Evaluation of antimicrobial activities of powdered cuttlebone against Klebsiella oxytoca, Staphylococcus aureus, and Aspergillus flavus

Background: The presence of medicines in the environment is considered as a serious threat to the human health. The entrance of these substances into the water sources causes soil pollution, which eventually leads to the environmental pollution and it creates some problems for the public health. Also, increasing antibiotic resistant bacteria has attracted the attention of researchers to the use of natural resources such as marine products, for producing new antibiotics. The aim of this study was to evaluate antimicrobial activities of powdered cuttlebone against Klebsiella oxytoca, Staphylococcus aureus, and Aspergillus flavus. Methods: At first, cuttlebones were washed, dried, and powdered. Then, the powdered cuttlebone was characterized. In the next step, its antimicrobial activities were evaluated using agar well diffusion technique, and minimum inhibitory concentration (MIC) was calculated. Results: The powdered cuttlebone was found to be effective against K. oxytoca (24 mm, MIC: 10-1 mg/mL), but no antimicrobial response was found against S. aureus. Also, the powdered cuttlebone antifungal activity and MIC against A. flavus were recorded 23 mm and 10-1 mg/mL, respectively. Conclusion: The obtained results suggest antimicrobial activities of powdered cuttlebone, which are concentration dependent. Furthermore, cuttlebone can be used as an accessible natural source to provide novel, low cost, and safe antimicrobial agents.


Introduction
Staphylococcus aureus is a gram positive and roundshaped bacterium, which is a member of Firmicutes species. S. aureus is the leading cause of human infections, which have a wide range in the environment and food sources. It can be transferred through food contact surfaces, hands, air, dust, etc. It typically causes skin infections, pneumonia, endocarditis, and osteomyelitis (1). Klebsiella oxytoca is one of several Klebsiella bacteria. Klebsiella spp. are ever-present in the environment. They could isolate from the soils, plants, and water surfaces. These bacteria are naturally found in the intestinal tract, mouth, and nose. Klebsiella spp. are opportunistic gramnegative, non-motile, and rod-shaped bacteria with a prominent polysaccharide capsule which belong to the Enterobacteriaceae family. K. oxytoca is considered as an opportunistic pathogen and is recognized as a clinically significant nosocomial infection in children and neonates (2).
Aspergillus flavus is an opportunistic pathogen in immunosuppressed patients, which is found in the soils and causes diseases in the agricultural products. A. flavus is the major producer of aflatoxin. Aflatoxins are the second strong poisonous metabolites, which infect agricultural products and make serious threat to both humans and livestock health (3).
Due to the effects of pathogenic bacteria and toxinproducing fungi on the human health, it seems necessary to control them. The growth of dangerous bacteria and fungi is usually limited using chemical and synthetic preservatives. These materials have some side effects such as teratogenic and carcinogenic effects. The mentioned issues make concerns for the health officials. Today, because of the outbreak of new infectious diseases and antibiotic-resistant bacteria, it is necessary to use novel antimicrobial compounds with various chemical formations which have new action mechanisms. These reasons have forced researchers to look for new antibiotics from various renewable natural sources such as marine origin materials (4,5), citrus extract (6), natural oils (7), medicinal herbs like ginger (8), garlic (9), etc.
The antimicrobial activities studies supply valuable information about antibiotic discoveries, and also, provide a new insight into the extraction of bioactive compounds from natural sources (10). One of the natural resources that is considered these days is Mollusca marine animals. Cuttlefish is one of the Mollusca marine animals that belongs to the class Cephalopoda and order Sepioidea (11).
Recent studies have shown that cuttlefish is among the most intelligent species of invertebrates. They are nocturnal and hunt crabs and shrimps in the night. Cuttlefish have a unique internal shell, which is called cuttlebone and exists in all members of Sepiidae family. In the native dialect, it is called seabed (Figures 1a and b).
Cuttlebone is a hard brittle internal structure with high porosity in the back of the cuttlefish body, which is oval shaped and spongy. In fact, cuttlebone is a porous internal shell that is made primarily of aragonite. It is a rigid buoyancy tank and functionally similar to swim bladder in fishes. It plays a key role in the protection of vital organs. Cuttlebone is made up of two parts: Organic part (protein and β-chitin) and inorganic part (calcium phosphate, sodium, magnesium, phosphorus, and mineral salts). Actually, cuttlebone is a marine product that contains no toxins or contaminants (12)(13)(14)(15)(16).
Due to the unique structure, cuttlebone has many pharmaceutical and industrial applications. It can be applied in the treatment of bleeding and control of external infections. Also, it can be used as a low-cost and nontoxic adsorbent in dyes, toxic elements, and removal of heavy metals from water and wastewater (17,18). Its other applications include adjusting the bird's liver and kidney function, oil spill clean-up, healing of indomethacininduced acute gastric mucosal lesions in rats, biodiesel production, nanobiocomposite synthesis, preparing bone grafts, as an antioxidant, etc (15,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30).
It is noteworthy that previous studies have shown antibacterial and antifungal activities of polysaccharides and chitosan extracted from the cuttlebone.
According to the literature, antimicrobial activities of powdered cuttlebone have not been evaluated yet. In addition, because of several reasons such as easy availability, affordability, and naturalness of the cuttlebone, the present study was conducted to evaluate the antimicrobial activities of powdered cuttlebone against K. oxytoca, S. aureus, and A. flavus.

Preparation of cuttlebone powder
For this purpose, cuttlebones were collected from Bandar Lengeh county, Hormozgan, then, were washed by distilled water, and dried. After drying, the cuttlebones were powdered using an electric mill A320R1 (Moulinex, France). Then, the obtained powder was sterilized in an autoclave (Reyhan Teb, Iran) at 121°C for 15 minutes. Afterwards, the powder was characterized using a WQF-510 FT-IR spectrometer (Bio-Equip, China). Also, the morphology and chemical composition of the powdered cuttlebone were investigated by the field emission scanning electron microscope (FESEM) and EDS-mapping (MIRA3 TESCAN XMU).
In the next step, to prepare the concentrations of serial dilutions of cuttlebone in a range of 10 -1 -10 -4 mg/mL, 1 mg of the powdered cuttlebone was added to 10 mL of the ringer's solution (Merck, Germany) and was sonicated for 30 minutes.  agar (PDA) (Merck, Germany) were prepared and sterilized. Then, they were poured into the sterile plates. The lyophilized bacterial and fungal were revived. Then, the 24 hour-old cultures of K. oxytoca and S. aureus, and 72 hour-old fungal culture of A. flavus were inoculated to the MHA and PDA media cultures, respectively, using a sterile swab. In addition, the standard antibiotic tetracycline and amphotericin B were used as negative controls. Also, S. aureus, K. oxytoca, and A. flavus suspensions were used as positive controls.
Antibacterial and antifungal activities survey 0.1 mL of cuttlebone with different concentrations (10 -1 -10 -4 mg/mL) was transferred by a sterile pipette into the wells (6 mm diameter). The wells were created on the agar media using a sterile cork borer. The plates were incubated at 37°C for 24 and 72 hours, respectively.
The antibacterial and antifungal activities of cuttlebone were investigated by agar well diffusion technique (31).
To determine the minimum inhibitory concentration (MIC), the microbial cultures were checked for the presence or absence of growth zone around the wells and the growth inhibition halos were measured using a ruler. The experiments were repeated three times to prevent any errors in the results.

Cuttlebone characterization
The FT-IR spectrum of cuttlebone at 400-4000 cm -1 is demonstrated in Figure 2. To investigate the morphology and chemical composition of the cuttlebone, the FESEM image and elemental mapping were evaluated (Figure 3).

Antimicrobial activities
The powdered cuttlebone showed a good antibacterial activity against K. oxytoca (Table 1). However, no antibacterial response was observed against S. aureus (Figure 4a) at all concentrations. On the other hand, S. aureus was resistant against the powdered cuttlebone. It indicates that antibacterial activity was dependent on the cuttlebone concentration. In the negative control samples, no activity was observed. The highest and lowest inhibition zones against K. oxytocic were observed to be 24 and 11 mm, respectively ( Figure 4b).

Antifungal activity
The results shown in Table 1 summarize antifungal activity of cuttlebone. In the negative control samples, no antifungal activity was observed against A. flavus. The highest and lowest inhibition zones against A. flavus were observed to be 23 and 9 mm, respectively. In addition, Figure 5 shows the highest inhibition zone against A. flavus. Table 2 shows the MIC values of powdered cuttlebone against K. oxytoca and A. flavus. The measured inhibition zones were compared with the negative controls and no activity was recorded for all pathogens. The highest and   (b) (a) lowest inhibition zones were obtained to be 10 -2 and 10 -3 mg/mL against K. oxytoca and A. flavus, respectively.

Discussion
Regarding the chemical structure of cuttlebone, as shown in Figure 2, wide absorption band is observed at 3398 cm -1 , that can be related to O-H stretching vibrations on the adsorbent surface. The presence of the absorption bands at 2922 and 2522 cm -1 is because of the alkyl groups (C-H) and amine groups (N-H) in the cuttlebone structure, respectively. The absorption bands at 1457 cm -1 can be related to pyranose ring bending vibrations. Also, the absorption bands at 1082, 853, and 712 cm -1 can be attributed to C-O, C-H, and C-H 2 stretching vibrations, respectively. According to the results of FESEM image and elemental mapping, the presence of Ca, Mg, N, Na, P, S, C, and O in the chemical structure of cuttlebone was confirmed ( Figure 3).
The main aim of the present study was to evaluate antimicrobial activities of powdered cuttlebone against K. oxytoca, S. aureus, and A. flavus. The results clearly show that relatively good antimicrobial activities were obtained by the powdered cuttlebone against K. oxytoca and A. flavus (Tables 1 and 2 and Figures 4 and 5). Nevertheless, at the same time, no antibacterial activity was observed against S. aureus. In the present research, the maximum inhibition zones were observed against K. oxytoca (24 mm) and A. flavus (23 mm), which illustrate and support the antimicrobial activities of powdered cuttlebone.
The findings of the present study showed the antimicrobial activities of powdered cuttlebone that it could be due to the presence of some polysaccharides such as chitosan and inorganic materials such as CaCO 3 in the chemical structure of cuttlebone. Also, the results of previous studies have shown the antimicrobial function of the polysaccharides extracted from cuttlebone. In a study by Liu et al. (2006), the chitosan isolated from Sepioteuthis lessoniana showed an antibacterial activity against S. aureus, K. pneumonia, and V. cholerae (32). Chitosan is a polymeric macromolecule which is not capable to pass the bacteria outer membrane. Therefore, it does not have a direct access to the cell intracellular sections. Nevertheless, chitosan due to the amino group in C-2 position, which has a positive charge in its structure, can interact with the anionic components (lipopolysaccharides and proteins) of the bacterial surface. Nevertheless, it shows that the chitosan biological activity depends on the chitosan pH solution, the target microorganism, and physico-chemical properties of chitosan. In addition, chitosan is a water-soluble derivative that could increase the permeability of bacterial cell membrane, which leads to the bacterial death by releasing cellular contents. Also, chitosan can precipitate on the microbial cell surface. Then, it makes an impervious layer Note. Values are presented as mean inhibition zone (mm) ± SD of three replicates.  around the cell and blocks the channels, thereby, prevent from the transportation of essential nutrition into the bacterial cell, which leading to cell death (37)(38)(39)(40)(41)(42). CaCO 3 is an inorganic material which forms the main part of cuttlebone structure. This compound with different concentrations is able to inhibit the growth of some bacteria and fungi species. The CaCO 3 can prevents of bacteria cell wall formation. Failure of the cell wall synthesis process leads to cell death due to lysis. Also, when the membrane cell contact with the material, the bacterial metabolism is disrupted (43).
Therefore, the presence of chitosan and CaCO 3 was the possible reason for the antimicrobial activities of powdered cuttlebone against K. oxytoca and A. flavus. Also, it seems that chitosan and CaCO 3 which exist in the powdered form of cuttlebone, could not penetrate to the peptidoglycan cell wall of S. aureus and destroyed it. Perhaps, it could be due to the type of the bacterial cell wall or cuttlebone concentrations. The noteworthy point in the mentioned studies is that the antimicrobial activities are strongly dependent on the type of target microorganism, which is consistent with the results of the present study.
For comparative testing of novel drugs, MIC estimation is extensively used. It is applied to establish the organisms' susceptibility in the clinical laboratories (36). In the present study, MIC value for the powdered cuttlebone against both K. oxytoca and A. flavus was reported as 10 -1 mg/mL, which indicates the best cuttlebone concentration that has strong antimicrobial activities.
Recently, the bioactivity study of natural products such as marine microorganisms due to their pharmacological application potential has received considerable attention. The initial efforts to study the antimicrobial activity in marine organisms were made around 1950s (33). Antibacterial activity has formerly been evaluated in the widespread range in the molluscan species such as oyster, mussel sea hare, etc. The searching principium for drugs from the marine environment is that marine animals and plants have a constant competition for reproduction, space, predation, etc (44,45). Although, most of the agents isolated from the marine sources, shows antimicrobial activities but they were not strong enough to compete with all classical antimicrobials obtained from the other microorganisms. The larger part of marine organisms has not been investigated to identify effective antibiotics yet. But many researchers investigated the antibacterial activity of the extracted materials from the body tissue of marine animals (46,47).
Different studies have shown the frequency of scrutable antimicrobial activity in marine molluscs. So, according to the mentioned results, it can be concluded that cephalopods are a source in the discovery of new substances to the development of drugs especially new types of antibiotics which have a better efficiency than the synthetic antibiotics (46)(47)(48)(49)(50). Generally, there are a few studies on the antimicrobial activities of internal bone of cephalopods.
The conspicuous point in the mentioned studies is that the cuttlebone biological activity is significantly dependent on the target microorganism species, cuttlebone structure, and its concentration. In the present study, the activity was also dependent on the cuttlebone concentration and type of microorganisms, which is consistent with the results of previous studies.

Conclusion
In different studies with various methods, the high frequency of detectable antimicrobial activities in the marine molluscs was reported. In the present study, the powdered cuttlebone showed antibacterial and antifungal activities against K. oxytoca and A. flavus, respectively. Additionally, the present study showed that the cuttlebone in the powdered form, which is thrown out as waste in the industries, is a very good and encouraging accessible natural antimicrobial source. Therefore, it is concluded that cuttlebones can be considered as a natural source of new substances to provide novel, low-cost, and safe antimicrobial agents.