% hydrophobicity = [1 − OD after vortexing/OD before vortexing] × 100%

#### *2.11. Disruption of Preformed Biofilms*

Biofilms were allowed to develop for 24 h in the wells of microtiter plates. Non-adhering cells were washed away, new sterile TSB with or without sub-MICs of ZnO@XG was supplemented to each well and MTP was incubated at 37 ◦C for 24 h. Then, unbound cells were removed by washing and adhering cells were stained with crystal violet. Absorbance was recorded at 585 nm, as described earlier (Al-Shabib et al., 2020, [39]).

#### **3. Results and Discussion**

Nanocomposites of zinc oxide and xanthan gum were synthesized according to the scheme depicted in Figure 2. Synthesized nanomaterial was characterized by structurally and morphologically using various spectroscopic and microscopic techniques

**Figure 2.** Proposed scheme for the synthesis of ZnO@XG nanocomposite.

#### *3.1. FTIR*

The Fourier-transform infrared spectra of xanthan gum and ZnO@XG NPs are displayed in Figure 3. The FTIR spectra of XG (Figure 3a) are demonstrated the peak at 3417 (–OH stretching), 2932 (aliphatic –CH stretching), 1736 (–C=O stretching), 1614, 1414 cm−<sup>1</sup> (COO– symmetric and asymmetric stretching) and the peaks between 1049–1249 cm−<sup>1</sup> (pyranoid C-O-C ring stretching) [40]. The FTIR spectra of ZnO@XG NPs in Figure 3b represents all the characteristic peaks from XG and ZnO with vibrational frequency, e.g., 606, 774 (Zn–O bond stretching), 1049–1221 (C–O–C XG pyranoid ring), 1409, 1596 (COO– symmetric and asymmetric stretching), 1736 (–C=O stretching), 2923, 2850 (C–H aliphatic stretching of XG), 3307 cm−<sup>1</sup> (–OH stretching). The shifting in the carboxylic acid vibrational frequency suggests that the reduction as well stabilization of the Zn2<sup>+</sup> into ZnO was done through the donation of electrons (lone pairs) from oxygen from an XG–O–Zn type lattice [41,42].

**Figure 3.** FTIR spectra of (**a**) xanthan gum (XG) (**b**) ZnO@XG nanocomposite.

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## *3.2. XRD*

The XRD spectra of bulk ZnO (black line) and ZnO@XG NPs (blue line) is given in Figure 4. The XRD of bulk ZnO NPs represented the characteristic peaks of ZnO NPs at 2θ values of 31.68◦ , 32.82◦ , 36.16◦ , 47.48◦ , 56.49◦ , 58.64◦ , 62.71◦ , and 67.81◦ , which correspond to miller indices values of (100), (002), (101), (102), (110), (103), (112), (200) crystalline plane of ZnO (JCPDS 89–0510) [43]. While looking at the spectra of ZnO@XG NPs, the characteristics peaks from ZnO NPs appeared at 2θ value 30.75◦ (ZnO), 33.63◦ (ZnO), 35.96◦ (ZnO) and 52.77◦ (ZnO) with corresponding miller indices values of (110), (002), (101), (200) and (202). The spectrum reveals the peaks with shifted diffraction angle (2θ values) from the precursor values with reduced intensity of ZnO due to functionalization with XG biopolymer chains, which imparted a small amorphous character to the nanoparticles.

**Figure 4.** XRD spectra of ZnO (black line) and ZnO@XG (blue line).

Further information about the lattice structure, deformations on fusion and crystallite size can be obtained using Scherer's formula from Equations (1)–(4) (Scherrer, 1918, [44])

$$D = \frac{0.9\lambda}{\beta \cos \theta} \tag{1}$$

$$\text{DislocationDensity} (\delta) = \frac{1}{D^2} \tag{2}$$

$$\text{IntervalyerSpaceing}(d\_{200}) = \frac{n\lambda}{2\text{Sin}\theta} \tag{3}$$

$$\% \text{Crystalality} = \frac{\text{Area} \text{UndertheCrystallinePears}}{\text{TotalArea}} \times 100\tag{4}$$

2 where *D* is the crystal's size, λ is the wavelength used (i.e., 1.54 Å), β is the half-width of the most intense peak, and θ is the angle of diffraction. Using Equations (1)–(4), the average particle size of ZnO and ZnO@XG NPs was found to be 21.5 ± 1.5 and 14.7 ± 1.2 nm. The particle size of ZnO@XG NPs is also found to be in close agreement with the particle size (15.73 nm) obtained by TEM analysis. Hence, a decrease in particle size of ZnO NPs from 21.5 ± 1.5 to 14.7 ± 1.2 nm suggested the successful functionalization and reduction of Zn2<sup>+</sup> ions to ZnO@XG NPs. The formation of the nanoparticles is also supported by the interlayer spacing value, which decreases from 0.24 Å in bare ZnO NPs to 0.18 Å in ZnO@XG NPs given in Table 1. These interactions of ZnO NPs and XG biopolymer chains resulted in a decreased value of dislocation density from 3.75 <sup>×</sup> <sup>10</sup><sup>15</sup> to 2.95 <sup>×</sup> <sup>10</sup><sup>15</sup> <sup>m</sup>−<sup>2</sup> owing to contraction in size and change in crystallinity from 73% to 46% due to attachment of amorphous biopolymer chain. The XRD data analysis clearly suggested that there is a successful formation of ZnO NPs followed by surface functionalization by xanthan gum biopolymer chains.


**Table 1.** XRD parameters ZnO and ZnO@XG nanocomposite.

#### *3.3. Morphological Analysis: SEM and TEM*

Scanning electron microscopy (SEM) was employed to observe the surface morphological changes in the material during the solid-state reactions/interactions. Figure 5a,b represents the SEM image of ZnO@XG NPs at 7000×, 2 µm magnification ranges with EDX spectra within 1–20 KeV energy ranges. Figure 5a exhibits a highly porous surface morphology with a loosely agglomerated distribution of tiny nanowires of ZnO NPs on the surface (white dots) and black dots, represents the XG biopolymer matrix. Further, the atomic percentage of individual constituents used for the formation of ZnO@XG NPs was observed by the energy-dispersive X-rays (EDX) given in Figure 5b. The total output and conclusion received by EDX analysis express the composition of ZnO@XG NPs as C (72.16% ± 0.42%), O (26.42% ± 1.87%) and Zn (1.42% ± 0.48%). The transmission electron microscopy (TEM) was used for the elucidation of the optimized diameter and their variation in the XG biopolymer matrix. Figure 5c represents the TEM image of ZnO@XG NPs at 50 nm magnification range, which represents the loose agglomeration of tiny circular particles completely distributed along the XG matrix. The average size of nanorods was found to be 16.05 nm, which is in close concurrence with XRD and statistical Gaussian distribution analysis. Figure 5d was utilized to obtain the average particle size of ZnO NPs functionalized with XG biopolymer matrix using statistical domain tools like gaussian distribution. With a frequency of 16%, the average particle size was estimated as 15.73 nm, which is in close concurrence with XRD (14.7 nm) and TEM results (16.05 nm).

**Figure 5.** (**a**) SEM image of ZnO@XG nanocomposite (**b**) EDX spectra showing individual constituent elements comprising the material (**c**) TEM image of ZnO@XG nanocomposite showing the distribution of MSNs in the polymer matrix at 50 nm magnification range (**d**) Gaussian distribution of particle size for assessing the average particle size of nanomaterial.

## *3.4. BET*

The Brunauer–Emmett–Teller (BET) isotherm for ZnO@XG NPs was acquired by nitrogen adsorption–desorption method. The BET plot for ZnO@XG NPs given in Figure 6 shows a type IV pattern, which suggested that the synthesized BNC has a nearly mesoporous structure [45]. The value of BET specific surface area for ZnO@XG NPs was found to be 9.24 m<sup>2</sup> ·g <sup>−</sup><sup>1</sup> with a total pore volume of 0.045 cm<sup>3</sup> ·g <sup>−</sup><sup>1</sup> and pore diameter of 12.87 nm. The reported values of BET specific surface area of bulk ZnO NPs synthesized by different routes are given as 15.45, 34.5, 12.998 and 7.5 m−<sup>2</sup> ·g −1 [42,46–48]. Hence, the reduction in specific surface area for the current BNC material suggests the incorporation of organic moieties of XG, which leads to blocking some pores due to surface functionalization.

**Figure 6.** Low-temperature N<sup>2</sup> adsorption–desorption plot for ZnO@XG nanocomposite.

#### *3.5. MIC*

MIC of the synthesized ZnO@XG was determined against test pathogens *C. violaceum* and *S. marcescens*. MIC of ZnO@XG was recorded to be 256 µg/mL against both test pathogens. Since the current investigation was aimed to assess the quorum sensing and biofilm inhibitory of ZnO@XG, subinhibitory concentrations (0.0625–0.5xMICs) were selected for further microbiological assays.

#### *3.6. QS Interference in C. violaceum*

3 Sub-inhibitory concentrations (sub-MICs) of synthesized ZnO@Xanthan gum (ZnO@XG) nanocomposite were assessed for its quorum sensing (QS) inhibitory potential employing biosensor strain *C. violaceum* ATCC 12472 (CV12472). Production of violacein in *C. violacein* is regulated by the CviR-dependent quorum sensing system [49]. Impaired violacein production in CV12472 in a concentration-dependent manner is depicted in Figure 7B. Statistically significant (*p* ≤ 0.05) reduction of 15%, 33%, 47% and 61% was over untreated control was recorded at 16, 32, 64, 128 µg/mL concentration of ZnO@XG nanocomposite, respectively. Tested sub-MICs (16–128 µg/mL) did not have any significant effect on the growth of the bacteria (Figure 7A), and thus, it is envisaged that the observed violacein reduction by nanocomposite is due to the quorum sensing interference rather than growth inhibition. Zinc oxide nanoparticles synthesized from plant extracts of *Nigella sativa* and *Ochradenus baccatus* have been reported with similar significant violacein inhibition activity (Al-Shabib et al., 2016, 2018, [50]). Chitinase production in *C. violaceum* is also QS regulated [29]. Chitinolytic activity in *C. violaceum* treated with sub-MICs of ZnO@XG was quantified using dye-release enzyme assay. The chitinolytic activity was reduced considerably with increasing concentration as compared to the control (Figure 7C). At 16, 32, 64, 128 µg/mL ZnO@XG treatment, 19%, 28, 54%, 70% reduced chitinolytic activity was observed, respectively. This is probably the first report demonstrating quorum sensing inhibition in *C. violaceum* by polysaccharide-based zinc oxide nanocomposite.

**Figure 7.** Effect of sub-MICs of ZnO@XG on (**A**) growth (**B**) violacein production and (**C**) chitinase production in *C. violaceum* ATCC 12,472. \* denotes significance at *p* ≤ 0.05, and \*\* denotes significance at *p* ≤ 0.005.

#### *3.7. QS Interference in S. marcescens*

ZnO@XG was assessed for its QS inhibitory activity against virulence factors (prodigiosin and protease) produced by *S. marcescens*. Prodigiosin is a red pigment produced under the control of QS by *S. marcescens*. It is a vital virulence factor, playing a crucial role in the invasion, survival and pathogenicity of *S. marcescens* [51]. Figure 8B shows a concentration-dependent decrease in prodigiosin upon treatment with sub-MICs (16–128 µg/mL) of ZnO@XG. At 128 µg/mL concentration of ZnO@XG, prodigiosin declined by 71%, while at the lowest tested concentration (16 µg/mL) 25% decrease was recorded. Synthesized bio-nanocomposite did not affect the growth of the bacteria significantly at 128 µg/mL (Figure 8A). Our findings are in accordance with the results published on AgNPs synthesized from the extract of *Carum copticum*. At the highest tested concentration, AgNPs induced a 75% reduction in prodigiosin as compared to the untreated control [37].

**Figure 8.** Effect of sub-MICs of ZnO@XG on (**A**) growth (**B**) prodigiosin production and (**C**) protease production in *S. marcescens* ATCC 13880. \* denotes significance at *p* ≤ 0.05, and \*\* denotes significance at *p* ≤ 0.005.

Protease is another important QS regulated virulence factor produced by *S. marcescens*. Agents that can suppress the production of protease can be useful in potentiating the innate immune response of the host [52]. Therefore, we evaluated the effect of subinhibitory concentrations of ZnO@XG on protease production. The obtained results demonstrated that the production of protease decreased significantly (*p* ≤ 0.05) at all tested concentrations (Figure 8C). Exposure to 128 µg/mL of ZnO@XG resulted in 72% less protease production as compared to the untreated control.

#### *3.8. E*ff*ect on Biofilm and Biofilm-Related Virulence Functions*

Considering the results of the virulence assays, we selected 64 (0.25xMIC) and 128 µg/mL (0.5xMIC) for further biofilm-related assays in *C. violaceum* and *S. marcescens*.

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4

#### 3.8.1. Inhibition of Biofilm Formation

The potential of the bacteria to cause infections and survive under stressed environments is often related to its capability to form biofilms. Biofilms are complex but organized structures of adherent bacteria forming microcolonies that are enveloped in a self-secreted matrix of EPS [53]. The role of QS in the regulation of various stages of biofilm formation like attachment and maturation is very well documented [54]. Biofilm inhibitory potential of ZnO@XG against both pathogens was evaluated using micro-titer plate (MTP) assay. ZnO@XG exposure caused a significant reduction in the biofilm-forming ability of both pathogens, viz. *C. violaceum* and *S. marcescens* (Figure 9A). At 64 µg/mL concentration, biofilm formation in *C. violaceum* and *S. marcescens* was inhibited by 49% and 53%, respectively, while at 128 µg/mL, it was further reduced by 67% (*C. violaceum*) and 77% (*S. marcescens*). Visual confirmation of the quantitative MTP biofilm inhibition assay was obtained using confocal laser scanning microscopic (CLSM) analysis (Figure 9B). CLSM image of the untreated control showed a closely-knit structure having dense aggregation of cells. ZnO@XG treatment resulted in considerably reduced biofilm formation marked by decreased surface coverage, scattered appearance of cells and disturbed integrity of biofilm (Figure 9B). Consistent with this report, Ravindran et al. (2018) reported significantly reduced biofilm formation in *S. marcescens* treated with AgNPs synthesized from the root extract of *Vetiveria zizanioides*.

**Figure 9.** (**A**) Effect of sub-MICs of ZnO@XG on the biofilm formation of *C. violaceum* 12472, and *S. marcescens* ATCC 13880. Data are represented as mean values of triplicate readings, and the bar is the standard deviation. \*\* denotes significance at *p* ≤ 0.005. (**B**) confocal laser scanning microscopic images of *C. violaceum* 12472 and *S. marcescens* ATCC 13880 biofilm in the absence and presence of sub-MIC ZnO@XG.

#### 3.8.2. EPS Production

EPS is one of the most vital components of biofilm and plays a critical role in the attachment of cells to the substratum, maintenance of biofilm architecture, obtaining nutrients for cells, and protection of cells from the entry of antimicrobials [55]. EPS extracted from ZnO@XG treated and untreated cultures of *C. violaceum,* and *S. marcescens* was quantified, and the concentration-dependent decrease was recorded in both pathogens (Figure 10A). ZnO@XG at 128 µg/mL impaired the EPS production by 66% and 78% in *C. violaceum* and *S. marcescens*, respectively. Since ZnO@XG effectively reduces EPS production, it could possibly render the biofilm cells susceptible to the action of antibiotics. The observed results are in agreement with the findings of Hasan et al. (2019), [56], wherein dextrin-based poly(methyl methacrylate) grafted silver nanocomposites reduced EPS production significantly in drug-resistant bacteria and *Candida albicans*.

5

**Figure 10.** Effect of sub-MICs of ZnO@XG on (**A**) EPS production (**B**) cell surface hydrophobicity (**C**) swarming motility in *C. violaceum* 12472 and *S. marcescens* ATCC 13880. Data are represented as mean values of triplicate readings, and the bar is the standard deviation. \* denotes significance at *p* ≤ 0.05, and \*\* denotes significance at *p* ≤ 0.005. (**D**) plates demonstrating swarming behavior of *C. violaceum* 12472 and *S. marcescens* ATCC 13880 in the absence and presence of sub-MIC ZnO@XG.

#### 3.8.3. Cell-Surface Hydrophobicity (CSH)

5 Cell surface hydrophobicity is another important factor that contributes positively to the adhesion of microbial cells to the substratum. CSH facilitates adhesion by enhancing the hydrophobic interactions between the bacteria and biotic or abiotic surfaces [32]. MATH assay was employed to assess the effect of 64 and 128 µg/mL of ZnO@XG on CSH of the test pathogens. CSH of untreated controls of *C. violaceum* and *S. marcescens* was observed to be 54% and 63%, respectively (Figure 10B). CSH declined significantly with increasing concentration of ZnO@XG in both pathogens, and at 128 µg/mL, 21% and 16% CSH was demonstrated by *C. violaceum* and *S. marcescens* (Figure 10B). This drop in CSH upon treatment with sub-MICs of ZnO@XG could be responsible for the reduced biofilm formation by the test pathogens. In a study conducted on *P. aeruginosa* biofilm, it was envisaged that inhibition of CSH by copper nanoparticles was responsible for its biofilm inhibitory potential (LewisOscar et al., 2015, [57]).

#### 3.8.4. Swarming Motility

Swarming motility is flagella-driven distinctive migration behavior in bacteria that plays a vital role in the inception of nosocomial infections. Furthermore, swarming motility is accountable for the enhanced biofilm formation of the pathogenic bacteria by facilitating the attachment of cells to the substratum [58]. Therefore, any interference in swarming behavior could lead to diminished biofilm formation. The diameter of the swarm of *C. violaceum* and *S. marcescens* was measured on 0.5% LB agar plates with or without sub-MICs of ZnO@XG. In *C. violaceum*, motility was reduced by 44% and 61% at 64 and 128 µg/mL concentrations, respectively (Figure 10C,D). Similarly, swarming was impaired by 42% and 77% in S. marcescens at 64 and 128 µg/mL concentrations, respectively (Figure 10C,D). Our findings corroborate well with a previous report demonstrating impaired swarming behavior in *P. aeruginosa*, *C. violaceum*, *S. marcescens* and *L. monocytogenes* upon treatment with 0.5xMICs of biologically synthesized tin oxide nanoflowers (Al-Shabib et al., 2018).

#### *3.9. Disruption of Preformed Biofilm*

Bacteria residing in the biofilm mode are many-fold more resistant to antibiotics, disinfectants and other bactericidal agents than their planktonic forms [59]. Therefore, disruption of preformed biofilms is rather difficult, and an effective biofilm inhibitory agent must be able to eradicate preformed mature biofilms. ZnO@XG was assessed for its ability to disrupt preformed mature biofilms at sub-MICs (0.25–0.5xMIC). Preformed biofilms were inhibited significantly at the tested concentrations in both pathogens (Figure 11). Observed results demonstrated that at 0.5xMIC (128 µg/mL) of ZnO@XG, preformed biofilms were eradicated by 54% in both *C. violaceum* and *S. marcescens*. EPS matrix envelopes the biofilm cells making them resistant to all kinds of bactericidal and bacteriostatic agents by blocking their entry. The obtained results demonstrate significant disruption of preformed biofilms upon treatment with ZnO@XG, indicating that the nanocomposite could breach the EPS matrix, disturb biofilm architecture and expose the bacterial cells rendering them susceptible to antimicrobials. This is probably the first report demonstrating inhibition of preformed biofilm of *C. violaceum* and *S. marcescens* by ZnO-biopolymer nanocomposite.

**Figure 11.** Effect of sub-MICs of ZnO@XG on preformed biofilm of *C. violaceum* 12472 and *S. marcescens* ATCC 13880. Data are represented as mean values of triplicate readings, and the bar is the standard deviation. \* denotes significance at *p* ≤ 0.05, and \*\* denotes significance at *p* ≤ 0.005.

#### **4. Conclusions**

In conclusion, the study reports the successful formation of ZnO NPs followed by surface functionalization by xanthan gum biopolymer chains. Synthesized ZnO@XG were structurally and morphologically characterized using FTIR, XRD, BET, SEM and TEM. Subinhibitory concentrations of the biopolymer-based ZnO nanocomposite mitigated QS controlled virulence functions such as violacein, chitinase, prodigiosin, protease, EPS, swarming motility and CSH in pathogens, *C. violaceum* and *S. marcescens*. Further, ZnO@XG impaired biofilm formation and eradicated preformed biofilms, eventually leading to reduced pathogenicity of the test pathogens. Thus, it is envisaged that by targeting QS-regulated virulence, the likelihood of development of resistance is less, as no pressure is exerted on the growth of these pathogenic bacteria. Inhibition of virulence functions by ZnO@XG will disarm the bacteria so that they can be more easily eliminated by the host immune response. Thus, the synthesized ZnO@XG nanocomposites could be exploited to combat the threat of persistent bacterial infections and may also prevent the development of drug-resistance. Furthermore, the nanocomposite can be utilized in the food industry to prevent biofilm-based food contamination.

**Author Contributions:** Conceptualization, F.M.H. and I.H.; methodology, F.M.H., I.H., F.A.Q., R.A.K. and P.A.; software, I.H., R.A.K., and P.A.; validation, F.M.H., I.H., and A.A.; formal analysis, F.M.H. and I.H.; investigation, F.M.H., F.A.Q., and I.H.; resources, A.A.; data curation, F.M.H., F.A.Q., P.A., and I.H.; writing—original draft preparation, F.M.H., F.A.Q., and I.H.; writing—review and editing, F.M.H., F.A.Q., P.A., R.A.K., and I.H.; supervision, A.A.; project administration, A.A.; funding acquisition, A.A. All authors have read and agreed to the published version of the manuscript.

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**Funding:** This research received no external funding.

**Acknowledgments:** The authors extend their appreciation to the Deputyship for Research and Innovation, "Ministry of Education" in Saudi Arabia, for funding this research work through project no. IFKSURG-1438-006.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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