Synthesis and characterization of chitosan oligosaccharide-capped gold nanoparticles as an effective antibiofilm drug against the Pseudomonas aeruginosa PAO1

Microbial Pathogenesis 135 (2019) 103623

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Synthesis and characterization of chitosan oligosaccharide-capped gold nanoparticles as an effective antibiofilm drug against the Pseudomonas aeruginosa PAO1

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Fazlurrahman Khana,1, Jang-Won Leeb,1, Panchanathan Manivasagana,1, Dung Thuy Nguyen Phamb, Junghwan Oha,c, Young-Mog Kima,b,* a b c

Marine-Integrated Bionics Research Center, Pukyong National University, Busan, 48513, Republic of Korea Department of Food Science and Technology, Pukyong National University, Busan, 48513, Republic of Korea Department of Biomedical Engineering, Pukyong National University, Busan, 48513, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Antibiofilm Chitosan oligosaccharide Nanoparticles P. aeruginosa Pathogen Virulence

The infection caused by Pseudomonas aeruginosa is a serious concern in human health. The bacterium is an opportunistic pathogen which has been reported to cause nosocomial and chronic infections through biofilm formation and synthesis of several toxins and virulence factors. Furthermore, the formation of biofilm by P. aeruginosa is known as one of the resistance mechanisms against conventional antibiotics. Natural compounds from marine resources have become one of the simple, cost-effective, biocompatible and non-toxicity for treating P. aeruginosa biofilm-related infections. Furthermore, hybrid formulation with nanomaterials such as nanoparticles becomes an effective alternative strategy to minimize the drug toxicity problem and cytotoxicity properties. For this reason, the present study has employed chitosan oligosaccharide for the synthesis of chitosan oligosaccharide-capped gold nanoparticles (COS-AuNPs). The synthesized COS-AuNPs were then characterized by using UV–Visible spectroscopy, Dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), Field emission transmission electron microscopy (FE-TEM), and Energy dispersive X-ray diffraction (EDX). The synthesized COS-AuNPs were applied for inhibiting P. aeruginosa biofilm formation. Results have shown that COS-AuNPs exhibited inhibition to biofilm as well as eradication to pre-existing mature biofilm. Simultaneously, COS-AuNPs were also able to reduce bacterial hemolysis and different virulence factors produced by P. aeruginosa. Overall, the present study concluded that the hybrid nanoformulation such as COSAuNPs could act as a potential agent to exhibit inhibitory properties against the P. aeruginosa pathogenesis arisen from biofilm formation.

Introduction During the last few decades, Pseudomonas aeruginosa infections have raised serious awareness about global health and food safety [1–4]. A wide range of chronic infections are resulted from the formation of biofilm structure when the pathogen colonizes on hospital settings in a form of biofilm [5–7]. Biofilm of P. aeruginosa is a matrix composed of polymeric substances such as exopolysaccharides, proteins and extracellular-DNA (e-DNA) [8–10]. These substances develop a multitude of mechanisms to restrain the entry of antimicrobial drugs to reach to the cellular level and kill the cells encased beneath the biofilm matrix, thereby giving rise to bacterial biofilm resistance against numerous conventional antibiotics [11,12]. These mechanisms include

electrostatic interaction, degrading enzymes, efflux pumps, lowered metabolism across bacterial cell layers and intrinsic mutations [13–15]. Along with biofilm formation, production of several toxins and other virulence factors is also known to contribute significantly to P. aeruginosa pathogenesis [11,16,17]. Overcoming different resistance mechanisms developed by P. aeruginosa is considered as an effective treatment for its infections, hence the ongoing practices of several alternative approaches [16–19]. Recently, nanoformulations using natural materials have been recognized as one of the simple, cost-effective, biocompatible and non-toxic alternatives for treating P. aeruginosa biofilm-associated infections [18–21]. Chitosan is a polysaccharide derived from chitin from shrimp and crab shells and is composed of Dglucose and N-acetylated glucose [22]. Numerous research have

*

Corresponding author. Department of Food Science and Technology, Pukyong National University, Busan, 48513, Republic of Korea. E-mail address: [email protected] (Y.-M. Kim). 1 These authors are equally contributed. https://doi.org/10.1016/j.micpath.2019.103623 Received 3 May 2019; Received in revised form 14 June 2019; Accepted 16 July 2019 Available online 17 July 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.

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exploited the potentials of chitosan in the biomedical field such as cancer and other fatal diseases [23–25]. However, chitosan applications have faced difficulties due to their limited aqueous solubility at neutral pH and high viscosity [23]. Different methods have been employed to overcome such limitations, including chemical modification as well as reduction of the biopolymer molecular weight. Chitosan oligosaccharide (COS) is one of the small-sized and more water-soluble derivatives of chitosan which has been extensively used as antimicrobial and antibiofilm agents for treating pathogenic infections [26–30]. Furthermore, hybrid formulation with a nanomaterial carrier is becoming highly preferable for increasing antibiofilm efficacy and minimal drug toxicity problem [31,32]. The metallic gold nanoparticles (AuNPs) are one of the highly explored and used agents for the treatment of microbial infections [19,33–35]. AuNPs have several advantages in the biological applications due to stability, large surface to volume ratio, controlled release and low toxicity [36–38]. Particularly, numerous studies have reported the effectiveness of AuNPs in inhibiting biofilm formation of various Gram-negative and Gram-positive bacteria as an individual or a nanocarrier [19,39,40]. Due to the aforementioned significance in the antibiofilm activity of both COS and AuNPs, the present study aimed for synthesizing and characterizing the chitosan oligosaccharide-capped gold nanoparticles (COS-AuNPs), followed by applying this hybrid nanoformulation in inhibiting various phenotypic traits such as biofilm formation, virulence factors production and motility properties of P. aeruginosa.

microscopy (FE-TEM) JEM-2100F (JEOL Ltd., Tokyo, Japan). For the analysis of crystalline nature of COS-AuNPs, the powder form was diffracted using X-ray diffraction (XRD; X'Pert-MPD system, Philips, Almelo, Netherlands). Energy dispersive X-ray spectroscopy (EDX; Hitachi, S-2400, Tokyo, Japan) was carried out in order to analyze the elemental composition of COS-AuNPs. 1.3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) determination of COS-AuNPs To determine the MIC and MBC of COS-AuNPs against P. aeruginosa, the overnight grown cell culture of P. aeruginosa was diluted at a turbidity of 0.05 at 600 nm optical density (OD600) and used for biofilm formation in the presence or absence of COS-AuNPs. A volume of 250 μl of diluted cell culture was transferred to a 96-well microtiter plate in triplicates and supplemented with COS-AuNPs at the concentrations range from 16 to 8192 μg/ml. The compound and culture containing titer plate was incubated at 35 °C for 24 h in shaking (567 cpm) using plate reader (BioTek, Winooski, VT, USA). After incubation, the optical density (OD) of the culture was measured at the wavelength of 600 nm using the same plate reader. The same MIC titer plate was used for MBC determination of COS-AuNPs by viable cell count method. Briefly, a volume of 100 μl of incubated cell suspension from all wells was serially diluted to 10−7 dilution factor with fresh TSB (200 μl). The mixed cell suspension (100 μl) was spread plated onto TSA agar plate and incubated at 35 °C for 24 h. After incubation, the colonies appeared on TSA agar plate were counted. The experiment was performed in triplicates.

1. Materials and methods 1.1. Bacterial strains, culture media, chemicals and growth conditions

1.4. Growth kinetics study under shaking condition The reference strain Pseudomonas aeruginosa PAO1 with the reference number of KCTC 1637 used in this study was obtained from the Korean Collection for Type Cultures (KCTC, Daejeon, Korea). The culture media used for P. aeruginosa cultivation included tryptic soya broth (TSB) as well as TSB agar plate. The temperature for cultivating P. aeruginosa throughout the study was maintained at 35 °C. Chitosan oligosaccharide (> 10 kDa in molecular weight) was obtained from Kitto Life Co., Ltd. (Seoul, South Korea). Hydrogen tetrachloroaurate (III) (HAuCl4·3H2O) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The chemicals and reagents used in all experiments were of analytical grade.

The bacterial growth kinetics of P. aeruginosa in the presence of COS-AuNPs at sub-MIC ranging from 16 to 1024 μg/ml was assayed in a 96-well microtiter plate using the bacterial culture grown overnight (1:100 dilution), as previously described by Khan et al. [19] with slight modifications. Briefly, the titer plate was incubated at 35 °C under orbital shaking (567 cpm) in the microtiter plate reader for 24 h. The OD value at 600 nm (OD600) of bacterial cell mass was determined every 2 h time interval. The experiment was performed in triplicates. 1.5. Quantification of biofilm inhibition and mature biofilm eradication properties of COS-AuNPs

1.2. Synthesis and characterization of chitosan oligosaccharide-capped gold nanoparticles (COS-AuNPs)

The antibiofilm efficacy of COS-AuNPs at sub-MICs was quantified using both crystal violet staining and viable cell counting. For the quantification of biofilm cells, the crystal-violet method was used following the previous protocol [18]. In this method, a 96-well polystyrene microtiter plate (SPL Life Sciences Co., Ltd. Korea) was used as the surface for the bacterial biofilm formation. The P. aeruginosa cell culture grown overnight was diluted at a turbidity of 0.05 (OD600) and used for biofilm formation in the presence and absence of COS-AuNPs. Various sub-MICs of COS-AuNPs were added to the 96-well microtiter plate containing 300 μl of cell culture and incubated at 35 °C for 24 h. The free-floating (planktonic) cells were discarded after incubation and washed with water three times, followed by 20 min of crystal violet staining. The biofilm cells were washed three times with water and resuspended in 95% ethanol and were measured at OD570 by micro-titer plate reader. The colony counting method was performed to evaluate the effect of COS-AuNPs at sub-MICs on the planktonic and biofilm cells viability [42] through measuring the colony forming unit (CFU). Briefly, overnight grown cell culture was supplemented with varying concentrations of COS-AuNPs ranging from 16 to 512 μg/ml in a 96well titer plate and incubated at 35 °C for 24 h. The planktonic cells (50 μl) were then removed and serially diluted up to 10−7 dilution factor with fresh and sterile TSB. The attached biofilm cells on the surface of the titer well were washed twice with fresh TSB media. The

The methodology for COS-AuNPs synthesis was adopted from the literature as described earlier [41]. Briefly, COS-AuNPs were synthesized at 80 °C under stirring condition by mixing > 10 kDa COS (50 mg) into 10ml aqueous solution of HAuCl4·3H2O (10−4 M). Preliminary confirmation for the synthesis of COS-AuNPs was by observing the appearance of dark ruby red color as well as by absorbance measurements by UV–visible spectrophotometer (Beckman Coulter, Fullerton, CA, USA). The soluble COS-AuNPs were pelleted down by centrifugation (12, 000×g for 30 min) and washed with water for several times. The remaining impurities were removed by dialysis using the size-selective dialysis tube (12 kDa molecular weight cut-off). The obtained pure COS-AuNPs were lyophilized in powder form and used for characterization as well as antibiofilm assays. Characterization of different functional groups present in COS-AuNPs was carried out using Fourier transform infrared spectroscopy (PerkinElmer Inc., USA). The wavelength ranged from 4000 to 500 cm−1 was used for scanning the spectra of COS-AuNPs in FTIR instrument. Determination of particle size of COS-AuNPs was carried out using Dynamic light scattering (DLS) and Electrophoretic light scattering spectrophotometer (ELS-800, OTSUKA Electronic Co. Ltd., Osaka, Japan). Determination of COS-AuNPs morphology was carried out using Field emission transmission electron 2

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Fig. 1. Synthesis and characterization of COS-AuNPs. (A) UV–visible absorption spectra of COS-AuNPs, (B) FTIR spectroscopy of COS and COS-AuNPs, (C) DLS histogram of COS-AuNPs, and (D) FE-TEM images of the COS-AuNPs.

Fig. 2. Characterization of COS-AuNPs. (A) XRD spectrum of the COS-AuNPs, (B) EDX spectrum of the COS-AuNPs and (C) Stability analysis of the six-month old COS-AuNPs by UV–visible absorption spectroscopy.

suspensions (100 μl) were spread plated onto TSA agar plate and incubated at 35 °C for 24 h. After 24 h of incubation, the colonies of bacterial cells appeared on all TSA agar plates were counted and

attached biofilm cells were again added with fresh TSB, scraped off with a sterile tip and serially diluted up to 10−7 dilution factor with fresh TSB. The diluted biofilm cells as well as planktonic cell 3

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Fig. 3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination of COS-AuNPs to P. aeruginosa. (A) MIC value and (B) representative TSA plate showing the visible colonies.

Fig. 4. Effects of COS-AuNPs at different sub-MIC levels on P. aeruginosa growth kinetics under shaking condition at 567 cpm. Each experiment was repeated two times using three independent cultures.

the 96-well microtiter plate at 35 °C. After 96 h of incubation, the CFU of the planktonic as well as the attached biofilm cells from three wells of the titer plate were determined, whereas from the remaining wells the planktonic cells had been discarded. The established mature biofilm cells were washed three times with fresh and sterile TSB media. Fresh TSB (300 μl) media was added to each well along with different sub-

calculated the CFU. The experiment was performed in triplicates. The quantification of established mature biofilm of P. aeruginosa in the presence of COS-AuNPs also employed crystal violet and bacterial colony counting methods in similar sequence as described in the above section. The establishment of mature biofilm for 96 h was carried out by incubating the grown overnight cell culture with initial OD of 0.05 in 4

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Fig. 5. Effect of sub-MICs of COS-AuNPs on P. aeruginosa biofilm formation, growth and cell viability (A) Biofilm assays, (B) Growth assay, (C) CFU of biofilm cells and (D) CFU of planktonic cells of P. aeruginosa. *p < 0.05 and **p < 0.01 indicated statistical significance while ns indicated no statistical significance as compared to the control.

MICs of COS-AuNPs (16–1024 μg/ml). The titer plate was incubated at 35 °C for 24 h. The planktonic cells were removed while the attached cells were washed with water three times, followed by 0.1% crystalviolet staining. The quantification of biofilm cells was carried out after suspending in 95% ethanol at 570 nm in titer plate reader. The eradicating effect of COS-AuNPs on established mature biofilm of P. aeruginosa was also shown by colony count assay. Firstly, the overnight grown bacterial cell culture (initial OD of 0.05) was allowed to form biofilm in a 96-well microtiter plate and incubated at 35 °C for 96 h. The free floating planktonic cells were removed while the attached cells were (1) washed twice with TSB, (2) supplemented with and sterile fresh TSB along with different sub-MIC levels of COS-AuNPs (16–1024 μg/ml) and then (3) incubated for 24 h at 35 °C. After 24 h of incubation, the residual planktonic cells were again removed and the biofilm cells were washed twice with TSB and scraped off using sterile tips from the surface of the microtiter plate. The biofilm cells were then serially diluted to 10−7 dilution factor with fresh TSB and the diluted cell suspension (100 μl) containing biofilm cells was plated onto TSA agar plate and incubated at 35 °C for 24 h. The colonies of bacterial cells appeared on TSA agar plate were counted and determined the CFU. The experiment was performed in triplicates.

cell culture (with an initial OD of 0.05) was allowed to form biofilm in the presence of COS-AuNPs. In the SEM analysis, the nylon membrane (0.5 × 0.5 cm in size) was placed in the 24-well titer plate along with cell culture and COS-AuNPs. After incubation at 35 °C for 24 h, the cells were fixed overnight at 4 °C by using formaldehyde (2%) and glutaraldehyde (2.5%). The fixed cells were washed and dehydrated with increasing concentrations (from 50 to 100%) of ethyl alcohol. After dehydration, the membranes with attached cells were freeze-dried using a freeze dryer (FD8518, ilShinBiobase Co. Ltd., Korea) and then affixed to stubs followed by coating with gold for 120s using ion-sputter (E−1010, Hitachi, Japan). The biofilm cells were visualized by using JSM-6490LV (JEOL, Tokyo, Japan) at the magnification of × 5000 and voltage of 15 kV. In fluorescence microscopic analysis, the cells were allowed to grow on the surface of glass pieces (1 × 1 cm in size) placed in the 6-well titer plate in the presence or absence of COS-AuNPs with the procedure as described earlier [18]. The cells were visualized after staining with acridine orange with a working concentration of 10 μg/ ml, followed by washing with PBS. The fluorescence microscope used for checking the stained cells was Leica DMI300B fluorescence microscope (Leica Microsystems, Wetzlar, Germany) at a magnification of value × 40.

1.6. Visualization of the biofilm cells by microscopes

1.7. Analysis of hemolytic activity and virulence factors

Microscopic visualization of biofilm cells using scanning electron microscopy (SEM) as well as fluorescence microscopy was carried out to observe the effects of the COS-AuNPs on biofilm architecture as described earlier [18]. In both cases, the overnight grown P. aeruginosa

The hemolytic activity and virulence factor-producing properties of P. aeruginosa were carried out according to the previous protocol [18,43]. In all experiments, the seed culture of P. aeruginosa was taken from the overnight grown cell culture with the OD value of 0.05. For 5

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Fig. 6. SEM, fluorescence microscopy analysis and relative fluorescence intensity measurement of biofilm architecture in the presence of COS-AuNPs (512 μg/ml). (A) SEM image (B) Fluorescence image (C) Relative fluorescence intensity measurement of biofilm cells in the presence of COS-AuNPs (512 μg/ml).

with a previous protocol with slight modifications [18,47]. Each motility assay was performed using solid agar media prepared from specific components. The solid media used for swarming was prepared by adding casamino acids (0.5%) in Luria Britani (LB) along with glucose (0.5%) and Bacto agar (0.4%). Swimming media was prepared by adding Bacto agar (0.3%) along with glucose (30 mM) and casamino acids (0.2%) to distilled water. The media for twitching prepared by adding of casamino acids (0.2%), Bacto agar (1.5%) and glucose (30 mM) to LB medium. After autoclaving the swarming and swimming media, different concentrations of COS-AuNPs were added and poured in the Petri plates. To perform these types of motility, the overnight grown P. aeruginosa cell culture (5 μl) was deposited in the center of swarming and swimming agar surface. There is a slight difference in twitching motility analysis, where cell culture was stubbed with the help of a toothpick in the center of the plate and then twitching agar media containing COS-AuNPs was poured. Each plate was incubated at 35 °C for 24 h. The diameter of the area of cells traveling on the surfaces of swarming and swimming agar plates was measured. To measure the cells diameter in case of twitching, the solid agar media was firstly removed and the adhered cells on the Petri plate were stained with crystal-violet (0.1%), washed, dried and then the diameter was measured.

hemolysis experiment, seed culture was incubated with different COSAuNPs concentrations at 35 °C for 24 h in shaking incubator (250 rpm). The treated cells (50 μl) were mixed with diluted sheep red blood cells (RBCs) and incubated for 60 s under shaking (250 rpm) at 35 °C. The lysed RBCs in the supernatant were determined at 543 nm in relation to the control (which was not treated with COS-AuNPs). The effect of different concentrations of COS-AuNPs on P. aeruginosa production of pyocyanin was determined following previous protocol [44]. Briefly, the estimation of pyocyanin was performed by growing P. aeruginosa in TSB along with different COS-AuNPs concentrations. After incubation at 35 °C under shaking condition (250 rpm) for 12 h, the supernatant was collected and the amount of pyocyanin (blue-green color) pigment was extracted by using chloroform, followed by acidification with HCl (0.2 N). The acidified extract which turned into pink color was estimated by measuring the OD at 520 nm. Similarly, quantification of rhamnolipid production in the presence of a different COS-AuNPs concentrations was carried out by following the orcinol colorimetric method [45]. The measurement of rhamnolipid was performed by estimating the OD of golden yellow color at 421 nm. The quantification of pyoverdine production in the presence of different concentrations of COS-AuNPs was carried out following the procedure as described earlier [46]. Pyoverdine from the supernatant was measured at 405 nm and comparatively analyzed with the control. Similarly, the effect of COS-AuNPs on the activity of the protease enzyme from P. aeruginosa was also checked using casein-agar plate method as described earlier [19].

1.9. Statistical analysis The graphs presented in this study were constructed by GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA) and the results were presented as means ± SD. Statistical analysis of each data was also performed using one-way ANOVA.

1.8. Motility assays The methodology for different motilities types such as swimming, swarming and twitching in P. aeruginosa was performed in accordance 6

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color were in close agreement with the previously reported COS-AuNPs [41]. The presence of different functional groups in the synthesized COS-AuNPs was detected by FTIR spectroscopy and the results were represented in Fig. 1B. The FTIR spectra showed the presence of different bands corresponding to the different functional groups such as 1088 cm−1 for C-O, 1154 cm−1 for C-O-C, 1641 cm−1 for C=O and 1526 cm−1 for N-H stretching (Fig. 1B). The size and morphology of the synthesized COS-AuNPs were also checked by using DLS and FE-TEM imaging (Fig. 1C and D). The particle size of COS-AuNPs was found at an average value of 56.01 ± 3.48 nm, which was almost evenly distributed as represented in the histogram (Fig. 1C). The FE-TEM analysis showed that COS-AuNPs had spherical shape (Fig. 1D). Furthermore, the nanoparticle nature of COS-AuNPs was also confirmed by XRD analysis. The XRD results showed the presence of different peaks such as (111), (200), (220), (311) and each peak was observed at 2 theta degrees with the values of 38.23°, 44.58°, 64.89°, and 77.81°, respectively (Fig. 2A). Based on this results, it can be clearly seen that COS-AuNPs were cubic crystalline in nature which was also evidenced from the previous report [58]. The synthesized COS-AuNPs were further characterized by analysis the EDX spectra as shown in Fig. 2B. The presence of gold (Au) in COS-AuNPs was reflected by various peaks in the spectrum, and the highest peak was observed at 2.2 keV. Other peaks which indicated the presence of carbon (labeled as C; 0.3 keV), oxygen (labeled as O; 0.5 keV) and copper (labeled as Cu; 8.2 keV) were distributed along the used grid. These observations agreed with the AuNPs capped with an algal compound named fucoidan employed in one of our previous studies [19]. The stability of nanoparticles directly determines their application as an antimicrobial agent, the synthesized COS-AuNPs were therefore evaluated for their stability by being stored in solution under 4 °C in refrigerator for six months and compared with the freshly prepared COS-AuNPs samples [59]. As shown in Fig. 2C, both freshly prepared and six-month old samples exhibited genuinely similar maximum absorbance spectra of value 532 nm. The stability of COS-AuNPs throughout six months of storage which possibly benefitted from capping with COS was supportive for the use of COS-AuNPs [60,61].

Fig. 7. Eradication of established mature biofilm of P. aeruginosa by different sub-MIC levels of COS-AuNPs demonstrated by OD measurement (A) and CFU counting (B). **p < 0.01 indicated statistical significance as compared to the control.

2.2. MIC and MBC values determination of COS-AuNPs 2. Results and discussion The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination of COS-AuNPs against the P. aeruginosa growth is considered as an essential step before the studies on their inhibitory activities against the bacterial biofilm formation and virulence factors production, as this step helps selecting the suitable COS-AuNPs concentration range that allowed the bacterial to grow and then form biofilm, thus enabling subsequent biofilm inhibition assays. The MIC and MBC values of COS-AuNPs against P. aeruginosa were found to be 4096 μg/ml and 8192 μg/ml, respectively (Fig. 3A). This result was also confirmed by viable cell count method which was demonstrated by Fig. 3B. In order to determine the sub-MIC of COS-AuNPs, the study of P. aeruginosa growth kinetics was conducted in the presence of different COS-AuNPs concentrations which were below the MIC (16–1024 μg/ ml) under agitation condition and determined by OD measurement at 600 nm. Results demonstrated by Fig. 4 revealed that the bacterial population growth responses were varied according to COS-AuNPs concentrations and the maximum growth rate was achieved at around 12 h–16 h under shaking condition (567 cpm). P. aeruginosa growth at 1024 μg/ml and 512 μg/ml were both slightly reduced after 24 h of incubation in comparison with control. The lag phase at these two concentrations was prolonged for almost 8 h whereas for other concentration (from 16 to 256 μg/ml) the lag phase were found to be 4 h. Overall, the concentrations which were below the MIC (ranging from 16 to 512 μg/ml) were selected as the sub-MIC for further biofilm inhibition and virulence factors attenuation studies in P. aeruginosa.

2.1. Synthesis and characterization of COS-AuNPs Biofilm-associated infections caused by P. aeruginosa have become a serious health issue [48]. The bacterial rapid resistance to antibiotics via multiple mechanisms has therefore urged for alternative antibiofilm therapeutic strategies [17]. As exploitation for the potentials of natural compounds is becoming increasingly attractive during the recent time, numerous compounds have been found having effective antibacterial and antibiofilm activities [49–53]. One of these compounds is known as chitosan and its derivatives, which can be in various forms and synthesized by various methods [54,55]. COS is a low molecular-weighed derivative of chitosan synthesized either chemically or enzymatically with the aim of improving the water solubility of chitosan [56]. Although previous studies have confirmed the bactericidal and biofilm inhibitory activities of COS and AuNP as an individual [57], the present study was the first to employ COS as a capping agent for AuNP and exploit the antibiofilm activity of the hybrid nanoformulation. Therefore, the characterization step was firstly performed. The synthesized COS-AuNPs were preliminary confirmed based on the UV–visible spectral scanning as well as checking the appearance of a new color. The UV–visible spectra of the synthesized COS-AuNPs demonstrated the maximum absorbance peak at the 532 nm wavelength (Fig. 1A). Furthermore, the appearance of ruby red color is another indicator of the synthesized COS-AuNPs. The results of UV-spectra (with maximum absorbance peak at 532 nm) and also the appearance of

7

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Fig. 8. Effect of COS-AuNPs on the hemolysis and virulence factors production in P. aeruginosa. (A) Hemolysis assay, (B) Pyocyanin production, (C) Rhamnolipid production and (D) Pyoverdine production. Each virulence factor production represented as relative amounts as compared with the control. All the experiments were performed in triplicates. **p < 0.01 indicated statistical significance as compared to the control.

respectively, (Fig. 5C). In contrast, these COS-AuNPs levels did not affect the planktonic cell viability in comparison with the control (Fig. 5D). Overall, combining with the crystal violet staining and CFU results, the presence of COS-AuNPs exhibited inhibitory activity towards the attachment of sessile cells to the surface of microtiter plate.

2.3. Biofilm inhibition properties of COS-AuNPs The chemically characterized COS-AuNPs were examined for its biological activity against P. aeruginosa. Crystal violet staining assay has shown that a statistically significant inhibition of P. aeruginosa biofilm in a concentration-dependent manner was achieved by COS-AuNPs at all tested sub-MIC concentrations (16–512 μg/ml) (Fig. 5A). No growth inhibitory effect on the bacterial cells of P. aeruginosa was observed in the presence of tested COS-AuNPs concentrations. (Fig. 5B). A number of past studies reported the biofilm inhibitory activity of each individual of the COS-AuNPs hybrid nanoformulation. Particularly, the antibiofilm property of chitosan and chitosan derivatives such as COS was resulted from the polycationic nature (given by the presence of amino groups) electrostatically interacting with the negative charge of biofilm matrix components such as exopolysaccharides, e-DNA and of bacterial cells membrane [62–65]. The antibiofilm activity of AuNPs, which is given by their electrostatic interaction with biofilm and activation of host immune response, has been recognized in either individual form [66] or combination form with other antimicrobial agents such as antibiotics [61], natural compounds [19,40,67] and small molecules [68,69]. The biofilm inhibitory activity of both individual COS and AuNPs was likely to maintain when they are hybridized into a nanoformulation. Results from bacterial colony counting methods which aimed to evaluate the viability of bacterial planktonic and biofilm-forming cells have also agreed with the crystal violet results discussed above (Fig. 5). The presence of COS-AuNPs at various sub-MICs from 16 to 512 μg/ml significantly suppressed the viability of biofilm-forming cells in a concentration dependent manner. Particularly, these sessile cells were the most significantly inhibited by COS-AuNPs at 256 and 512 μg/ml, with 2.7 and ~2.8 log CFU reduction in comparison with the control,

2.4. Microscopic analysis for biofilm architecture in the presence of COSAuNPs The biofilm inhibition properties of COS-AuNPs were also confirmed by the microscopic visualization of biofilm cells using SEM and fluorescence microscope. A significant inhibition in the attachment of bacterial cells to the surface of the nylon membrane was found when the cell was treated with COS-AuNPs (512 μg/ml) (Fig. 6A); whereas the non-treated control cells showed dense layers on the surface of the nylon membrane. Furthermore, the fluorescence microscopic analysis of the cells also showed similar results as the SEM results. The COSAuNP-treated cells showed significantly less green color fluorescence intensity in comparison with the control (Fig. 6B and C). 2.5. Eradication of COS-AuNPs to established mature P. aeruginosa biofilm Dispersion of the old mature biofilm of P. aeruginosa is also equally important as inhibition their formation. P. aeruginosa (one of the human pathogens) commonly colonizes on the surface of several medical devices such as urinary catheter and ventilators and form biofilm for longterm survival and resistance against environmental conditions, thereby causing nosocomial infections known as ventilator-associated pneumonia and urinary tract infection upon patient contact [70,71]. Hence, it is essential to evaluate the efficacy of COS-AuNPs at sub-MICs in eradicating the mature P. aeruginosa biofilm [8,12]. In the present 8

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Fig. 9. Effect of COS-AuNPs on swarming (A and B), swimming (C and D) and twitching (E and F) motilities of P. aeruginosa. Each experiment was performed in triplicates. **p < 0.01 indicated statistical significance as compared to the control.

aeruginosa is one of the up-to-date alternative approaches to combat the bacterial pathogenesis [43,74,75]. During the post-antibiotic era, targeting virulence factors which leads to ‘weakening’ the bacteria was proposed to reduce the pressure for mutation, emergence and selection of resistance strain in the bacterial population and revitalize the applications of numerous antimicrobial agents, especially the conventional antibiotics [76]. Currently, anti-virulence strategies involve inhibiting virulence factors that associate with (1) adhesion/attachment of biofilm cells, (2) toxins production, (3) type three secretion system and (4) quorum sensing [77]. P. aeruginosa produces several virulence factors such as pyocyanin, rhamnolipid, pyoverdine and protease enzymes which essentially help in the bacterial infection and survival in the host [74,78–81]. The production of pyocyanin (i.e. a blue-green pigment) was inhibited in a concentration-dependent manner by COSAuNPs (Fig. 8B). The pyocyanin production was found to be reduced up to 64.6% at 512 μg/ml concentration of COS-AuNPs (Fig. 8B). Similarly, all the tested concentrations of COS-AuNPs showed significant rhamnolipid reduction with the value of 54% at 512 μg/ml (Fig. 8C). However, the presence of COS-AuNPs enhanced the production of pyoverdine (a siderophore) as compared to the non-treated control (Fig. 8D). We have also checked the effect of different concentrations of COS-AuNP on the protease activity. However, there was no significant effects (data not shown). Overall, the present study has shown that COS-AuNPs were able to inhibit the production of some important virulence factors of P. aeruginosa, adding up to the diversity of potential anti-virulence agents. Different motility types displayed by P. aeruginosa are important to the searching for the biotic and abiotic surfaces followed by the attachment and formation of biofilm. There are two types of motility, one is flagellar-mediated (swarming and swimming) and the other is type IV pili/fimbriae-mediated (twitching) [82–84]. The flagellar-mediated motility is involved in the chemical sensing; whereas type IV pili-

study, crystal violet and bacterial colony counting assays were employed (Fig. 7A and B). Results from both assays have shown that the presence of COS-AuNPs at sub-MICs significantly dispersed the established mature (96 h) biofilm of P. aeruginosa, as compared to the control. Furthermore, as shown by CFU result, a significant amount of 96-h mature biofilm cells have been removed after further incubation with COS-AuNPs at all sub-MIC levels. In fact, the eradicating property of COS and AuNPs towards pathogenic bacteria has been reported previously [19,27,72]. Despite detailed mechanism of the hybrid formulation as well as these two individuals remained not well studied, it is the surface charge of AuNPs that was claimed to give rise to the biofilm eradication activity [73]. Combining with polycationic nature of COS, the two individual compounds have contributed to the biofilm eradication effect of their hybrid nanoformulation. Overall, based on the above findings, it can be concluded that the synthesized COS-AuNPs exhibited both biofilm inhibition and eradication to the established mature biofilm of P. aeruginosa. 2.6. Anti-hemolytic and anti-virulence properties of COS-AuNPs P. aeruginosa could infect the animal and human by disrupting the red blood cells (RBCs). We have checked the effect of bacterial hemolytic properties in the presence of different COS-AuNPs sub-MIC concentrations (32–512 μg/ml). The results showed a concentration-dependent inhibition of hemolytic property of P. aeruginosa (Fig. 8A). The reduction in the hemolytic properties of P. aeruginosa was found up to 59% at high COS-AuNPs concentration (512 μg/ml). Similarly, a genuinely equal percentage of bacterial hemolysis (59%) was also reduced and concentration-dependent manner was observed in the presence of fucoidan-gold nanoparticles (F-AuNPs) at sub-MIC level (256 μg/ml) [19]. In addition, the strategy to inhibit several virulence phenotypes of P. 9

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supported by the grants (No.16162MFDS607 in 2016 and 2020) from the Ministry of Food and Drug Safety, Republic of Korea.

meadiated motility acts as the mechanosensors and helps in the attachment to the biotic or abiotic surfaces [82,85]. After reaching and attaching onto the biotic or abiotic surfaces, the bacterial cells start producing the extracellular polymeric substances and make the biofilm structure enclosing different types of the cells with various physiological states [9,82,86]. Thus, attenuating these motility types is also considered as a potential approach to inhibit the infections caused by P. aeruginosa biofilm formation. In the present study, the effects of COSAuNPs towards swarming, swimming and twitching motilities have been examined. Except for swarming, the swimming and twitching motilities were found to inhibited by concentration-dependent (Fig. 9). However, the swarming motility was found to enhance at higher concentration of COS-AuNPs (Fig. 9A and B). The swimming inhibition at higher concentration (256 μg/ml) was found up to 69%; whereas the inhibition to twitching motility was approximately 38% at 256 μg/ml concentration of COS-AuNPs (Fig. 9C, D, E and F). There are several reports on the motility-modulating properties of the drugs where they exerted opposite effects on P. aeruginosa motility properties [43]. In addition, previous studies have shown that unlike swimming which is solely driven by flagellar function, motive force and chemotactic signal, swarming further requires iron limitation, production of wetness agent (rhamnolipid) and cis (3′–5′)-cyclic-diguanylate-monophosphate (c-di-GMP) and even the involvement of type IV pili [84,87–90]. Although the essentiality of type IV pili in swarming, as well as the interaction between type IV pili and the main swarming machinery – flagella remain unknown, the pili system has been recently reported to slow down the swarming motion and its role in swarming is optional depending on the bacterial strain [91,92].

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3. Conclusion In conclusion, we have reported that the water-soluble and highly stable COS-AuNPs synthesized from naturally derived chitosan oligosaccharide material can be a potential drug against P. aeruginosa. The activity included inhibition of biofilm at the initial stage and dispersion of established mature biofilm. These properties were confirmed by microscopic analyses using SEM and fluorescence microscope. Furthermore, COS-AuNPs were found to inhibit the bacterial hemolysis as well as reduce the production of some virulence factors from P. aeruginosa. Apart from the above properties, COS-AuNPs also attenuated the bacterial swimming and twitching motilities. For future studies, the above-mentioned phenotypic properties need to also be confirmed either by considering the mutant strain of respective phenotypes or at the molecular level in wild strain by studying the gene expression of biofilm forming, virulence factor-producing, motility properties and quorum sensing signaling genes. Additionally, the efficacy test using the animal models is required. Overall, the synthesized COS-AuNPs can be used as a potential agent to control the infections associated with P. aeruginosa biofilm formation. Conflicts of interest The authors declare that they have no conflict of interest. Author contribution The idea of the present study was conceived, designed the experiment, analyzed the data and wrote the paper by FK, DTNP, PM, OJ and YMK. The experiment was carried out by FK, JWL, PM and DTNP. Funding The present research work financially supported by Marine Biotechnology Program (Grant number 20150220) funded by Ministry of Oceans and Fisheries, Republic of Korea. This study was also 10

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