**Fabrication of Zinc Oxide-Xanthan Gum Nanocomposite via Green Route: Attenuation of Quorum Sensing Regulated Virulence Functions and Mitigation of Biofilm in Gram-Negative Bacterial Pathogens**

#### **Fohad Mabood Husain 1,\*, Imran Hasan <sup>2</sup> , Faizan Abul Qais <sup>3</sup> , Rais Ahmad Khan <sup>4</sup> , Pravej Alam <sup>5</sup> and Ali Alsalme 4,\***


Received: 14 November 2020; Accepted: 3 December 2020; Published: 5 December 2020

**Abstract:** The unabated abuse of antibiotics has created a selection pressure that has resulted in the development of antimicrobial resistance (AMR) among pathogenic bacteria. AMR has become a global health concern in recent times and is responsible for a high number of mortalities occurring across the globe. Owing to the slow development of antibiotics, new chemotherapeutic antimicrobials with a novel mode of action is required urgently. Therefore, in the current investigation, we green synthesized a nanocomposite comprising zinc oxide nanoparticles functionalized with extracellular polysaccharide xanthan gum (ZnO@XG). Synthesized nanomaterial was characterized by structurally and morphologically using UV-visible spectroscopy, XRD, FTIR, BET, SEM and TEM. Subinhibitory concentrations of ZnO@XG were used to determine quorum sensing inhibitory activity against Gram-negative pathogens, *Chromobacterium violaceum,* and *Serratia marcescens*. ZnO@XG reduced quorum sensing (QS) regulated virulence factors such as violacein (61%), chitinase (70%) in *C. violaceum* and prodigiosin (71%) and protease (72%) in *S. marcescens* at 128 µg/mL concentration. Significant (*p* ≤ 0.05) inhibition of biofilm formation as well as preformed mature biofilms was also recorded along with the impaired production of EPS, swarming motility and cell surface hydrophobicity in both the test pathogens. The findings of this study clearly highlight the potency of ZnO@XG against the QS controlled virulence factors of drug-resistant pathogens that may be developed as effective inhibitors of QS and biofilms to mitigate the threat of multidrug resistance (MDR). ZnO@XG may be used alone or in combination with antimicrobial drugs against MDR bacterial pathogens. Further, it can be utilized in the food industry to counter the menace of contamination and spoilage caused by the formation of biofilms.

**Keywords:** xanthan gum; zinc oxide; nanocomposite; quorum sensing; biofilm; virulence; *S. marcescens*; *C. violaceum*

#### **1. Introduction**

Xanthan gum is a natural anionic extracellular polysaccharide. The non-toxic and biocompatible nature of this polymer makes it quite useful for the food sector [1]. The inorganic particles, such as metals, can easily be adsorbed onto it to form a stable emulsion without altering the interfacial tension. This is a Food and Drug Administration (FDA)-approved biopolymer for the food industry [2,3]. Zinc is an essential micronutrient and therefore extensively prescribed for its nutritional supplement in case of deficiency for human health. As zinc is biocompatible, there is not much risk associated with public health and is used as food coating materials [4]. The unique property of zinc oxide nanoparticles is exploited as promising antibacterial, antibiofilm, or antivirulence candidates (Al-Shabib et al., 2018; Sirelkhatim et al., 2015, [5,6]).

Tremendous growth and spread of multidrug resistance (MDR) among microbial pathogens have become a global concern for human health countries [7]. The worldwide deaths of human-caused by antimicrobial resistance (AMR) is a major contributor to global mortality after cancer and cardiovascular diseases [8]. The problem caused by AMR has reached an alarming situation, and if not action is taken, it is expected to cause more global mortality than cancer by 2050 [9,10]. The infections caused by MDR pathogens are an epidemiological concern and worsen the treatment of infectious diseases by diminishing the therapeutic effectiveness of antibiotics [11,12]. Moreover, AMR is not only problematic for public health but also poses an extra burden on the environment and livestock. The first half 20th century is regarded as the golden era for the discovery and development of antibiotics. Nearly 70% of all antibiotics used so far were discovered by 1960. After this, there was poor progress in the antibiotic drug discovery and in the last four decades, only a few antibiotics exhibiting a novel mechanism of action are discovered [13]. The injudicious use of antibiotics in public health and health and environment creates a selection pressure that results in the development of AMR among microbial pathogens [14,15].

The risk of AMR development is so much that even the antibiotics of the last generation cannot be entrusted for prolonged applications. Hence, there are two major issues associated with the discovery or development of antibacterials; the first is to find or make new chemotherapeutic antimicrobials with novel modes of action, and the second is to minimize the risk of development of AMR against the discovered antimicrobials. This has led the researchers to focus on the development of alternative anti-infective strategies to combat AMR.

Quorum sensing (QS) is a communication system occurring via chemical signaling that operates as a function of the density of the bacterial population [16]. These chemical signal molecules are called autoinducers (AIs). Certain phenotypes of bacteria are only expressed when their population has reached a certain threshold value. Many clinically important traits of bacteria, such as virulence production, biofilms formation, expression of drug-resistant genes, are controlled via QS [17]. As the bacterial population increases, the concentration of AIs increases and triggers the expression of QS-regulated genes by transcriptional regulation when it reaches a certain limit [18]. Biofilms are the microbes residing in a biopolymeric matrix. Earlier, it was thought that bacteria live in a planktonic state. However, it was discovered that most of the bacteria form complex structures called biofilms [19]. A large number of bacterial infections are encouraged or caused by the formation of biofilms by the pathogenic or opportunistic pathogens [20,21]. One of the novel strategies in antibacterial drug discovery is to selectively target the bacterial quorum sensing and biofilms. Among new drug candidates, natural/biocompatible products and nanoparticles have proven as an important therapeutic antimicrobial alternative (Husain et al., 2019; Qais et al., 2018, 2019, [22–24]).

In this study, green synthesis of zinc oxide xanthan gum (ZnO@XG) nanocomposite was done. The antiquorum-sensing potential of ZnO@XG was tested against biosensor strains of Gram-negative bacteria. The QS controlled violacein production, and chitinase activity was tested in *C. violaceum*. QS regulated virulence traits of *S. marcescens* such as prodigiosin production, and protease production were also assessed at subinhibitory concentrations of ZnO@XG. Further, the effect on biofilm formation, preformed biofilm and factors such as EPS production, swarming motility, and cell

surface hydrophobicity that contribute to the development of biofilm against both the bacteria was also studied. This is probably the first study assessing the quorum sensing and biofilm inhibitory potential of green synthesized polysaccharide-zinc oxide nanocomposite.

#### **2. Materials and Experimental Methods**

#### *2.1. Chemicals*

Xanthan gum extracted from *Xanthomonas Campestris* biological grade was purchased from Sigma-Aldrich (Bangalore, India). Zinc nitrate (Zn (NO3)2·6H2O white crystals) and sodium hydroxide (NaOH) were purchased from Merck (Mumbai, India). All the chemical materials were used without any purification or refinement. All the aqueous solutions were prepared using deionized water.

#### *2.2. One-Pot Green Synthesis of ZnO@XG Nanoparticles*

The Nanoparticles were consolidated by using a chemical coprecipitation scheme using an ecological green route [25]. In a 3 necked round-bottomed flask, a 100 mL solution of 0.45 M Zn (NO3)2·6H2O was taken and placed under magnetic stirring (900 rpm) for 30 min to obtain homogeneity. A solution of 2.3% xanthan gum was prepared by dissolving 2.3 g powder in 100 mL deionized water with vigorous stirring at 40 ◦C for 2 h to obtain a complete bubble-free homogeneous solution. Now the blended solution of xanthan gum with 20 mL of 0.1 M NaOH solution was added drop-by-drop to the aqueous ionic solution of Zn2<sup>+</sup> in order to extend the reducing character of xanthan gum to the bulk of Zn2+. The mixture remained on vigorous stirring under observation at 40 ◦C, and the progress of the reaction was checked by taking small aliquots of the reaction mixture at different time intervals to verify using UV-vis spectroscopy (Figure 1). Finally, after 8 h, a white precipitated colloid was obtained from which the product was isolated using a centrifuge (REMI rpm 8500). The product was squeezed using deionized water seven to eight times for the efficient removal of nonreactive species and dried in a hot air oven for 3 h at 60 ◦C.

**Figure 1.** Time-dependent UV-vis spectra for green synthesized ZnO@XG nanocomposite.

#### *2.3. Analytical Techniques Used for Characterization*

The prepared nanocomposite and its crystal structure were characterized by several characterization techniques such as FTIR, XRD, SEM-EDX, TEM, BET and UV-Vis. The type of bonding and functional groups present in the synthesized material was investigated by using Fourier-transform infrared spectroscopy (FTIR) PerkinElmer (PE1600, PerkinElmer, Waltham, MA, USA) in the frequency range of 400–4000 cm−<sup>1</sup> with transmission mode. The crystal phases of the synthesized material were collected on an X-ray diffractometer (A Rigaku Ultima 1 V, Woodlands, TX, USA). The morphologies of

1

the sample were analyzed by using scanning electron microscopy (SEM; JEOL GSM 6510LV, JEOL, Tokyo, Japan). The elemental size and dispensation of the sample were examined by JEM 2100 (Tokyo, Japan) transmission electron microscopy (TEM). For the analysis of aliquots of ZnO@XG samples during the synthesis process, Shimadzu UV-1900 UV-vis double beam spectrophotometer was taken into consideration. The specific surface areas of the synthesized material were tested on Micromeritics Tristar II (Micromeritics, Atlanta, GA, USA) and calculated using the Brunauer–Emmett–Teller (BET) method.

#### *2.4. Bacterial Strains*

*Chromobacterium violaceum* ATCC 12472 and *Serratia marcescens* ATCC 13880 were used to evaluate the QS and biofilm inhibitory property of the synthesized nanocomposites. Stock cultures of all the test bacteria were maintained on nutrient-agar under refrigeration and subcultured in Luria–Bertani (LB) broth for 24 h. Overnight culture (1%) was added to the fresh LB medium [the final optical density (OD) was adjusted to 0.1 at 600 nm].

#### *2.5. Determination of Minimum Inhibitory Concentration (MIC)*

The minimum inhibitory concertation of ZnO@XG against test bacteria was assessed by microbroth dilution assay as described previously [26,27]. Briefly, bacteria were cultured in the presence of different concentrations (1024–0.125 µg/mL) of ZnO@XG. Post incubation, TTC (10 µL) was added to each well and incubated at room temperature for 20 min to observe a change in color. The lowest concentration at which the development of pink color was not observed was termed as the MIC.

#### *2.6. Violacein Inhibition Assay*

Violacein pigment production was quantified spectrophotometrically using the previously described protocol [28]. Briefly, *C. violaceum* 12,472 (CV12472) was grown overnight in the absence and presence of sub-MICs (16–128 µg/mL) of ZnO@XG at 30 ◦C. Post incubation, 1 mL culture was centrifuged (10,000 rpm) for 5 min, and violacein pigment was extracted from the pellet using 1 mL DMSO. The extracted mixture was centrifuged to pellet out the bacterial cells. The optical density (OD) of supernatant was recorded at 585 nm using a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan).

## *2.7. Chitinolytic Activity*

A dye-release assay involving chitin azure was employed to quantify the chitinase produced by CV12472 [29,30]. Briefly, 100 mL bacterial supernatant obtained from treated and untreated bacteria was mixed with 1 mL phosphate buffer containing 10 mg chitin azure and incubated overnight at 37 ◦C. The insoluble substrate was pelleted out, and absorbance was read at 585 nm.

#### *2.8. Prodigiosin Assay*

Production of red-colored prodigiosin in ZnO@XG treated and untreated *S. marcescens* was quantified using the method described previously [31]. Briefly, pellets obtained from the overnight grown cultures of *S. marcescens* were resuspended in 1 mL acidified ethanol and vortexed vigorously. The mixture was centrifuged at 13,000 rpm for 5 min, and the resulting supernatant was read for absorbance at 534 nm.

#### *2.9. Protease Assay*

The proteolytic activity in cell-free supernatant of *S. marcescens* was assessed using azocasein as the substrate [32]. ZnO@XG treated and untreated cultures of *S. marcescens* were grown overnight on shaking, and cell-free supernatants (CFS) were collected by centrifugation (7000× *g* for 10 min). Subsequently, CFS (75 µL) was added to 2% azocasein (125 µL) in 0.25 mol Tris (pH 8.0) and incubated for a half-hour at 37 ◦C. The reaction was stopped with the addition of 10% Trichloroacetic acid, and the

mixture was centrifuged for 10 min. The absorbance of the resultant supernatant was measured at 440 nm.

#### *2.10. Biofilm Inhibition Studies*

#### 2.10.1. Microtiter Plate Assay

Overnight grown test pathogens *C. violaceum* and *S. marcescens* were diluted in wells containing fresh Tryptic soy broth, sub-MICs of ZnO@XG was added to wells and incubated at 37 ◦C for 24 h duration. Wells were decanted to get rid of unattached cells and washed with sterile water. Then bound cells were stained with crystal violet and incubated. After 15 min incubation, the dye was removed from wells, and thorough washing was done to remove excess stain. The absorbance of each well was measured at 585 nm to quantify the biofilm inhibition (Al-Shabib et al., 2020, [33]).

#### 2.10.2. Confocal Laser Scanning Microscopic (CLSM) Visualization of Biofilm Structure

CLSM analysis of biofilm of ZnO@XG treated and untreated test pathogens formed on glass coverslips were performed using the protocol described previously (Al-Shabib et al., 2020, [34]). Biofilm was developed on glass coverslips placed in 24 well tissue culture plates as described above. CLSM imaging was done on coverslips stained with 0.1% acridine orange in the dark under JEOL-JSM 6510 LV confocal laser scanning microscope.

#### 2.10.3. Quantification of EPS

EPS was quantified from test pathogens grown in the presence and absence of sub-MICs of ZnO@XG at 30 ◦C for 24 h. Incubated cultures were centrifuged, ice-cold ethanol (3 volumes) was added to the resultant supernatant, and the mixture was left at 4 ◦C for 18 h. Subsequently, the mixture was centrifuged, and the pellet was dissolved in 1 mL deionized water [35]. EPS was quantified using the standard method for the estimation of sugars, as described previously [36].

#### 2.10.4. Swarming Motility

Effect of sub-MICs of ZnO@XG on the swarming motility of *C. violaceum* and *S. marcescens* was determined by point inoculating the bacteria on LB soft agar plates and incubating the plates at 30 ◦C for 18 h. Briefly, LB soft agar plates (% agar *w*/*v*) were prepared, containing ZnO@XG. No treatment was given to control plates. Bactria were point-inoculated in soft agar plates and incubated under static conditions. Post incubation, the diameter of the swarm was measured in treated and control plates to evaluate the inhibition of swarming motility [37].

#### 2.10.5. Microbial Adhesion to Hydrocarbon (MATH) Assay

MATH assay was used to evaluate the effect of ZnO@XG on cell surface hydrophobicity of the test pathogens [38]. Optical density (OD) of treated and untreated bacteria was recorded at 600 nm. Then, toluene was added to each set and vortexed for 10 min, and the mixture was left for separation of phases. The aqueous phase was collected, and the absorbance was recorded at 600 nm. Percent of hydrophobicity was calculated using the following formulae:
