Investigation on anti-quorum sensing activities of Chitosan AgNP’s-chitosanase against MDR pathogens

doi:10.21203/rs.3.rs-2626954/v1

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Investigation on anti-quorum sensing activities of Chitosan AgNP’s-chitosanase against MDR pathogens

https://doi.org/10.21203/rs.3.rs-2626954/v1

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Marine bio-nanotechnology is a new promising field with having high perspective in the area of biological research. In 2018 the production of crustacean shells especially from shrimp is about 54,500 tons on South East coast of India. The current study focuses on the use of extracted chitosan (Squilla shells) polymer in silver nanoparticle synthesis along with immobilized chitosanase synergistically improves the antimicrobial and quorum quenching effects against the multi drug resistant (MDR) pathogens. The main objective of the project is to synthesize the chitosan AgNPs and to immobilize the enzyme chitosanase with it and to study the anti quorum sensing (quorum quenching) activity against MDR pathogens. This study will render a new ideology to eliminate biofilm formation and suppress the pathogenicity of planktonic MDR pathogens. Since the combinations of chitosanase, as well as Chitosan silver nanoparticles, are very efficient in eliminating them.

Applied & Industrial Microbiology

Chitosan AgNPs-Chitosanase

MDR pathogens

Biofilms

Antimicrobial effects

Quorum Quenching

Chitosan an extracted product of chitin is a homopolymeric linear polysaccharide [1]. It is infrequent in the environment acquired by alkali deacetylation [2]. It is a semi-crystalline polysaccharide. The felicitous utilization of these crustacean shell wastes forms the basis of raw materials used in various fields owing to their idiosyncratic properties such as bio cooperative, biodegradation and low toxicity [3]. It also helps to control the nanoparticle size which is highly sensitive in the applications of nanotechnology [4].

Silver nanoparticles have distinctive physiochemical properties which make them play a vital role as antimicrobial, anti-biofilm, and anti-quorum sensing [5]. Silver nanoparticles influence strong inhibitory effects against various pathogens even MDR pathogens hence it is been used as an antibacterial agent for decades [6]. The various physicochemical methods used in the synthesis of silver nanoparticles make use of highly reactive chemicals making the silver nanoparticles cytotoxic in nature. To overcome this stumbling block biomimetics came into action.

The chitosan mediated silver nanoparticle synthesis is being highly recognized due to their biocompatibility, biodegradability and hydrophilic properties, which endow them with various opportunities in diverse fields [7].

Chitosanase causes endo hydrolysis in between D-glucosamine which is now well known to play an efficient role in controlling pathogens. They act as a good bioactive material [8]. As the enzymes are highly vulnerable and the cost for production and purification of chitosanase is high, the immobilization technique helps to overcome all those disadvantages [9, 10]. Now a day’s nanoparticles are even more successful immobilizing agents than other macromolecular substrates. The main reason behind this is their nano-sized structure, then charge in them, the surface area of the nanosized particles and they also serve to be satisfactory in enzyme loading. Due to the magnetic properties of nanoparticles, they can be easily separated from the enzyme without undergoing any harsh treatments.

Increased use of antibiotics is directly proportional to the increased number of resistance by more number of pathogens against the particular antibiotics in developing scenarios and is referred to as Multi-Drug Resistance. In recent years there is an emergence of MDR UPEC which is difficult to diagnose and treat. The MDR S. aureus is considered a scandalous pathogen and it acts as a major source of nosocomial infections. The MDR E. coli strains are now placed under the category of critical priority pathogen list and S. aureus strains are now designated under the category of high priority pathogen list released in 2019 by WHO in the document of IPPL. They are easily been transmitted via food and water and in hospitals, they are transmitted via indwelling catheters and other medical instrumentation [11].

Biofilms provide adherence of the microorganisms to a substratum like indwelling medical devices, biomedical types of equipment and implant animate objects [12] and protect them from the host immune system (cause frustrated phagocytosis) and anti-microbial therapy [13]. It is estimated that the MDR S.aureus which is capable of forming biofilms caused most circulatory system infections and even more by indwelling catheter biofilm infections [14]. The exact mechanism of Chitosan AgNP as a quorum quencher is not yet discovered.

Antifungal resistance serves as a major challenge for medical practitioners. Antifungal resistance can be Microbiological resistance, Clinical resistance and the Combination of clinical and microbiological resistance [15]. Aspergillus is one of the broad spectra of the human pathogen that causes several types of Aspergillosis. These diseases are already difficult to and so if the affected fungi are drug-resistant then it is highly dreadful and very difficult to treat. These fungi are mostly resistant to most of the azole drugs and are the top mold infection among the critical priority pathogen list release by WHO in 2022. [16, 17].

The motto of this investigation is to study whether the immobilized chitosanase with chitosan silver nanoparticle synergistically improves the antimicrobial, anti-quorum sensing and anti-antifungal activity against drug-resistant pathogens.

The integrative methodology is represented in Fig. 1.

2.1. Chitosan extraction:

The shrimp shells were collected from the Nagapattinam coastal regions. From the collected shrimp shells the chitosan was extracted using Anand et al., 2014 with slight modifications (Supplementary Fig. 1).

2.2. Characterization of extracted Chitosan:

2.3.1. Dry weight:

The dry weight of extracted chitosan was measured and the final yield was calculated.

2.3.2. FTIR Analysis:

The FTIR spectra of the extracted chitosan were measured on Spectrum 100, Perkin Elmer, (USA).

2.3. Biological synthesis of chitosan silver nanoparticle (Chitosan AgNPs)

The synthesis of chitosan AgNPs was performed as reported by Kalaivani et al., 2018 with slight modifications. The synthesis begins with dissolving 0.33g of chitosan in 1.5% acetic acid. The chitosan acetic acid solution is kept under continuous stirring without the application of heat for about half an hour. The undissolved substances are removed by filtration. To that collected clear solution, 5 ml of 0.1M silver nitrate (AgNO₃) [purchased from Sigma- Aldrich] 166µl of 0.1M sodium hydroxide is added. The solution is kept in a magnetic stirrer at 90°C for 10 hours. The color change is from colorless to faded yellow then finally to the brown color solution which indicates the precipitation formation of chitosan-silver nanoparticles (Supplementary Fig. 2).

2.4. Characterization of Chitosan AgNPs:

The UV- visible spectrophotometer is the preliminary analysis that was characterized in Evolution 201 UV-visible spectrophotometer at a resolution of 1nm with a scan speed of 400 nm min^− 1, in a 1 cm path quartz cuvette. The wide range for silver nanoparticles for peak formation is from 200 to 800 nm. [18].

In FTIR spectrum of samples was recorded on Spectrum 100, Perkin Elmer, (USA). For this analysis, the synthesized chitosan AgNPs were mixed with potassium bromide in a ratio of 5:95 [5].

The synthesized chitosan AgNPs was measured by Bruker AXS Diffractometer (D8 Focus, Germany) for X-ray diffraction. The diffraction peaks were recorded between 2Ɵ ranges 10°-80° at a fixed time. This operation was carried out at a voltage of 40KV and a current of 30 MA with CuKɑ radiation [5].

The Particle size analysis was carried out in a particulate system particle size analyzer [5].

FE- SEM is used to get clear topographical micrographs from lower to higher resolution. The use of FE- SEM is it will provide a clear magnified image and also shows if any impurities present [19].

2.5. Bacterial and fungal cultures:

The Gram-positive bacterial strain Staphylococcus aureus SA1199 and gram-negative bacterial strain Escherichia coli MG1655 were used as a control strains in antimicrobial testing for making the comparative study with MDR pathogens. MDR Staphylococcus aureus I₁, MDR Staphylococcus aureus I₂, MDR Escherichia coli and MDR Aspergillus spp. (Supplementary Figs. 3, 4 and 5), were clinically isolated from the human samples. They were cultured in their respective selective media. The gram-negative strain Chromobacterium violaceum MTCC 2656 is used as the reporter strain. Bacillus cereus MTCC 1305 and Lactobacillus plantarum MTCC 6160 were used for enzyme production.

2.6. Enzyme production:

2.6.1. Media formulation:

Two different media were formulated and their composition is given in Table 1 and Table 2. Media A and Media B were formulated in 250 ml of Erlenmeyer flask. After sterilization the flak are inoculated with Bacillus cereus MTCC 1305 and Lactobacillus plantarum MTCC 6160, incubated at 37℃. The flask is then incubated in a rotary shaker at 37°C for 3 days which is 72 hours. After 24 hours the turbidity starts increasing which shows the growth of the microorganisms [9].

Table 1

Composition of Media A
S.no	Composition	Grams/litre
1.	Peptone	0.1% [w/v]
2.	Glucose	0.1% [w/v]
3.	Chitosan	0.2% [w/v]
4.	Magnesium sulphate	0.05% [w/v]
5.	Di potassium hydrogen phosphate	0.1% [w/v]

Table 2

**Composition of Media B**
S.no.	Composition	Grams/litre
1.	Peptone	0.6% [w/v]
2.	Chitosan	0.2% [w/v]
3.	Magnesium sulphate	0.05% [w/v]
4.	Di potassium hydrogen phosphate	0.1% [w/v]

2.7. Enzyme purification:

2.7.1. Ammonium sulfate precipitation:

At the end of the stipulated period, the production media is centrifuged at 6000 rpm for 15 minutes and the supernatant is used for ammonium sulfate precipitation. In the supernatant, ammonium sulfate precipitation is carried from 20–70% [20].

2.7.2. Dialysis:

The precipitate phosphate buffer mixture is now dialyzed against the same buffer for 48 hours at room temperature by changing the phosphate buffer intermittently [20].

2.8. Chitosanase assay:

The chitosanase activity was estimated by enumerating the reducing sugars that are D- glucosamine from the chitosan substrate. The optical density was measured at 540 nm in a colorimeter [21].

2.9. Estimation of proteins by Lowry’s assay:

The protein content of the enzymes was determined by using Lowry’s method with BSA (1 mg/ml) as standard and with O.D 660nm. The slope of the linear calibration curve has been measured and reported [22] [23].

2.10. Antibacterial and antifungal assays:

2.10.1. Kirby Bauer disc and well diffusion method:

The agar disc diffusion and well diffusion method were performed with 24 hour MDR broth cultures of S. aureus and E. coli using Muller Hinton agar. Different concentrations of chitosan AgNPs were used in each sterile disc and well. After incubation at 37℃ for 24 hours the diameter of the zone of clearance was measured [5].

The antifungal assay was done with antifungal assay agar. Different concentrations of chitosan AgNPs were used in each well. After incubation at 37℃ for 24 hours the diameter of the zone of clearance was measured

2.10.2. Minimal inhibitory concentration (MIC) assay:

For each bacterial culture, MIC was performed with different concentrations of chitosan AgNPs, and 0.1ml of 24 hours NB cultures of MDR S. aureus and E. coli were inoculated and incubated at 37℃ for 24 hours. The optical density was measured at 600nm in a colorimeter [18].

For the fungal culture, MIC was carried out with increasing concentrations of chitosan AgNPs and 0.1ml of 24 hours PDB cultures of Aspergillus species were inoculated and incubated at 37℃ for 24 hours. The optical density was measured at 600nm in a colorimeter

2.11 Anti-quorum sensing assays:

2.11.1 Qualitative assay- Congo red agar assay:

In this assay, the Congo red agar medium is formulated, and the 24 hour broth culture was transferred to the plates and incubated at 37°C for 24 hours [24].

2.11.2 Semi-quantitative assay – Crystal violet tube assay:

For the semi-quantitative assay, a batch of 7 test tubes was used. One tube for the untreated sample, one tube for media control and 5 tubes for the test samples were the increasing concentration of chitosan AgNPs and a standard quantity of MDR pathogens were inoculated and incubated at 37℃ for 24 hours. After the period of incubation, the contents of the tubes were aspirated, washed with PBS and dried. The 0.1% crystal violet stain is poured into the tubes to stain the biofilms. After the stipulated time the excess stain was removed and washed with Milli-Q water [24].

% biofilm inhibition= [1- (O.D of cells treated with Chitosan AgNPs/ O.D. of non-treated control)*100]

2.11.3 Swarming assay:

For this assay, LB agar was used in untreated samples and LB agar blended with the synthesized chitosan AgNPs was used for treated samples. The MDR E.coli is pocked at the center of each plate. In this experiment, Chromobacetrium violaceum MTCC2656 acts as a reporter strain. [25].

2.11.4 Pigment inhibition assay:

Overnight culture (14-18 hours) of Chromobacterium violaceum MTCC 2656 was inoculated in fresh LB broth with various concentrations of chitosan AgNPs. After the period of incubation, the cultures were centrifuged and the collected pellet is treated with 0.5 ml of DMSO to dissolve the violet pigment from the bacterial cells. The cell debris in the resulting solution was removed by centrifugation at 10,000 rpm for 10 minutes. The absorbance was read at 570nm in the colorimeter. [26].

Inhibition rate (%) = O.D _{570 nm control} – O.D_{570 nm sample}/ O.D_{570 nm control} *100%

3.1. Characterization of extracted chitosan:

3.1.1. Dry weight of chitosan:

The chitosan extracted from the squilla shells appears white and flaky after several treatments with a strong alkali. The final yield of chitosan is about 21%. Anand et al., 2014 stated that the chitosan extracted by the method he followed give a higher yield (17.8%) than the previously followed method by Yildiz Bolat, Şengül Bilgin, Ali Günlü, Levent Izci, Seval Bahadır Koca and University 2010; Thirunavukkarasu, Dhinamala, and Moses Inbaraj 2011.

3.1.2. FTIR characterization:

The Fourier transform of the infrared spectroscopic pattern of extracted chitosan was presented in Fig. 2. The FTIR showed characteristic bands at 3300, 3010, 2854, 2339, 1615, 1488, 1045, 851 and 597 cm^− 1. The FTIR is done to find out the biomolecules present in the characterizing solution. The band at 3300 cm^− 1 showed the O-H stretch band. The characteristic band at 3300 cm^− 1 showed characteristic stretch at N-H. The characteristic band at 3010 cm^− 1 is been assigned as C ≡ O which corresponds to the amide compound. The intense band at 3300 and 3010 cm^− 1 are characterized by hydroxyl groups in alcohol and phenol compounds. The band at 2854 and 2339 cm^− 1 were assigned to the carbonyl group and secondary amines. This characteristic band shows that chitosan is surrounded by some proteins and secondary metabolites. The absorption band at 1615 cm^− 1 corresponds to C = O amide and the peak at 1045 corresponds to C-O stretching. Thus the characteristic peak confirms that the extracted biomolecule was chitosan. [1].

3.1.3. XRD characterization:

The X-ray diffraction pattern of the extracted chitosan was presented in Fig. 3. The extracted chitosan from the squilla represented a characteristic diffraction peak at 2θ = 19.2°. Anand et. al., 2014 stated that the diffraction peak intensity of chitosan extracted from squilla was high when compared to the diffraction peaks of commercial chitosan. The XRD pattern of extracted chitosan in his paper matches the diffraction pattern obtained. The diffraction peak at 19.2° is known to be the typical fingerprint of semicrystalline chitosan. The crystallinity of chitosan is initiated by hydrogen bonds between analogous hydroxyl and N- acetyl groups. Hence the characteristic peak confirms the presence of chitosan in the extracted biomolecule.

3.2. Characterization of synthesized chitosan AgNPs:

3.2.1. UV- visible spectrophotometer:

The biosynthesized chitosan AgNPs was characterized preliminarily by a UV visible spectrophotometer in the broad range of 300- 700nm. The characteristic peak of absorbance maximum (λ max) of the chitosan-silver nanoparticle was found to be 412 nm and it is depicted in Fig. 4. Kalaivani et al., 2018 stated that the increased intensity of color from pale yellow to dark brown was because of the Surface Plasmon Resonance (SPR) of AgNO3. The control sample which is incorporated with AgNO₃ without chitosan showed no change in color. The observed peak is designated to a particular SPR of polymer-metal nanoparticle and the broadness of the peak specifies that the nanoparticles are polydispersed.

3.2.2. FTIR:

The FTIR spectrum of chitosan AgNPs was shown in Fig. 5. The FTIR spectrum of chitosan-silver nanoparticles disclosed several intensive peaks at 3421.01, 2924.98, 2426.82, 2169.36, 1563.69, 1384, 1273.61, 1024.96, 660.63, 616.33 cm^− 1. The bands attribute to 1384 and 1226 cm^− 1 are due to CO and CH bending vibrations. The bands formed in the range 3450 − 3200 cm^− 1 correspond to OH stretch vibrations. The bands composed in the range 1635 cm^− 1 are correlated with amide I a carbonyl stretch. The bands framed between 2851–2924 cm^− 1 are compatible with CH stretch vibrations. The band lying in the range 1024 cm^− 1 resembles COH, COC, and CH₂OH stretch vibrations. The band in the range of approximately 800 − 600 cm^− 1 matches to C-Br alkyl halides. The sequel distinctly identified the intricate of primary amino groups in the interaction with the surface of the metal nanoparticle, and the amino groups also acted as capping sites for AgNP stabilization [29].

3.2.3. XRD:

X-ray diffractometer is used to study and analyze the crystalline structure and size of the chitosan AgNPs. The XRD model of chitosan AgNPs was shown in Fig. 6. The number of Bragg reflections with 2θ values of 38.53°,44.67°,65.08° and 78.06° corresponding to (111), (200), (220) and (311) respectively designated that the AgNPs were spherical structured and crystalline in nature. The intense peak is due to some bioorganic compounds produced during the period of synthesis [30]. The prevailed XRD pattern exemplified that the silver ions were been reduced to AgO by the stabilization of chitosan. The pattern of the peak obtained confirmed the presence of a face-centered cubic form of metallic silver without any impurities [31].

3.2.4. PARTICLE SIZE DISTRIBUTION:

Generally, the chitosan AgNPs are mostly spherical and the size distributions fluctuate mostly between 60–100 nm in diameter. The particles having a size higher than this are also found. The present method of nanoparticle synthesis allowed the nanoparticle synthesis in a very smaller size in contrast to other different studies which used many other biological compounds as reducing and stabilizing agents [32]. The DLS image also confirmed the size ranges of silver nanoparticles in Fig. 7. In this, the size distribution of CsAgNP and its structure were assessed where the majority of AgNP are embedded in the chitosan matrix.

3.2.5. FE- SEM:

Field emission-scanning electron microscopy was used to scrutinize the topological morphology of the synthesized chitosan AgNPs. The secured image unveiled the fact that the synthesized chitosan AgNPs are almost spherical and they are all polydispersed and non-agglomerated. This might be because some of the paramount capping reagents present in chitosan seem to act as a ligand that stabilizes the AgNPs. The magnified views of chitosan AgNPs are shown in Fig. 8. FE- SEM also showed that the silver nanoparticles are been surrounded by chitosan polymer [33]. Such nanoparticles have high solidity. It is perceived that the silver nanoparticles are encompassed in a matrix of chitosan and importantly the size of such nanoparticles is confined to the nano range [19].

3.3. Screening of chitosanase-producing strains:

The screening for chitosanase was carried out with Bacillus cereus and Lactobacillus plantarum which were grown at 37℃ in plates with the newly formulated media A and media B with 1% agar-agar. The growth of the organisms in such media indicates that the organism is capable of forming chitosanase as the media contain chitosan as a sole carbon source. Among the two formulated media, media A showed good growth and high production of enzymes.

3.4. Purification of chitosanase:

In ammonium sulfate precipitation the culture supernatants were subjected to ammonium sulfate fractionation which gets precipitated at a 70% saturation point. The protein gets precipitated in water due to the higher affinity of salt to the water. The precipitated proteins were subjected to dialysis against the same buffer overnight. This will result in the removal of ammonium sulfate salt from the protein mixture. In this process of dialysis, the protein kind of macromolecules was entrapped inside the dialysis bag whereas the salt comes out of the dialysis bag. The overnight dialyzed dialysis bags were removed from the phosphate buffer and kept treated with polyethylene glycol to remove the excess phosphate buffer inside the bag. After the volume was reduced to 1/10th of the bag the dialysis bag was again dialyzed against the phosphate buffer. The contents of the tubes were dried and subjected to Lowry’s assay and DNS assay. The amount of protein estimated from 50 ml of broth containing B. cereus (extracellular enzyme) is about 26 mg, B.cereus (intracellular enzyme) is about 16 mg, and L. plantarum (intracellular enzyme) is about 55 mg. The amount of reducing sugar produced by each will be 17.77 IU/ml, 6.66 IU/ml and 33.33 IU/ml. The highest reducing sugar generated is from the chitosanase produced from L. plantarum (intracellular)

3.5. Antibacterial assays:

The bactericidal activities of chitosan AgNPs were exhibited in three different methods like disc diffusion, well diffusion method and MIC. The antibacterial assays were done against the clinical isolates of MDR E.coli, MDR S. aureusI₁ and MDR S.aureusI₂. The concentrations used in the plates were 1–50 µg/ml. The concentrations below 5µg/ml there is no bactericidal activity and from 5–50 µg/ml the zone of inhibition gets increased in both disc diffusion and well diffusion methods (Fig. 9 and supplementary table 1). The MIC value which is the lethal concentration of each MDR pathogen were represented in (Fig. 10 and Supplementary Tables 2, 3, and 4). At first, the MIC was carried out with antibiotics like vancomycin and amikacin for MDR S.aureus I₁, MDR S.aureus I₂ and MDR E.coli. The MIC values obtained by treating the organisms with antibiotics showed that the MDR strains exhibit high resistance even at 400µg/ml. When they are treated with chitosan AgNPs MDR S.aureus I₁ showed sensitiveness at 200 µg in which no growth was found, For MDR S.aureus I₂ the sublethal concentration is 150 µg and for MDR E.coli deleterious concentration is 180 µg. The main reason for the antibacterial effectiveness of chitosan AgNPs is due to the positive charge of the silver ions that were exercised through electrostatic attraction that exist between the bacteria and the nanoparticles [34]. This resulted in the successive destabilization of the bacterial cell wall and altered cell permeability which resulted in the death of pathogens [35]. Hence, imperil the permeability of the cell wall resulting in cell death. The first bonded silver nanoparticle caused the generation of gaps known as “pits” on the surface of the cell wall resulting in the accumulation of nanoparticles [36]. The invaded nanoparticles interact with sulfur and phosphorus-containing biomolecules such as the bases of DNA and degrade the DNA which led to cell death [30].

3.6. Anti-quorum sensing assays:

The development of biofilms and the bacterial quorum sensing mechanisms are closely interconnected social behavior expressed by most of the microorganisms in nature. In fact, quorum sensing promotes biofilm formation.

3.6.1. Congo red agar assay:

The Congo red assay is a qualitative assay for determining the presence of biofilm (exopolysaccharide) production by a particular microorganism. Under controlled conditions (37° for 24 hours in aerobic conditions) the microorganism having the gene icaAB capable of producing the biofilms exhibit continuous dry black crystalline colonies after the period of incubation. Such color indicates the presence of strong biofilm producers. This assay was done for MDR S.aureus I₁, MDR S.aureus I₂ and MDR E.coli which exhibited a strong biofilm formation that was illustrated in (Fig. 11). [37] [38].

3.6.2. Crystal violet tube assay:

The analysis was carried out to check the influence of chitosan AgNPs on the MDR pathogens at various concentrations for their biofilm formation. After 24 hours of incubation, the samples were treated with chitosan AgNPs, and showed weak biofilm formation and also the absence of biofilm formation at high concentrations In MDR E.coli and MDR S.aureus I₂ the biofilm inhibition efficiency is 100% at 20 µg of chitosan AgNPs and MDR S.aureus I₁ the biofilm inhibition efficiency is 100% 40 µg of the chitosan AgNPs. The biofilm formation of the treated sample is compared with the biofilm formation in the untreated sample which was shown in (Fig. 12, Supplementary Fig. 6 and Supplementary table 5) [26][39].

3.6.3. Swarming assay:

Swarming motility takes place via the synthesis of biosurfactants as a result of quorum sensing an intracellular communication system. It is an episode of group translocation of bacterial population transversely in molten or liquid media [40, 41]. In this assay, it is predicted that at 20 µg of chitosan AgNPs the migration of MDR E.coli was completely inhibited. The result obtained for E.coli was the same as for the C. violaceum MTCC 2656 which was portrayed in (Fig. 13).

3.6.4. Pigment inhibition assay:

Violacein is a violet-colored pigment produced by several strains of C. violaceum as a result of the quorum-sensing system network. This reporter strain showed that at 40 µg of chitosan AgNPs, the violacein pigment was completely inhibited and is characterized in (Figs. 14, 15, supplementary Fig. 7 and supplementary Tables 6 and 7). Compared to the untreated sample at 10 µg of nanoparticle itself the pigment was reduced to an extent. From this it is interpreted as the inhibition of violacein pigment is due to the nanoparticle interruption in the quorum-sensing system [26].

3.7. Antifungal assay:

The fungicidal activities of chitosan AgNPs were exhibited in two different methods like agar diffusion and MIC. The Aspergillus species that live in a natural environment acts as an opportunistic pathogen while they typically enter the respiratory tract causing serious pathology. The concentrations used in the plates were 100, 500 and 1000 µg of the nanoparticle. The concentration below 100 µg shows no fungicidal activity and from 100–4000 µg and the zone of inhibition get increased in agar diffusion methods. For MDR Aspergillus species the growth inhibitions were determined and presented in (Fig. 16). The MIC value which is the sublethal concentrations of each MDR pathogen were represented in (Fig. 17 and Supplementary table 8). At first, the MIC was carried out with antibiotics like Fluconazole for MDR Aspergillus species. The MIC values obtained by treating the organisms with antibiotics showed that the MDR strains exhibit high resistance even at 4000µg. When they are treated with chitosan AgNPs the MDR Aspergillus species showed sensitiveness at 1000 µg in which no growth was observed. The antifungal assay was also implemented with various concentrations of chitosanase and chitosanase immobilized chitosan AgNPs. With this, it is predicted that the activity of chitosanase immobilized chitosan AgNPs had greater efficiency which is displayed in (Figs. 18 and 19) than if they acted separately on the MDR pathogens.

In our investigation, at first, the chitosan was successfully extricated and characterized by shrimp waste. With the extricated chitosan AgNPs were successfully synthesized by biomimetics and it was characterized. Such biomimetically synthesized chitosan AgNPs unveiled exceptional antibacterial, anti-quorum sensing, anti-biofilm and antifungal activity with 100% efficiency against MDR pathogens. L. plantarum was affected in the mass production of chitosanase enzyme and such produced evinced good antifungal activity. Hence when the enzyme was immobilized in chitosan AgNPs it synergistically improvised the microbicidal effect on both MDR bacteria and MDR fungi.

Future work:

Accordingly, such enzymes immobilized in chitosan AgNPs will be used as coating agents in surgical equipment and indwelling catheters to prevent nosocomial infections by MDR pathogens, food packaging industries, and cosmetics. Consequently, our detection confirmed that the chitosanase immobilized chitosan AgNPs have excellent antifungal activity and chitosan AgNPs exhibited great antibacterial, antifungal, antibiofilm and anti-quorum sensing activity.

Acknowledgment

The authors are grateful to SASTRA Deemed University for their kind support and encouragement. We extend our sincere gratitude to the Microbiology lab at SASTRA Deemed University.

Conflict of Interest

No conflict of interest.

Author’s contribution

All authors contributed equally.

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S1.Chitosan.tiff
Extraction of chitosan from squilla shells A) Drying and grinding of shrimp shells, B) Demineralization, C) Deproteinization, D) Decolorization, E) Deacetylation and F) Drying
S2.CsAgNP.tiff
Synthesis of Chitosan AgNPs A) Solution of dissolved chitosan in 1.5% acetic acid, 0.1 M AgNO3 and 1M NaOH, B, C, D, E and F shows the change of color due to nanoparticle formation during the stipulated time of 10 hours
S3.MDRS.aureus.tiff
MDR S. aureus I1 and S. aureus I2 A) and C) Isolation of S. aureus from an infected sample, B) and D) Isolated S. aureus I1 showing resistance to Vancomycin and Isolated S. aureus I2 showing resistance to methicillin
S4.MDRE.coli1.tiff
MDR E.coli A) Isolation of E. coli from infected sample B) Isolated E. coli showing resistance to tetracycline, streptomycin, amikacin and erythromycin
S5.MDRAspergillus.tiff
Isolated Aspergillus spp. showing resistance to amphotericin and fluconazole
S6CVAssay.tiff
Semi-quantitative assay for biofilm formation A) Untreated S. aureus I1, B) Treated S. aureus I1, C) Untreated S. aureus I2, D) Treated S. aureus I2, E) Untreated E. coli and F) Treated E. coli
Supplementarytable1.docx
Antibacterial activity of CsAgNPs by agar disc diffusion method
Supplementarytable3.docx
MIC of MDR S. aureus I2
Supplementarytable4.docx
MIC of MDR E. coli
Supplementarytable5.docx
Biofilm inhibition in MDR S. aureus I1, S. aureus I2, E. coli
Supplementarytable6.docx
Pigment inhibition assay by agar well diffusion assay
Supplementarytable7.docx
Pigment inhibition assay (Tube test)
Supplementarytable8.docx
MIC of MDR Aspergillus spp.

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Investigation on anti-quorum sensing activities of Chitosan AgNP’s-chitosanase against MDR pathogens

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Abstract

Figures

1. Introduction

2. Methods

2.1. Chitosan extraction:

2.2. Characterization of extracted Chitosan:

2.3.1. Dry weight:

2.3.2. FTIR Analysis:

2.3. Biological synthesis of chitosan silver nanoparticle (Chitosan AgNPs)

2.4. Characterization of Chitosan AgNPs:

2.5. Bacterial and fungal cultures:

2.6. Enzyme production:

2.6.1. Media formulation:

2.7. Enzyme purification:

2.7.1. Ammonium sulfate precipitation:

2.7.2. Dialysis:

2.8. Chitosanase assay:

2.9. Estimation of proteins by Lowry’s assay:

2.10. Antibacterial and antifungal assays:

2.10.1. Kirby Bauer disc and well diffusion method:

2.10.2. Minimal inhibitory concentration (MIC) assay:

3. Result And Discussion

3.1. Characterization of extracted chitosan:

3.1.1. Dry weight of chitosan:

3.1.2. FTIR characterization:

3.1.3. XRD characterization:

3.2. Characterization of synthesized chitosan AgNPs:

3.2.1. UV- visible spectrophotometer:

3.2.2. FTIR:

3.2.3. XRD:

3.2.4. PARTICLE SIZE DISTRIBUTION:

3.2.5. FE- SEM:

3.3. Screening of chitosanase-producing strains:

3.4. Purification of chitosanase:

3.5. Antibacterial assays:

3.6. Anti-quorum sensing assays:

3.6.1. Congo red agar assay:

3.6.2. Crystal violet tube assay:

3.6.3. Swarming assay:

3.6.4. Pigment inhibition assay:

3.7. Antifungal assay:

4. Conclusion

Declarations

References

Supplementary Files

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