ZnO/CdS Nanocomposite: An Anti-Microbial and Anti-Biofilm Agent

Microbial infectious are becoming a global threat, which is a reason for rise in mortality of human beings. One of the reasons for this mortality has been the drug resistance in microbes. The drug resistance poses a major challenge for effective control of microbial infections, and this threat has prompted us to search for alternative strategy to control the microbial infections. Recently, nanomaterials have emerged as an alternative to conventional platforms because they combine multiple mechanisms of action into one platform due to the distinctive properties of nanosized materials. In the present research we have attempted to synthesize ZnO/CdS nanocomposite for its application as an antimicrobial agent. We have characterized the synthesized nanocomposites by X-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV-Vis), photoluminescence spectroscopy (PL), field emission scanning electron microscopy (FESEM) and high-resolution transmission electron microscopy (HRTEM). The nanocomposites have exhibited good antibacterial property against Gram positive and Gram-negative organisms by virtue of the generation of reactive oxygen species (ROS) inside the cells, as reflected by ruptured appearances in the FESEM micrographs. Apart from antimicrobial activity, it also inhibited biofilm formations in Pseudomonas aeruginosa, a causative organism in lung infection and burn associated infections.

bacterial infections are resistant to one or more of the antibiotics that are generally used to eradicate the infection [1]. There are in general two reasons for antimicrobial resistance; one being the misuse or overuse of antibiotics and second being the ability of microorganisms to form biofilm, a more prominent issue [2].
Biofilms are defined as conglomerations of bacterial cells and it remains with extra polymeric substance (EPS). EPS behaves as diffusion barrier and does not allow an entry of antibiotics inside the biofilm, thereby protecting cells residing inside the biofilm. The consequences of the restricted entry of antibiotics inside biofilm allow cells to grow and proliferate by drawing nutrients from biofilm, eventually emerging as multidrug resistant pathogens [3]. In the medical science, a recent survey shows that up to 60% of all human infections are owing to the biofilm. Therefore, effective strategies which will eradicate the microorganisms and biofilm have become a need of an hour [4]. In this regard, nanomaterials owing to their nanosized effect (higher surface to volume ratio) showed unique physical and chemical properties different from bulk materials which offer wide applications in various fields. Recently, metal nanoparticles have been reported for antimicrobial and antibiofilm activities [5]. However, semiconductor metal oxide nanostructures are better over all existing nanocomposites owing to very good property of photon absorption as well as efficient transport of photogenerated electron hole charge carriers [6]. Among semiconductor metal oxide, e.g. zinc oxide (ZnO) is tremendously explored due to wide band gap energy of 3.37 eV and large exciton binding energy (60 meV) [7]. Another reason for wide applications of ZnO nanoparticles in antimicrobial and antimicrobial research has been its constant photo-catalytical activities under harsh processing conditions [8]. Upon illumination, ZnO nanoparticles generate the electron hole pairs, and produce reactive oxygen species (ROS), which oxidizes organic matter and, thus, imparts biocidal property to ZnO [9]. However, ZnO nanoparticles are having a very low efficiency for the separation of electron hole pairs due to fast recombination of charge carriers [10]; therefore, efforts have been made to suppression of the recombination of photogenerated electron-hole pairs in ZnO nanoparticles. In this regard, ZnO nanoparticles have been doped or conjugated with other nanoparticles such as ZnO/CdTe for antimicrobial applications [11], ZnO/CdSe for photoelectrode for splitting water [12], ZnO/CdS for enhanced field emission behavior [13], ZnO/CdS for enhanced photocatalytic activity [14], ZnO/CdS for antibacterial activity [15], ZnO/CdS for enhanced photocatalytic H 2 evolution [16], and ZnO/CdS nanocomposite for Solar cell [17]. Herein, we have attempted to enhance the photocatalytic efficiency ZnO nanoparticles by synthesizing a composite with CdS nanoparticles, and further evaluated its antimicrobial and antibiofilm.

Chemicals
The as-synthesized ZnO/CdS nanocomposites were synthesized by using analyt-

Synthesis of ZnO Nanostructure
In the typical of ZnO nanostructures, 1 mmol of Zn (NO 3 ) 2 •6H 2 O and 5 mmol of KOH were dissolved in 100 mL aqueous solution. Then mixture immediately transferred into a Teflon-lined stainless-steel autoclave. The hydrothermal synthesis was carried out at 120˚C etc. for 2 h duration. After completion of the synthesis duration, product washed with distilled water and dried in oven at 70˚C.

Synthesis of ZnO-CdS Heterostructures
In addition to ZnO synthesis process 0.63 mM Cd(NO 3 ) 2 , 1.9 mM PVP and 0.0150 g SC(NH 2 ) 2 were added. Then mixture immediately transferred into a Teflon-lined stainless-steel autoclave (200 mL). The hydrothermal synthesis was carried out at temperatures 120˚C etc. for 2 h duration. After completion of the synthesis duration, product washed with distilled water and dried in oven at 70˚C.

Antimicrobial Study
Antimicrobial study was performed by measuring growth inhibition ability of

Antibiofilm Study
Antibiofilm study of ZnO and ZnO/CdS nanocomposite was studied in Pseu-domonas aeruginosa at 60 -100 Lux intensity of visible light. It was carried out by using crystal violet retention assay because crystal violet has an affinity towards polysaccharides of biofilm. In summary, the P. aeruginosa was grown overnight in Luria Bertaini (LB) medium at 37˚C with agitation. Later, the culture was diluted with LB medium (OD600-0.02), and 50 µL of the diluted culture was added to 950 µL of LB medium and allowed to form biofilm. After formation of biofilm on polystyrene plastic surfaces, the planktonic cells (cells which are suspended in the medium) were replaced with fresh medium supplemented with 0 -125 µg/mL ZnO and ZnO/CdS nanocomposite, separately, and incubated statically for 20 h at 37˚C. After incubation, planktonic bacteria were removed, and the biofilms were washed 2 -3 times with phosphate buffered saline buffer. Washed biofilms were fixed with 1 mL of methanol. After 15 min, the methanol was removed, and the plates were dried at room temperature. Crystal violet (0.1% in water) was then added to each well (1 mL/well), and the plates were incubated for 15 min at room temperature. Crystal violet was then removed, and stained biofilms were washed three times with 1 mL of water. Acetic acid (33% in water) was added to the stained biofilms in order to solubilize the crystal violet, and the absorbance of the solution was read at 590 nm with a spectroscopy (Schimadzu, Japan) [18].

Optical Properties
The optical properties of ZnO and ZnO/CdS nanocomposite samples were studies by UV-visible diffused reflectance (UV-Vis-DRS) and photoluminescence spectroscopy (PL) at taken at room temperature. The UV-visible diffused reflectance (UV-Vis-DRS) of ZnO/CdS nanocomposite was shown in Figure 1(d). The spectrum of only ZnO nanostructure which illustrates the sharp absorption band

Photoluminescence Spectroscopy (PL)
In order to shows the influence of CdS nanoparticles on the electronic properties of ZnO, the PL spectra of the ZnO in the range of 350 -650 nm at room temperature is shown in Figure 1(c). In the case of pure ZnO, a narrow UV band emission at 393 nm was observed, which can be assigned to exciton recombination, a strong blue emission at ~400 -450 nm, and a broad green band at about 554 nm as well as a strong emission at 596 nm in the yellow region, which can be attributed to intrinsic defects in ZnO as oxygen interstitials dominates the PL spectrum [19]. The intensity of the characteristic peaks in the PL spectrum of ZnO/CdS nanocomposite was smaller than that for the ZnO. The observed decrease in the overall intensity of the PL spectrum is attributed to the decrease in the oxygen vacancies [20]. We also assume that flakes like structure on the sur-

Morphology
The morphology of ZnO nanoparticles was studies by FESEM ( Figures  2(a)

Antibacterial
The  2 ) in B. subtilis and a preferred action on B. subtilis [23].
Several suggestions have been proposed to explain the mechanism of antimicrobial action of ZnO nanocomposites. The release of Zn 2+ has been suggested as one of the reasons for the antimicrobial activity [24]. According to this theory, in the permeability of membrane allowing the internalization of nanocomposites and induces the oxidative stress. However, when we assayed the molecular marker enzymes of oxidative stress viz., Catalase and Superoxide dismutase, we found that the expression levels of these two enzymes were like control. Secondly, the pore size in the cells wall of bacteria under study were small, approximately, 4 to 10 nm, so it was not practical possible to ZnO/CdS nanocomposite to enter the cell through cell membrane [25].
When the nanostructures interact with the cellular membrane of microorganisms in presence of sunlight, the electron and hole generated in which hole remains in the valence band and electron move towards the conduction band.
Hence, while, electrons in the conduction band shows higher reducing power, where holes in the valence band shown higher oxidizing power [26].

Anti-Biofilm
Biofilm is a severe problem in biomedical sector; the medical implants and indwelling devices are severely affected due to bacterial colonization and biofilm formation. The conventional antibiotic therapies have become ineffective against device-associated biofilm [29]. Thus, to overcome the inherent resistance of biofilms to antimicrobial agents, we explored ZnO/CdS nanocomposite on the biofilm of P. aeruginosa. The most distinguishing phenotype of the biofilm mode of growth is its intrinsic resistance to antimicrobial treatment and immune response killing [30]. The cells of P. aeruginosa in presence of nutrient components of media tend to grow and form biofilm after 36 -40 h. We assayed the biofilm by measuring the amount of crystal violet retained by biofilm, because crystal violet has an affinity towards the biofilm. Biofilm retains 100% crystal violet at 0 g/mL, however, biofilm retention was reduced to less than 35% at 50 μg/mL of ZnO/CdS nanocomposites. The formation of biofilm decreased with increase in the concentrations of ZnO/CdS nanocomposite. The disruption of biofilm is liable to extend the antimicrobial action of ZnO/CdS nanocomposite to the cells protected inside biofilm. Therefore, in order to know the viability of these cells inside biofilm, viable count of cells inside biofilm was performed. As can be seen from Figure 5, the cell viability inside biofilm after exposure ZnO/CdS nanocomposite decreased. At 0 g/mL, cells viability was 100%, however, viability decreased to 40% at 125 μg/mL of ZnO/CdS nanocomposites. The antibiofilm activity in P. aeruginosa was also supported by topographical observation of biofilm under FESEM at 50 μg/mL ZnO/CdS nanocomposite ( Figure   5). Under FESEM, the biofilm of P. aeruginosahas been reported as a smooth layer of matrix with an under-covered uniform cell i.e. cells embedded in the polysaccharide's matrix [18], a typical of biofilm structure. However, at 50 μg/mL ZnO/CdS nanocomposite, a truncated biofilm was observed, with cells exposed to outer surfaces ( Figure 5). The antibiofilm study is significant because the cells in the biofilm are not viable, and therefore, will not form biofilm. Earlier reports co-relate antibiofilm activity of nanomaterials due to anti-quorum sensing (anti-QS) sensing activity [31] [32]. In present study we rule out the QS mediated antibiofilm activity because our nanostructures also have brought about a reduction in the number of viable cells. We do not rule out the ROS mediated antibiofilm activity in P. aeruginosa because ROS species have been reported to carry out the oxidation of polysaccharides, proteins, and lipids biomolecules.

Conclusion
ZnO/CdS nanocomposites are promising applicant for the photocatalytic demolition of bacterial cells. ZnO/CdS nanocomposites are effective alternative to organic based drugs. Structural, optical and morphological data confirm the successful synthesis of ZnO/CdS nanocomposite. ZnO/CdS nanocomposites have shown antimicrobial activity against B. subtilis and E. coli, and the enhancement in antibacterial property by ZnO/CdS nanocomposite was concentration dependent. The ruptured appearances on bacterial cell surfaces indicate cellular membrane damage by ZnO/CdS nanocomposites. The decline in the development of biofilm in P. aeruginosa in the presence of ZnO/CdS nanocomposite shows that it is one of the excellent materials for inhibiting biofilm formations in other clinically pathogenic biofilm forming organisms. ZnO/CdS nanocomposites not only showed antibiofilm activity in P. aeruginosa, but it also inhibited the microbial population inside the biofilm and eliminates the biofilm forming ability of the microorganisms.