Nanomaterials, especially the metal oxide nanoparticles like ZnO NPs have been studied widely during the last few years for various clinical and commercial applications mainly due to their antimicrobial properties. This has made ZnO NPs to have applications in the medical industry for wound dressing, development of surgical instruments, bone prosthetics production, and in other industries for textile production, food packaging, cutting board materials, wastewater treatment, etc (Jin et al. 2019; Rambabu et al. 2021; Huang et al. 2021; Saddik et al. 2022). Medical devices like urinary catheters have also been fabricated by incorporating ZnO NPs for efficient clinical applications (Ivanova et al. 2021). ZnO NPs exhibit multiple antimicrobial mechanisms which include membrane-damaging abrasiveness, generation of reactive oxygen species, and release of antibacterial zinc ions from the nanoparticle surfaces with impact on the bacterial glycolysis, and transmembrane proton translocation (Mendes et al. 2022). However, in most cases, microbial populations can get exposed to sub-MIC levels of ZnO NPs either due to slow release or dilution that happens at sites apart from its point of administration. The impact of such a phenomenon is greatly unexplored which makes the current study to be an important initiative in that direction. This is because, instead of preventing the growth of pathogens, the antimicrobial agents when present at levels below the minimum inhibitory concentration (MIC) may act as inducers of microbial antibiotic resistance and virulence.
At sub-MIC concentrations, the antimicrobial agents can expect to act as key signalling molecules which alter the biochemistry and structure of bacteria to make them ready to resist even the increased concentrations of the drug. These global changes can also alter their pathogenicity, physicochemical properties, and crucial bacterial cell functions including adhesion, surface hydrophobicity, fimbriation, motility, and host-bacterial interactions including phagocytosis (Yuan et al. 2023). Exposure to the sub-inhibitory concentrations of drugs might also be linked to an enhanced formation of biofilm which is a visible and easily detectable change in bacterial physiology and metabolism activated by the low concentration of the applied antimicrobial agent.
The formation of biofilm is a major adaptative response strategy frequently used by bacteria and the mechanistic insight into the biofilm has already been described in various pathogenic microorganisms (Kelly et al., 2020.). In general, bacterial contact with any surface can lead to biofilm formation naturally when the surface is immersed in water or slightly moist, and also in environments with abundant or limited nutrients. Diverse factors have already been described to regulate the formation and structural complexity of biofilm. In the clinical context, biofilm can serve as a pathogen reservoir and is linked to the severity of conditions like bloodstream and urinary tract infections (Pinto et al. 2021). As biofilm-associated bacteria are highly resistant to antimicrobial treatments and host immune response mechanisms, factors favouring biofilm formation by clinically important bacteria generate much clinical concern.
Many previous studies have already reported the ability of sub-MIC concentrations of different antibiotics such as chlorhexidine, tetracycline, kanamycin, spectinomycin, streptomycin, and neomycin to have an inducible effect on the bacterial biofilm (Omer and Aka 2022). Here, the sub-inhibitory doses can consider to execute selective pressure among bacteria for the predominant growth and biofilm formation by resistant organisms. This promotion of biofilm development could ultimately be due to the transcriptional activation of genes involved in virulence and bacterial homeostasis. Various nanomaterials have already been investigated widely for their antimicrobial applications by projecting them as superior solutions even to manage drug-resistant microorganisms (Allahverdiyev et al. 2011; Zhao et al. 2022). However, just like the antibiotics, the sub-MIC levels of nanoparticles can also have an inducible effect on bacterial biofilm formation. As the ZnO NPs are one of the most commonly used antimicrobial agents, the current study hence has been designed to identify the impact of sub-MIC concentrations of ZnO NPs on the biofilm formation of Klebsiella pneumoniae and Staphylococcus aureus. Here, the microtiter plate-crystal violet assay was carried out for the direct quantification of biofilm formation by selected organisms at different sub-MIC concentrations of ZnO NPs and atomic force microscopy was carried out to study the pattern and morphological features of modulated biofilm formation. Results of the study provide novel insights into the role of nanomaterials as inducers of biofilm in clinically important organisms at sub-MIC concentrations.