank the colleagues from the Public Health Institute of Dubrovnik-Neretva County for providing the reference bacteria.

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. List of bacterial strains obtained from environmental sources (supplementary materials, https://www.ncbi.nlm.nih.gov/genbank/, accessed on 28 March 2024).
Table 1. List of bacterial strains obtained from environmental sources (supplementary materials, https://www.ncbi.nlm.nih.gov/genbank/, accessed on 28 March 2024).
SourceBacteriaGenBank Accession Number
Soil originPaenibacillus cookii strain L1.2PP549971
Bacillus licheniformis strain MA-42PP549972
Geobacillus sp. HBB272PP549973
Marine originCitrobacter sp. strain YB074PP549974
Citrobacter freundii strain EF55PP549975
Citrobacter sp. strain EB-C-4PP549976
Table 2. Diameters of the inhibition zone (mm) of extracts from marine invertebrates against environmental bacteria.
Table 2. Diameters of the inhibition zone (mm) of extracts from marine invertebrates against environmental bacteria.
Marine Invertebrates
BacteriaPatella sp.Gibula sp.Mytilus galloprovincialisArbacia lixula
Paenibacillus cookii strain L1.20000
Bacillus licheniformis strain MA-422231
Geobacillus sp. HBB2720310
Citrobacter sp. strain YB0746545
Citrobacter freundii strain EF554443
Citrobacter sp. strain EB-C-44533
Table 3. Diameters of the inhibition zone (mm) of extracts from marine invertebrates against reference bacteria.
Table 3. Diameters of the inhibition zone (mm) of extracts from marine invertebrates against reference bacteria.
Marine Invertebrates
BacteriaPatella sp.Gibula sp.Mytilus galloprovincialisArbacia lixula
Escherichia coli0000
Klebsiella aerogenes0000
Staphylococcus aureus0000
Staphylococcus aureus subsp. aureus Rosenbach 1000
Salmonella enterica subsp. enterica serovar Enteritidis1210
Salmonella enterica subsp. enterica serotype Typhimurium11.510
Listeria monocytogenes33.542
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Brailo Šćepanović, M.; Maršić-Lučić, J.; Beloša, R.; Tomšić, S. Preliminary Findings on Antibacterial Activity of Selected Marine Invertebrates. Appl. Sci. 2025, 15, 3101. https://doi.org/10.3390/app15063101

AMA Style

Brailo Šćepanović M, Maršić-Lučić J, Beloša R, Tomšić S. Preliminary Findings on Antibacterial Activity of Selected Marine Invertebrates. Applied Sciences. 2025; 15(6):3101. https://doi.org/10.3390/app15063101

Chicago/Turabian Style

Brailo Šćepanović, Marina, Jasna Maršić-Lučić, Romana Beloša, and Sanja Tomšić. 2025. "Preliminary Findings on Antibacterial Activity of Selected Marine Invertebrates" Applied Sciences 15, no. 6: 3101. https://doi.org/10.3390/app15063101

APA Style

Brailo Šćepanović, M., Maršić-Lučić, J., Beloša, R., & Tomšić, S. (2025). Preliminary Findings on Antibacterial Activity of Selected Marine Invertebrates. Applied Sciences, 15(6), 3101. https://doi.org/10.3390/app15063101

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