# Biosynthesized Zinc Oxide Nanoparticles Disrupt Established Biofilms of Pathogenic Bacteria
## 3 Results and Discussion
### Inhibition of the Biofilm Development
#### 3.2.1 Quantitative Analysis of Biofilms Inhibition  
The effect of ZnO-NPs on the biofilms development of test bacteria was quantitatively studied using crystal violet assay in a 96-well polystyrene plate. The dose-dependent inhibition of biofilms formation is presented in Figure 3. Treatment with 1/16 \(\times\) MIC, 1/8 \(\times\) MIC, and 1/4 \(\times\) MIC of ZnO-NPs reduced the biofilms development of _E. coli_ by 15.80%, 27.48%, and 48.21%, respectively. In presence of highest sub-MIC (1/2 \(\times\) MIC of Zn-NPs), there was more than 60% inhibition of _E. coli_ biofilms. Likewise, the biofilm formation of _P. aeruginosa_ was reduced by 30.86%, 48.83%, 54.59%, and 77.69% by the treatment of 1/16 \(\times\) MIC, 1/8 \(\times\) MIC, 1/4 \(\times\) MIC, and 1/4 \(\times\) MIC of ZnO-NPs, respectively. We also tested the effect of ZnO-NPs on the biofilms of a Gram + ve bacteria. At lower concentration  
Figure 2: (**A**) Transmission electron micrograph of ZnO\(-\)NPs. (**B**) Particle size distribution histogram of ZnO\(-\)NPs. (**C**) Fourier-transform infrared spectrum of ZnO\(-\)NPs.  
(1/16 \(\times\) MIC), there was an insignificant inhibition of the biofilms of _S. aureus_. However, at higher sub-MICs (1/8 \(\times\) MIC, 1/4 \(\times\) MIC, and 1/2 \(\times\) MIC of ZnO-NPs), the biofilms were significantly reduced where percent inhibition reached more than 70%. The results clearly show the biofilm inhibition potential of ZnO-NPs against the test bacteria.  
The incidence and worldwide spread of antimicrobial resistance both in clinical settings and the environment has posed serious concerns. The biofilms are considered as reservoirs of drug-resistant genes. Moreover, the horizontal gene transfer in biofilms leads to the rapid spread of antibiotic resistance [33]. In biofilms, there is limited entry of antibiotics and hence bacteria residing in biofilms are more tolerant and resistant to chemotherapeutic agents [15]. Considering the importance of biofilms in antibiotics resistance and clinical settings, targeting the bacteria biofilms is considered as a novel strategy in modern antimicrobial drug discovery. Our findings have shown that ZnO-NPs successfully inhibited the biofilm development of both Gram-negative and Gram-positive bacteria at sub-MICs. Previously, we have reported that zinc oxide nanoparticle synthesized using leaf extract of _Ochradenus buccalus_ inhibited the biofilms development of _C. violaceum_, _P. aeruginosa_, _E. coli_, _K. pneumoniae_, _S. marcescens_, etc. at the sub-inhibitory concentrations [28]. Similarly, another study has reported that zinc oxide nanoparticles produce reactive oxygen species within cells of _P. aeruginosa_ that also inhibit the biofilms development [34]. The nanoparticles are capable of interacting and diffusing on biofilms through the water channels that serve as transport passage for nutrients [35]. Additionally, the nanoparticles are adsorbed onto bacterial membrane and penetrate it, resulting in intracellular accumulation and killing of bacterial cells [36]. This explains the possible mechanism of antibiofilm activity of green synthesized ZnO-NPs.  
Figure 3: Inhibition of biofilm formation of _E. coli_, _P. aeruginosa_, and _S. aureus_ by ZnO-NPs. Data is presented as average of three replicates and error bar is standard deviation. One-way ANOVA was performed using Tukey test at significance level of 0.05. Different letters in treatment groups represent different significance groups at _p_-value = 0.05 and are in ascending/descending order of values starting from the letter “a”.