Anti-biofilm Activity of Chitosan from Crab and Shrimp Species Indigenous to the Philippines on Established Biofilms of Pseudomonas aeruginosa and Staphylococcus aureus

Biofilms are structures produced by bacteria by attaching themselves together in a surface to form a protective matrix, rendering them resistant to antimicrobial treatments. The objective of this study was to examine the antibiofilm activity of chitosan from crab and shrimp species on the established biofilms of Pseudomonas aeruginosa and Staphylococcus aureus. The test groups were treated with chitosan solutions of varying concentrations (2.5 g/L and 10 g/L) chitosan from shrimp shells extract, 2.5 g/L and 10 g/L crab shells extract and a mixture of both shell extracts with the same concentration mixed in a one-to-one volume by ratio. Three different analysis were conducted involving color intensity test through TotalLab software, spectrophotometric analysis and microplate reader analysis. The highest percent anti-biofilm formation inhibition was observed with the 2.5 g/L mixed (1:1) chitosan solution against established biofilms of P. aeruginosa with a 62.90 ± 12% inhibition. On the other hand, S. aureus showed no percent inhibition with the 2.5 g/L shrimp chitosan and 10 g/L crab chitosan but was most sensitive to 10 g/L mixed (1:1) chitosan solution with an inhibition of 39.47 ± 19%. From the results, the 1:1 combination of shrimp and crab chitosan solutions resulted to a higher percent anti-biofilm formation inhibition than when given separately.


Introduction
According to the World Health Organization (2016), antimicrobial resistance continues to be a global problem due to wide range of infections caused by bacteria, virus, fungi or parasites. Microorganisms develop certain mechanisms to counteract the medical treatments such as the enzymatic inactivation of the antibiotic or alteration and modification of the antibiotic target site which would render the antibiotic useless [1]. As a result, patient's illness would be prolonged and there would be difficulties in curing the acquired infection. Biofilm formation is one of the mechanisms acquired by microbes to counterattack the effects of the antibiotics at hand. Biofilms are structures produced by bacteria by attaching themselves together in a surface (inert or living) to form a protective matrix. To address this problem, this study was conducted to combat the microbial biofilms formed by destroying it permanently. As a result of this, the microbe will then be exposed and will be susceptible to any antimicrobial agent which leads to the death of the microbial cell.
Not all microorganisms are capable of forming biofilms. Pseudomonas aeruginosa, a Gram-negative bacterium, is known to be resistant to antibiotic treatments due to formation of biofilms. This opportunistic pathogenic bacterium is known to be responsible for various infections, particularly in immunocompromised patients [1]. Another bacterium capable of biofilm formation is the Staphylococcus aureus. S. aureus is considered to be the most frequent cause of nosocomial infections and infections on indwelling medical devices along with S. epidermidis [2]. These two pathogenic bacteria were the focus of this research due to the different studies conducted that proved the ability of the bacteria to form biofilms [3,4]. Also, they pose a serious threat which would lead to chronic infections that are not susceptible to any treatments leading to long-lasting infections or even death.
Chitosan is a polysaccharide derived from chitin and is abundant in crustaceans. It is found to have a broad antimicrobial activity against a wide range of microorganisms [5]. Being a natural polysaccharide, chitosan exhibits notable biological properties including non-toxicity and biocompatibility which makes it a good candidate for biomedical applications [6]. This led to studies on chitosan being an effective antibiofilm agent by penetrating biofilms present, particularly in medical devices. By doing so, chitosan will be able to disrupt the microbial cell membrane [7]. Thus, biofilm formation will be inhibited due to the disruption of the attachment of the bacteria's cell membrane to the solid surfaces. By destroying the biofilm, the bacteria will then be susceptible to antibiotics which will consequently lead to bacterial cell death.
The objective of this study was to examine the anti-biofilm activity of chitosan from crab and shrimp species on the established biofilms of Pseudomonas aeruginosa and Staphylococcus aureus.

Limitations of the study
This study was done with limitations during the experimental procedures due to the availability of the resources. The degrees of deacetylation of the chitosan samples were not determined. The chitosan samples were only stored in a regular transparent containers and were placed in a refrigerator without controlling temperature. There were only two concentrations (2.5 g/L and 10 g/L) used for this particular study.

Materials and Methods
Staphylococcus aureus and Pseudomonas aeruginosa were treated separate. Three trials were conducted in triplicates. Two chitosan extracts were used as the treatment for the test groups in the experiment: crab chitosan extract, shrimp chitosan extract and a mixture of both in a one-to-one volume by ratio. Two concentrations were prepared for each extract (2.5 g/L and 10 g/L). Acetic acid was used for the treatment of the control group.

Extract preparation
Extraction of chitosan from Blue Swimming crab and Whiteleg shrimp was done separately. For the extraction of chitosan from Whiteleg shrimp, the shells were washed, air dried and refrigerated overnight. It was then oven dried for 4 days at 65°C. After, it was treated with 2000 mL of 4% NaOH at room temperature for 24 hours. The deproteinization of Whiteleg shrimp shells was done by draining the alkali solution from the shells and the shells were washed with distilled water repeatedly until the pH turned neutral. The deproteinized shells were then treated with 2000 mL of 4% HCl for 12 hours for demineralization to yield chitin. The resulting acid solution was drained off from chitin, washed with distilled water and dried at room temperature. The processes were repeated with 2000 mL of 2% NaOH and 2000 mL of 1% HCl. Further decolorization of the chitin was obtained by soaking it in 2000 mL of 1% KMnO 4 for 30 minutes followed by 2000 mL of 1% oxalic acid for 30 minutes to 2 hours. The decolorized chitin was then deacetylated to form chitosan by treating with 2000 mL of 65% NaOH for 3 days at room temperature. The alkali solution was drained off and washed repeatedly with distilled water until the pH was neutral. The extracted chitosan was further dried at room temperature [8].
The extraction of chitosan from blue swimming crab was done using the procedure by Sujeetha et al., 2015. The blue swimming crab shells were washed thoroughly in running tap water to remove the debris and were sun dried. The sun dried shells were then crushed and soaked in 4000 mL of 9% HCl in the ratio 1:14 for 40 hours at room temperature. After, 4000 mL of 5% NaOH was used for further treatment. After the neutralization process, it was then sun dried for two days and was grounded afterwards. The deacetylation process was done using 4000 mL of 70% NaOH solution in a ratio of 1:15 (w/v) for 72 hours at room temperature. The resulting mixture was then filtered and was oven dried at 80°C [9].

Preparation of chitosan treatments
The chitosan was dissolved in acetic acid. To make the 2.5 g/L chitosan solution, 0.5 g of the chitosan was dissolved in 250 mL of 0.1 M of acetic acid. 2.5 g of the chitosan was dissolved in 250 mL of 0.1 M of acetic acid to make 10 g/L chitosan solution. The chitosan solution was then stirred for 8 hours using a magnetic stirrer set at 55°C, 100 rpm. Afterwards, the solution was filtered using cheesecloth to remove the impurities. The solution was then stored inside the refrigerator.

Growing bacteria culture
The microorganisms from the agar slant, which is the source of the bacteria, were allowed to grow in nutrient broth. The nutrient broth served as a media for the bacteria to grow, which was then used in the in the determination of the bacterial population and in the preparation for the biofilms on the microtiter plates.
Ten milliliters of broth was transferred to each of the test tube. P. aeruginosa and S. aureus were then inoculated under laminar flow hood, following aseptic technique. The bacterial cultures were then incubated for 24 h at 37C and were allowed to grow.

Determination of bacterial population
Serial dilution was done to determine the bacterial population in the liquid culture. After serial dilution, one hundred microliters was then pipetted into agar plates and was allowed to grow in the incubator for three days at 27°C. An estimation of the number of bacteria per mL of the original culture or Colony forming unit (CFU) was then computed using the same method as the study by Ednalino et al.

Preparation of biofilm on microtiter plates
Biofilms were cultured in a 96-well microplate. Twenty one wells were used and filled with one hundred microliter (100 μL) of the diluted culture of S. aureus. One hundred microliter (100 μL) of the diluted culture of P. aeruginosa was used to fill another twenty one wells of the microplate. The microtiter plate was covered and was placed in an incubator for six days at 37°C.

Application of chitosan treatments
Two concentrations of the chitosan solution were prepared (2.5 g/ L and 10 g/L) from the shrimp shells extract, crab shells extract and with both shell extracts mixed in a one-to-one ratio by volume.
One hundred microliter of each concentration was introduced into 42 wells, with three wells allotted for each concentration per bacteria. This served as the test group (or assay group). One hundred microliter of acetic acid was then pipetted onto three wells for each microorganism that served as the control group. The microtiter plate was covered with the lid, sealed and placed inside an incubator at 37°C for 4 days.

Evaluation and analysis of biofilms
Biofilm quantification was carried out resembling the procedures done by Costa et al., 2014. The contents from the wells was discarded and washed three times with sterile water in order to visualize the adhesion of the biofilm as well as to remove the non-adherent cells. 200 μL of ethanol was then added to the wells to ensure the fixed attachment of the remaining microorganisms at the surface of the wells. After 15 minutes, ethanol was discarded and the wells were airdried. After the drying procedure, 200 μL of crystal violet solution was added to the wells for 5 minutes. Excess stain was removed by rinsing the plate with water and air drying was followed.
The microplate was then subjected in a microplate reader in order to measure the optical density at 625 nm. The degree of biofilm formation inhibition was calculated using the equation by Costa et al., 2014. %Biofilm Formation Inhibition=100 -(OD assay /OD control ) × 100 Where OD assay is the optical density of the test group and OD control is the optical density of the control group.
The microplate was also subjected to a qualitative analysis. It was then scanned using a printer with a resolution of 600 dpi. Using the digital images obtained, the biofilms were evaluated using image analysis software called TotalLab Array v10 software.
The results were analyzed based from the color intensities which were measured in pixels per inch (ppi). It is the pixel density that will determine the color intensity of the stain. The intensity of the color of each well is proportional to the biofilm present in the solution.
A spectrophotometer was also used to quantify and validate the results by measuring the absorbance of the solutions. 125 µL of the solution from the wells was then transferred to the cuvettes. It was then diluted with 3 mL of 95% ethanol. The spectrophotometer was then set at 435 nm for the absorbance of the violet color and each of the cuvette was analyzed for its absorbance values. Absorbance reflected in the instrument will be used as measures of the effectiveness of the treatment especially when compared to the baseline (Ednalino et al., 2012).

Statistical analysis
Two-way ANOVA with a significance level of 0.01 and 99% CL was used to determine the effectiveness of different chitosan solutions from P. pelagicus, P. vannamei, and the mixture of both in a one-to-one ratio by volume against S. aureus and P. aeruginosa biofilms. Two independent factors were determined to be the absorbance values and the percent formation inhibition activity of the chitosan. In ANOVA, the null hypothesis states that there is no significant difference in the mean pixel densities of the treatments. The alternative hypothesis states that the mean pixel densities of the treatments has a significant difference.
The correlation coefficient between the absorbance values and the color intensity values was also determined to quantify the relationship between these two parameters.

Results
Chitosan extracted from shrimp shells which are white and flaky solid material. On the other hand, the chitosan extracted from crab shells which are light orange and hard solid material.
To verify verifies the presence of the chitosan from both shrimp and crab shell samples. Chitosan was added with Lugol's iodine solution and resulted to a yellowish-brown material. Upon addition of the H 2 SO 4 , the color changed to red-violet. Bacterial population determination through serial dilution revealed that that the tenth plate of Staphylococus aureus with a dilution factor of 10 was found to contain 208 colonies and 2.08 x 1011 CFU's. On the other hand, the tenth plate of Pseudomonas aeruginosa with a dilution factor of 10 was found to contain 54 colonies and 5.40 × 1010 CFU's. Figure 1 shows the color intensities of the violet stains of Staphylococus aureus biofilms. As compared to the one treated with 0.1 M acetic acid (control group) which has 11673 ± 16318.51 ppi, the least color intensity value was seen in the wells treated with 10 g/L of crab chitosan for S. aureus which has 87.57 ± 43.10 ppi. It was followed by the one treated with 2.5 g/L mixed (1:1) chitosan solution with 209.44 ± 182.59 ppi. The 2.5 g/L crab chitosan solution followed which has 617.0 ± 664.66 ppi. The S. aureus biofilms treated with 10 g/L mixed (1:1) chitosan solution followed which has 1883 ± 2269.88 ppi.

Color intensity test
The well containing S. aureus biofilms treated with 2.5 g/L shrimp chitosan solution has the highest color intensity value which has 11411 ± 15350.93 ppi.
The color intensities of the violet stains of Pseudomonas aeruginosa for the different chitosan types and concentrations were shown in Figure 1. The graph below shows the least color intensity value which was seen in the well treated with 10 g/L of crab chitosan having 87.81 ± 81.47 ppi as compared to the one treated with 0.1 M acetic acid (control group) which has 17244 ± 24274.01 ppi. It was then followed with the well treated with 2.5 g/L mixed (1:1) chitosan solution with 446.52 ± 461.83 ppi.

Microplate reader analysis
From the results of the microplate reader analysis, optical densities of each well were determined. Following the equation by Costa et al., the percent biofilm formation inhibition of each chitosan solution against S. aureus and P. aeruginosa was calculated as shown in Figure   2. The 2.5 g/L mixed (1:1) chitosan solution has 18.42 ± 9% and 62.90 ± 12% biofilm formation inhibition against S. aureus and P. aeruginosa, respectively. On the other hand, 39.47 ± 19% and 44.55 ± 23% biofilm formation inhibition were demonstrated by 10 g/L mixed (1:1) chitosan solution against S. aureus and P. aeruginosa, respectively.
Based from the results, the two mixtures of the chitosan solution from shrimp and crab shells in two different concentrations were found to have a higher percent anti-biofilm formation inhibition. Figure 3 shows the absorbance values of the violet stains of S. aureus and P. aeruginosa biofilms, respectively, after the chitosan treatment.

Spectrophotometric analysis results
The one treated with 2.5 g/L mixed (1:1) chitosan solution has the lowest absorbance which has 0.065 ± 0.000471 for S. aureus while 0.065 ± 0.00216 for P. aeruginosa. This means that 2.5 g/L mixed (1:1) chitosan solution effectively destroyed the established biofilms of both bacteria. S. aureus biofilms treated with 0.1 M acetic acid (control group) has the absorbance of 0.098 ± 0.022 as depicted in Figure 3. When compared to the control group, no activity was seen in 2.5 g/L shrimp chitosan solution and 10 g/L crab chitosan solution against S. aureus biofilm, which has 0.099 ± 0.023 and 0.099 ± 0.024 respectively.
In Figure 3, 10 g/L shrimp chitosan showed no activity against established biofilms of P. aeruginosa. Its absorbance is 0.084 ± 0.021 which is higher compared to the control group which has the absorbance of 0.081 ± 0.022.   Table 2: Two-way ANOVA analysis for P. aeruginosa.

Source of variation
On the other hand, the p-value between absorbance and percent formation inhibition values for both bacteria is less than 0.01 which is significant.
The correlation coefficient between the absorbance and color intensity values is 0.459 (as depicted in Table 3) while the p-value is 0.042. These two parameters have insignificant positive correlation.

Absorbance
Color intensity Color intensity 0.459113177 1 Table 3: Determination of the correlation coefficient between absorbance and color intensity.

Discussion
Chitosan was extracted through three major processes which include deproteinization, demineralization and deacetylation. Deacytelation is the last procedure in the extraction of chitosan. It is a process that would convert chitin to chitosan [10,11].
To test for the biofilms left after chitosan treatment, color intensity test through TotalLab Image Analysis and spectrophotometric analysis were conducted. According to a study conducted by Ednalino et al. (2012), the amount of biofilm is proportional to the color intensity and the absorbance of the well.
The optical density analysis through microplate reader was done to calculate the percent biofilm formation inhibition of the different chitosan solutions. Based from the results shown in Figure 2, P. aeruginosa biofilms are more susceptible to chitosan treatment when compared to S. aureus. In a study conducted by Orgaz et al., Pseudomonas biofilms are highly susceptible against chitosan treatment even though it has thick matrix [12].
Based from the results of the spectrophotometric analysis and the validation through microplate reader, the two mixtures of the chitosan solution from shrimp and crab shells in two different concentrations (both crab and shrimp chitosan mixed in 1:1 by volume) were found to be more effective in the inhibition of the S. aureus and P. aeruginosa biofilms. The insufficient results for the chitosan inhibition of S. aureus biofilms can be explained because of the ability of S. aureus to produce a multilayered biofilm which is embedded within a glycocalyx [13]. It is generally known that biofilm-embedded cells are protected from certain conditions that may be damaging to a cell, such as lack of water or nutrients or even if an antimicrobial agent is introduced into it [12]. With these mechanisms, it will be difficult for the chitosan to penetrate the matrix of the bacteria resulting to insusceptibility.
The variety of chitosan and the different concentrations are considered to be insignificant in this study. This can be attributed to the fact that both chitosan sources are marine crustaceans which produce α-chitin in general [14]. Chitin can be subdivided according to the derived material: α-chitin, β-chitin and γ-chitin [15]. α-chitin can be extracted from marine crustaceans, arthropods, fungi and the cysts of Entamoea while β-chitin can be seen from the pen of the Loligo squid. γ-chitin is very rare and it can only be extracted from cocoon fibers of the Ptinus beetle and the stomach of the Loligo squid [14].
On the other hand, the absorbance values and percent formation inhibition values are considered to have a significant correlation. The significant difference reflected in the statistical analysis can be attributed to the inverse relationship of the two parameters.
For this particular study, absorbance values and color intensity values have insignificant positive correlation. This means that the two parameters have a direct relationship with each other. The Beer-Lambert Law states that the absorbance is proportional to the concentration, which was measured with the color intensity test, and the path length through which the light passes through the sample [2].

Conclusion
The study concluded that Pseudomonas aeruginosa appeared to be more sensitive to the chitosan treatments than Staphylococcus aureus.
It has been demonstrated that the hydrophilicity in gram-negative bacteria is significantly higher than in gram-positive bacteria. This, in turn, makes P. aeruginosa, a gram-negative bacterium, more sensitive to chitosan. S. aureus also forms a multilayered biofilm making it more difficult for the chitosan treatment to penetrate its biofilm. Also, the study concluded that the 1:1 combination of shrimp and crab chitosan solutions resulted to a higher percent anti-biofilm formation inhibition than when given separately.