Evaluation of Antibacterial and Antifungal Activity of Chitosan in Integument of Cockroaches

Introduction: Due to drug resistance and toxic side effects observed following administration of many antibacterial drugs, novel strategies are highly needed for treatment of bacterial diseases. Chitosan, as an important immune system stimulator, is found in cockroaches that are living in contaminated sites. The aim of this study was to extract the chitosan from nymph and adult stage of both Blattella germanica and Periplaneta americana and to evaluate its antibacterial and antifungal activities. Methods: The mature form of B. germanica and the nymph and adult forms of P. americana were killed by CO2 gas, and washed and dried at 60oC. Then, they were mechanically ground in a mixer and passed through 20 mesh size. Finally, chitosan was extracted from shrimp processing discards. Chitosan was dissolved in lactic acid 1% and its effects against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, methicillin-resistant S. aureus, Candida albicans, and Aspergillus were evaluated. Results: Chitosan extracted from cockroaches showed antibacterial effects on gram-positive bacteria especially S. aureus and E. coli. However, it had no impact on fungi. Conclusion: The findings of the current study revealed that chitosan had excellent antibacterial


Introduction
Since a vast majority of bacterial species developed resistance against antibacterial drugs which are highly toxic, novel strategies are required to be employed in this respect.The annual cost of drugresistant infections is estimated to be $20 billion in surplus health care costs and 8 million extra hospital days in the United States. 1 Because the majority of insects such as cockroaches live in contaminated areas, they appear to have developed effective strategies to deal with pathogens. 2Chitosan as a natural polysaccharide has been widely used in pharmaceutical excipients. 3This material is produced by the deacetylation of chitin.Chitin is a natural polymer composed of randomly dispersed β-(1-4)-linked D-glucosamine.This biopolymer is a major component of not only organisms like crustaceans, fungi, and insects, 4 but also of invertebrates' integument. 5Chitin can also be produced by chemical and biological methods. 6,7Chitin annual turnover was estimated to range from 10 10 to 10 11 tones. 8,9Additionally, it can be readily obtained by simple extraction. 6In addition, it contains proteins, minerals, lipids, and pigments. 10Chitosan has characteristic features such as bioadhesive nature and hydrophilic properties.Besides, it can be used as potent carrier for braintargeted drugs, transdermal films and wound healing biodegradable grafts, as well as antimicrobial and stabilizing constituent of liposomes.Due to these properties,

Evaluation of Antibacterial and Antifungal Activity of Chitosan in Integument of Cockroaches
chitosan, as one of the most promising renewable polymeric materials for a variety of applications, has received greater attention than before.2][13] Notably, it facilitates the transportation of polar drugs through the epithelial surfaces and is regarded as both biocompatible and biodegradable. 14ecently, various studies have focused on the strong antimicrobial effects of chitosan against different groups of microorganisms, from bacteria 15 to fungi, 16 parasites, [17][18][19][20] and yeasts. 213][24][25][26] Despite numerous studies on the antimicrobial effects of chitosan, researchers have not yet come up with a consensus result in this respect.Therefore, further studies should be conducted accordingly.
8][29][30] Moreover, this material can be produced from exoskeleton of arthropods as well as the cell walls of fungi and yeasts. 5Insects like Periplaneta americana and Blattella germanica are the potential sources of chitosan due to their high reproduction rate. 31ecause of the low content of minerals and total protein in insects' body, less acidic and alkaline materials are required to extract the chitin and chitosan. 32According to what has previously been mentioned, this study aimed at extracting chitin and chitosan from nymph and adult cuticle of cockroaches (B.germanica & P. americana) and assessing the anti-bacterial and anti-fungal properties of the extracted chitosan.

Preparation of Insect Corpses
In this study, the nymph and adult forms of B. germanica and P. americana were obtained from the Laboratory of Medical Entomology, Tehran University of Medical Sciences.Adult and nymph forms were killed by CO2 gas.Subsequently, insect corpses were washed by distilled water (DW) and dried at 60°C.Then, they were mechanically crushed in a mixer and passed through 20 mesh size.

Demineralization
Chitosan was extracted from shrimp processing discards using a method described by Chang et al 27 Demineralization step was accomplished by mixing 5 g of B. germanica and P. americana integument dry powder with 1M HCl at 100°C for 30 minutes followed by rinsing with DW until natural pH was obtained.This demineralized insect sample was subsequently washed (with 99% ethanol) and air dried at room temperature.

Deproteinization
This step was performed using alkaline treatment.The de-mineralized integuments dry powder was deproteinized with 1M NaOH solution at 80°C.This step was repeated several times during 24 hours.The chitin product was filtered through 20 µM mesh and washed with DW until the pH became neutral.

Deacetylation
In order to deacetylate the chitin, NaOH 50% was used at 100°C for 4 hours.Then, the alkali was drained off and washed thoroughly with DW until the pH was less than 7.5 and subsequently dried at ambient temperature (30±2°C).

Antimicrobial Susceptibility Test
The suspensions of bacteria were prepared in sterile phosphate-buffered saline (PBS) and standardized to 1 × 10 6 CFU/mL.The antimicrobial activities of the chitosan solution were tested against Staphylococcus aureus (ATCC; 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Vibrio cholerae (ATCC; 39315), and methicillin-resistant S. aureus (ATCC: 33591) by disk diffusion method (Bauer et al, 1996).Bacterial suspension was initially prepared and inoculated onto the entire surface of a Mueller-Hinton agar (MHA) (diameter, 90 mm) with pH of 6.9 using a sterile cotton-tipped swab to form an even lawn.
The plate was air-dried for 15 minutes and five sterile paper disks (6 mm in diameter; BD, Becton Dickinson Diagnostic Systems) impregnated with 20 μL diluted chitosan solution (0.4% & 0.8% for chitosan concentration, respectively) were loaded on the surface of each MHA plate using a sterile pair of forceps.Then, the plates were incubated aerobically at 37°C for 24 hours.The diameter of inhibition zone was measured after 24 hours of incubation in the usual manner.Disc Diffusion Method for Fungi Disk diffusion method was performed for fungi against Candida albicans and Aspergillus species on yeast nitrogen base glucose detection (YNBG).Five disks papers, impregnated with chitosan solution, were placed over the MHA medium.

Data Analysis
All steps of this work were carried out in triplicate.Data were analyzed using SPSS (statistical package for the social sciences) software, version 18.In addition, ANOVA and independent student t test were used.

Demineralization
Five grams of the yielded powder of integument from B. germanica and P. americana were treated with 1M HCl in order to extract the chitin (Table 1).The average of the remaining material were 2.12 and 2.17 mg after demineralization step for nymph and adult forms of B. germanica while being 2.13 and 2.33 mg for nymph and adult forms of P. americana.

Deproteinization
The average amounts of the remaining chitin after deproteinization from B. germanica in nymph and adult forms were 0.28 and 0.31 mg whereas in P. americana, nymph adult forms, they were 0.22 and 0.74 mg, respectively.Deacetylation Following deacetylation of B. germanica and P. americana, to extract the chitosan in NaOH 50 %, the average amounts of the remaining chitosan were 0.25 and 0.26 mg in nymph and adult forms of B. germanica and also 0.18 and 0.55 mg in nymph and adult forms of P. americana, respectively.

Antibacterial Test
In this test, both gram-positive and gram-negative bacteria were tested.The chitosan showed different degrees of antibacterial activity against S. aureus, E. coli, and P. aeruginosa.However, no significant antibacterial activity was observed against V. cholerae and methicillinresistant S. aureus.Table 2 shows inhibition zone for different concentrations of chitosan against the tested bacteria.The chitosan did not show any activity against C. albicans and Aspergillus species.

Discussion
Chitin is a major part of the insect's cuticle, which always covalently binds to minerals and proteins.The most frequently used method for chitin extraction from insects has two steps: 1) an acidic step, to eliminate minerals (also named demineralization step); and 2) a basic step, to eliminate the proteins from cuticle (also referred to as deproteinization step). 4n this study, the amount of chitosan extracted from adults and nymphs of the two spices was completely different.Compared to the results obtained in previous studies on crab and shrimp shells, 27 based on the percentage of the obtained compounds, these results suggest that the methods used for chitosan production from crab and shrimp shells was also an effective method for chitosan extraction from adult and nymph forms of P. americana and B. germanica as well.
There are various studies focused on the antimicrobial activity of different chitosans and their derivatives from different sources.4][35][36] In fact, different concentrations of both types of chitosan used in the present study demonstrated antimicrobial activity on the selected bacteria. 37owever, chitosan could not inhibit the development of methicillin-resistant S. aureus species.Applying chitosan in cooperation with Ag + nanoparticles has rendered antibacterial activities. 38In fact, the use of nanoscale compounds because of the availability to the drug target, could increase its effect.There are some examples in this regard. 20,39reliminary studies demonstrated that chitosan extracted from P. americana was more effective than what extracted from B. germanica 40 while in the current experiment, the chitosan extracted from B. germanica showed better level of efficacy.Furthermore, chitosan was shown to have better effects on gram-positive than on gram-negative bacteria.
In general, there has been little research on antifungal activity of chitosan.Kendra et al assessed the antifungal effects of different acetylation grade of chitosan against Fusarium solani in an in vitro study.The results indicated that chitosan had maximum antifungal activity especially the high-molecular-weight chitosan. 41In the current study, no inhibitory activity of chitosan was observed in fungi.Chitosan was found to have a greater antifungal activity at pH = 4.0. 42However, in order to be passed through the anti-bacterial filter, chitosan solution was prepared at pH = 7.0.It is worth to mention that this solution is extremely dense at pH = 4.0 and will not pass through the anti-bacterial filter.

Conclusion
According to the results of the recent study, chitosan had antimicrobial effects.For this reason, it is expected that further studies to be carried out on this compound to determine its antibacterial or antimicrobial effects on other bacteria and microorganisms.Additional studies should also be undertaken to determine the toxic effects of this combination in order to eventually introduce it as an antimicrobial agent.

Ethical Approval
Considering that human and animal samples were not used in this study, there was no need for moral confirmation.

Competing Interests
The authors declare that there is not any conflict of interests.

Table 1 .
The Integument Composition of Blattella germanica and Periplaneta americana

Table 2 .
Inhibition Zone Diameter (mm) Following Administration of Different Concentrations of Chitosan for Selected Bacteria