Wild type Escherichia coli (DH5α) (Monk et al., 2016) and ESBL producing isolates of E. coli (16, 36, 105, and 153) were used in this study as representatives of bacterial pathogens. The E. coli isolates (16, 36, 105, and 153) were from Pathological and Diagnostics Laboratory (Gitanjali Patho Diagnostics Laboratory, Patna, India). Antibiotic susceptibility testing of the E. coli isolates was performed in accordance with the recommendations of Clinical Laboratory Standard Institute (CLSI) using Kirby-Bauer Method (Pouladfar et al., 2017). Kirby-Bauer Method is based on agar disc diffusion assay and is a widely used method for antimicrobial susceptibility testing. Herein, the filter paper discs impregnated with antimicrobial agents are applied onto the agar surface already inoculated with test microbe. On incubation, the antimicrobials diffuse out from the disc into the agar creating a concentration gradient and the area of no microbial growth around the disc is called zone of inhibition. The Antibiogram for the isolates of E. coli used in this study was performed using proper quality control protocols on Muller-Hinton agar disc diffusion plate and is shown in Table 1.

Wild type Candida albicans (SC5314) and three isogenic matched pairs of isolates (G1 & G5, F1 & F5, and GU4 & GU5) of C. albicans, used in this study are listed in Table 2 and the strain descriptions are already reported in many studies previously (Fonzi and Irwin, 1993; Franz et al., 1998, 1999). These three isogenic matched pairs of strains were provided by Joachim Morschhauser, University of Wurzburg, Germany and originally isolated from three different AIDS patient suffering from recurrent episodes of OPC. The azole therapy given over a period of time to these AIDS patients for OPC resulted in the acquisition of MDR by the sensitive strains and subsequent evolution of the azole sensitive G1, F1, and GU4 to highly resistant G5, F5, and GU5, respectively (Franz et al., 1998, 1999).

MIC of ZnO QDs for E. coli (DH5α) and C. albicans (SC5314) was determined by broth microdilution assay, which was performed in 96 well microplate, following the protocol described by CLSI (Clinical and Laboratory Standards Institute, 2006, 2008; Oh et al., 2012; Preeti et al., 2020). The exponentially growing log phase cells of E. coli and C. albicans were resuspended in 0.9% saline to give an optical density of 0.1 at 600 nm (OD₆₀₀), which correspond to cell number of 5 × 10⁷ CFU/ ml for E. coli and 0.5–1 × 10⁶ CFU/ ml for C. albicans. The cells were subsequently diluted 100-fold in LB and YEPD media, respectively for E. coli and C. albicans to achieve the final concentration of cells 5 × 10⁵ and 0.5–1 × 10⁴ CFU/ ml. Thereafter, different concentrations of ZnO QDs (0–400 μg/ ml) were added to the respective media containing cells and incubated at 37°C for E. coli and at 30°C for C. albicans. Respective controls for growth of the microbial cells were maintained without ZnO QDs. Readings were then recorded at 600 nm and the differences in optical densities were compared with the control grown without ZnO QDs.

Spot assay was used to confirm the susceptibility of wild type, drug sensitive and resistant isolates of E. coli and C. albicans toward ZnO QDs (Mukhopadhyay et al., 2004; Clinical and Laboratory Standards Institute, 2006; Thomas et al., 2015; Wang et al., 2017; Radhakrishnan et al., 2018b).

Overnight grown microbial strains of E. coli and C. albicans were resuspended in 0.9% saline and the OD₆₀₀ of the cell suspension was first adjusted to 0.1 and CFU/ ml was calculated. Then, 5-fold serial dilutions of cell suspension containing 5 × 10⁵ cells/ ml of E. coli and 1 × 10⁶ cells/ ml of C. albicans were prepared. Subsequently, 5 μl of each serially diluted cell suspension were spotted onto LB plates (containing 0, 200, and 250 μg/ ml of ZnO QDs) for bacterial and YEPD plates (containing 0, 200 and 400 μg/ ml of ZnO QDs) for fungal strains, respectively. The LB plates were incubated for E. coli at 37°C for 24 h and for C. albicans at 30°C for 48 h. Growth controls without ZnO QDs were maintained for both E. coli and C. albicans separately. The plates were then imaged and reduction in cell viability in presence of ZnO QDs was evaluated by comparing with the control grown without ZnO QDs.

Microbicidal activity of ZnO QDs was evaluated against MDR isolates of E. coli and C. albicans. Antibiotic susceptibility of ESBLs producing E. coli (16, 36, 105, and 153) revealed their resistance toward penicillins, cepahlosporins and cross-resistance to quinolones (Table 1). ESBL producing MDR isolates of E. coli acquire resistance to β-lactam antibiotics such as penicillin derivatives (penams), cephalosporins (cephems), monobactams, carbapenems, and carbacephems etc. through degradation of β-lactam rings of the antibiotics by production of extended spectrum beta lactamase (Shaikh et al., 2015). The three isogenic matched pairs (G1 & G5, F1 & F5, and GU4 & GU5) were originally collected from three different AIDS patient suffering from OPC (Franz et al., 1998, 1999). Due to continuous administration of high doses of azoles for recurrent episodes of OPC, the azole sensitive G1, F1, and GU4 acquired resistance during therapy and evolved to become highly azole resistant G5, F5, and GU5, respectively (Table 2). The isolates of Candida used in this study were reported to acquire resistance due to increase in amount of cellular ergosterol and overexpression of drug efflux pumps (Franz et al., 1998, 1999).

The microbicidal activity of ZnO QDs was further confirmed by spot assay. ZnO QDs exhibited dose dependent, broad spectrum microbicidal activity against all MDR isolates of E. coli and C. albicans tested (Figures 4, 5). Here it clearly showed significantly reduced cell viabilities for MDR strains of both microbial pathogens in comparison to the control plate (without ZnO QDs), which was indicated by the disappearance of cells on agar plates containing ZnO QDs. No such microbicidal effect or reduction in cell viability was observed on respective Luria Bertani (LB) and Yeast Extract, Peptone and Dextrose (YEPD) control plates, maintained without ZnO QDs. Spot assay involves spotting of dilutions of microbial cells at concentrations of antimicrobial agents determined by broth microdilution assay. In this study, the spot assay was performed at different concentrations between MIC values and four times MIC values. Of these, the best results for spot assay obtained at two concentrations (i.e., MIC concentration and the next higher concentration are represented in Figures 4, 5, to demonstrate significant loss of cell viability which is indicated by the significant disappearance of cells in presence of ZnO QDs). Studies evidence that microbicidal concentration is usually either equal to/ 2-fold/ 4-fold > MIC values (Microbicidal concentration ≥ 1x or 2x or 4x MIC) (Morrissey et al., 2014). This validated our results that highly reduced cell viability was observed for MDR isolates of both E. coli and C. albicans cells at MIC concentration of 200 μg/ ml ZnO QDs, which was further reduced in presence of 250 and 400 μg/ ml of ZnO QDs for E. coli and C. albicans, respectively (Figures 4, 5). Complete growth inhibition was observed at 4x MIC₇₀ of ZnO QDs for E. coli and at 4x MIC₉₀ of ZnO QDs for C. albicans cells, thus confirming the microbicidal potential of ZnO QDs (data not shown). The data represented was reproducible in at least three independent set of experiments. Therefore, our spot assay results demonstrated that ZnO QDs were able to counter MDR in microbial isolates tested and could render susceptibility to the drug resistant microbials cells.

Several studies have documented size dependent microbial toxicity of ZnO NPs (Sirelkhatim et al., 2015). In our study, the synthesized 5–6 nm ZnO QDs exhibited significant microbicidal activity against both drug sensitive and resistant isolates of E. coli and C. albicans. The small size of ZnO QDs might be responsible for this promising microbicidal activity. It is possible that antimicrobial or microbicidal activity of metal oxide NPs results from the interaction of metal ions (resulting from dissolution of metals ions from the surface of NPs) with cells, which lead to reactive oxygen species (ROS) generated oxidative stress. The dissolution of metal and metal oxide nanoparticles remains highly dependent on NPs type, concentration, size, coatings and pH of aqueous media. Higher dissolution of Zn²⁺ ions was reported from 4 nm ZnO QDs than larger NPs, resulting in presence of more Zn²⁺ ions in ZnO QDs solution (Mudunkotuwa et al., 2012), which in turn might be responsible for the observed higher antimicrobial activity of QDs against both drug susceptible and resistant pathogens (Siddiqi et al., 2018). Moreover, the quantum confinement of ZnO QDs creates surface defects on ZnO QDs and produces large number of electron donor or acceptor active sites, which react with oxygen and hydroxyl ions (in aqueous suspension) to produce highly reactive superoxide and hydroxyl radicals. These highly reactive free radicals contribute to enhanced ROS generation and result in peroxidation of lipids, nucleic acid and proteins, disruption of cell membrane and leakage of cytoplasmic content, thus leading to cell death (Asok et al., 2015; Gold et al., 2018; Radhakrishnan et al., 2018a; Tiwari et al., 2018).

In addition, the small size of ZnO QDs also provide larger surface area for their higher interaction with microbial cells. Furthermore, the surface defects of ZnO QDs might also increase the density of positive surface charge as particle size decreases, which might increase interaction with microbial cells (possessing negative charge). This can lead to higher agglomeration of ZnO QDs on the surface of microbes leading to altered metabolic activity and increased antimicrobial activity (Asok et al., 2015). Thus, small size of ZnO QDs might exert excellent antimicrobial activity through multiple mechanisms.

However, the major limitation of microbial therapeutics is that each class of antimicrobial drug has a particular mode of action and definite cellular target such as cell wall, membrane lipids, translational machinery, or DNA replication (Liwa and Jaka, 2015; Silver, 2016). For example, the mode of action of β-lactam antibiotics is through inhibition of bacterial cell wall biosynthesis and that of antifungal azoles is via inhibition of fungal membrane ergosterol biosynthetic pathway (Prasad et al., 2011; Shaikh et al., 2015). But on emergence of MDR in microbial pathogens, drug therapy becomes ineffective and poses a bottleneck in microbial therapeutics. MDR being multifactorial, microbial pathogens acquire resistance by various adaptive strategies such as drug target modifications, drug degradation, target amplification, alterations in cell wall, modifications in cell membrane, and overexpression of drug efflux pumps (Shaikh et al., 2015; Munita and Arias, 2016). The challenges faced due to acquisition of microbial drug resistance can likely be overcome by favoring multi-targeted action of antimicrobial agents. In view of the previous reports that interactions of nanoparticles with microbes lead to membrane lipid peroxidation, cell membrane disruption, leakage of cytoplasmic contents, and cell death (Jalal et al., 2018; Radhakrishnan et al., 2018b), it appears that small sized ZnO QDs can address the multifactorial challenges of microbial drug resistance (Graphical illustration in Scheme 1) by favoring multi targeted action, broad spectrum activity and lower host toxicity. In the light of such multi targeted action, it is unlikely for the microbes to simultaneously develop a host of cellular changes or mutations to exhibit resistance.