# Biosynthesized Zinc Oxide Nanoparticles Disrupt Established Biofilms of Pathogenic Bacteria
## 3 Results and Discussion
### Inhibition of the Biofilm Development
#### 3.2.2 Biofilm Inhibition by ZnO-NPs on the Glass Surface  
The inhibition of biofilm development by ZnO-NPs of test bacteria was further explored on glass surface. The detailed analysis of the changes in biofilm architecture was studied using confocal microscopy, electron microscopy, and light microscopy. The findings are discussed below. The preliminary qualitative examination of the biofilms inhibition on glass surface was performed using light microscopy. The light microscopic images of biofilms of test bacteria without and with highest respective sub-MICs of ZnO-NPs are shown in Figure 4. The micrographs show that there was dense biofilm formation on glass surface in the control groups of all bacteria. The bacteria were heavily colonized and can be seen as huge clumps of cells (Figure 4A,C,E). However, the treatment with 1/2 \(\times\) MIC of ZnO-NPs remarkably reduced the biofilms formation on surface glass coverslips. Treatment with ZnO-NPs resulted in less colonization of cells to the solid support, and the bacteria were observed as scattered cells. The confocal microscopic images of biofilms in the absence and presence of ZnO-NPs are shown in Figure 5. In control slides, all tested bacteria were found to be greatly colonized on the surface of glass coverslips. The bacteria were observed as thick mat-like structures of biofilms in many layers. On the contrary, ZnO-NPs reduced the bacterial biofilm development of solid support in which their colonization was extensively diminished. The bacteria in the treated group were found to be in scattered form and mostly as a single layer of cells on the surface of glass coverslips. These results agree with previous findings, where zinc oxide-xanthan gum nanocomposites reduced the biofilms formation of _C. violaceum_ and _S. marcescens_ as observed under confocal microscope [37]. The detailed examination of the changes in biofilm architecture by ZnO-NPs treatment was studied using scanning electron microscopy (SEM). SEM provides more detailed picture at higher magnification and enhanced resolution. The electron micrographs of the biofilms without and with ZnO-NPs is shown in Figure 6. Untreated _E. coli_ was found to form strong biofilms on the surface of glass coverslips in which the bacterial morphology was observed to be smooth and normal. Moreover, some bacterial cells were also observed to be enclosed within the copolymeric substances. However, the treatment with 1/2 \(\times\) MIC of ZnO-NPs  
Figure 4: Light microscopic images of the _E. coli_, _P. aeruginosa_, and _S. aureus_ biofilms in the absence and presence of ZnO-NPs. **(A)** untreated control _E. coli_; **(B)**_E. coli_ treated with 1/2 \(\times\) MIC of ZnO-NPs; **(C)** untreated control _P. aeruginosa_; **(D)**_P. aeruginosa_ treated with 1/2 \(\times\) MIC of ZnO-NPs; **(E)** untreated control _S. aureus_; **(F)**_S. aureus_ treated with 1/2 \(\times\) MIC of ZnO-NPs. resulted in reduced adherence of _E. coli_ as evident from the limited colonization of bacterial cells. The presence of exopolymeric substances was also not observed. Similarly, in control slides of _P. aeruginosa_ and _S. aureus_, the bacteria were found to be colonized and forming biofilms on the solid support. The presence of 1/2 \(\times\) MIC of ZnO-NPs reduced the biofilms of both the bacteria in which a limited or reduced bacterial colonization was found. Based on the microscopic results, it became clear that ZnO-NPs not only inhibited the biofilms formation but also distorted the biofilm's architecture. The significance of the biofilms in clinical settings can be assessed from the National Institute of Health (NIH) and Centers for Disease Control and Prevention (CDC) United States data which estimates that the majority of microbial infections, including chronic infections, are associated with, and encouraged by, the biofilms [38]. Moreover, biofilms reduce the therapeutic efficacy of antibiotics and are also known as reservoirs of drug-resistant genes when horizontal gene transfer takes place [33].