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. Author manuscript; available in PMC: 2022 May 4.
Published in final edited form as: J Nanopart Res. 2020 Feb 6;22:1–8. doi: 10.1007/s11051-020-4767-z

Impact of the changes in bacterial outer membrane structure on the anti-bacterial activity of zinc oxide nanoparticles

Priyanka Surwade 1, Todd Luxton 2, Justin Clar 3, Fan Xin 4, Vishal Shah 5
PMCID: PMC9067412  NIHMSID: NIHMS1793559  PMID: 35517915

Abstract

Metal and metal oxide nanoparticles (NPs) have been increasingly utilized in many industries to harness their documented antibacterial properties. However, the mechanism(s) of action is still debated in the literature. The aim of this study is to understand how changes in outer membrane charge of a test bacteria Haemophilus influenzae alter the antibacterial activity of ZnO NPs of average sizes of 20 nM and 60 nM. H. influenzae outer membrane charge was altered through use of the wild strain (Rd) and mutant lines H543 and H446. Results indicate that antibacterial effects are both concentration and size dependent, with smaller NPs causing increased antibacterial response. Most critically, antibacterial assays and collected TEM images demonstrate that increasing negative charge on the outer membrane of bacteria decreased the antibacterial activity of the ZnO NPs. Finally, this work demonstrates the possibility of using ZnO NPs to treat H. influenzae infection in clinical settings.

Keywords: Antibacterial activity, Zinc oxide nanoparticles, Haemophilus influenzae, Outer membrane, Bacteria, Toxicity, Environmental and health impacts

Introduction

Use of metal nanoparticles (NPs) as an antibacterial agent have been gaining momentum in industries ranging from food additives (Sirelkhatim et al. 2015a; Berekaa 2015) to drug administration (Sahoo et al. 2017; Surwade et al. 2018). The metal and metal oxide NPs known to have profound antibacterial activity are silver, copper, copper oxide, titanium dioxide, cerium oxide, and zinc oxide (Slavin et al., 2017). While NPs have been finding increasing uses across the society, till date, the mechanism of antibacterial action is unclear. Various mechanisms have been proposed for the anti-bacterial action of different NPs including disruption of cell membrane, intracellular reactive oxygen species (ROS) generation, biomolecule interaction and regulation, internalization, and ATP depletion (Slavin et al., 2017; Sirelkhatim et al. 2015a; Durán et al., 2016). However, questions remain on the molecular mechanism how NPs would cross the outer membrane and cell wall before eliciting damage to the bacteria. Gram-negative bacteria are more susceptible to NPs than gram-positive bacteria (Feng et al. 2000; Mukha et al. 2013; Cavassin et al. 2015). It is hypothesized that the differing cell wall structures could be reason for the difference in susceptibility. Gram-negative bacteria have thin peptidoglycan layer, making it more susceptible to cell wall destruction through interaction with NPs. Studies suggest that adsorption of NPs on the cell wall is the primary mechanisms of toxicity. For example, antibacterial activity studies with Ag NPs against E. coli have shown that NPs cause irregular pits in the cell wall. These pits facilitate Ag ions to enter the cell and cause intracellular damage (Sondi and Salopek-Sondi 2004; Raffi et al. 2008).

Additionally, gram-negative bacteria have a negatively charged outer membrane composed of lipopolysaccharide (LPS). Positive-charged NPs can buildup in the outer membrane, leading to increased uptake of leached ions inside the bacterial cell, eventually causing cell damage (Slavin et al. 2017). Studies have further shown that the anionic surface domains are not uniformly distributed across the membrane, but rather are present in highly concentrated domains (Slavin et al. 2017). NPs will be found in highly concentrated amount in these areas, resulting in focal toxicity against the bacteria. For example, in studying the toxicity of gold NPs against Shewanella oneidensis, Jahnke et al. (2016) showed preferential attachment of the NPs to the subpolar area of the cell. Goal of the current study is to understand the impact of changes in outer membrane charge and structure of Haemophilus influenzae on the antibacterial activity of ZnO NPs of varying sizes.

ZnO NPs have been reported to have anticancer, antioxidant, wound healing, antidiabetic, cryoprotective, and antimicrobial activity—making it highly attractive for clinical considerations (Mishra et al. 2017). In addition to the biological properties, ZnO NPs are also excellent candidates for drug carriers (Mishra et al. 2017). Advantages of using ZnO NPs in biomedical studies include its properties of being bioavailable, biocompatible, and soluble (McNeil 2009). This allows ZnO NPs to mimic the activity of biomolecules, and can selectively localize within the human body (Bisht and Rayamajhi 2016).

H. influenzae is a gram-negative cocobacillary, facultative anaerobic, pathogenic bacterium that normally colonizes the upper respiratory tract of humans. Approximately 20–40% of adults carry H. influenza and young children are colonized at an even higher rate (Kuklinska and Kilian 1984). Because of the prevalence of its asymptomatic carriage in human population, H. influenzae presents substantial risk in cardiovascular treatment as a frequent cause of ventilator-associated pneumonia and septicemia acquired during surgery (Fan 2004).

H. influenzae has multiple virulent factors that contribute to the pathogenesis (Fan 2004). Surface expressions of phosphorylcholine (ChoP) on LPS are one of the major virulence factors. ChoP [(CH3)3N+CH2CH2PO4−] is a zwitterionic lipid moiety that is covalently attached to LPS through its phosphate group (Clark et al. 2012). Presence of ChoP on the outer membrane allows for a greater positive charge under the testing conditions. ChoP is not uniformly decorated on the LPS and its attachment is stochastic, creating high degree of phase variation in the membrane structure (Clark 2013). H. influenzae cannot synthesize choline and thus must import it from the environment (Fan et al. 2003). Choline is used through licA-D pathways to decorate H. influenzae LPS with ChoP in a phase-variable manner. glpQ facilitates acquisition of choline from host sources. In this study, we use two different mutants of H. influenzae—H543 and H446, in addition to the wild type strain (Rd) to investigate the influence of difference in LPS structure on antibacterial activity of NPs. When grown in brain-heart infusion broth medium, the glpQ mutant (H543) would have sufficient amounts of choline to express ChoP, whereas the licD mutant (H446) would constitutively have no ChoP present in the LPS (Fan et al. 2001; Lysenko et al. 2000). H543 and Rd. strain have varied phase variations (changes in the level of expression) of ChoP on the LPS (Clark 2013). Thus, strain Rd. would have maximum ChoP present on its outer membrane, followed by strains H543 and H446, respectively. Increased presence of ChoP leads to increased positive charge, and we hypothesize that ZnO NPs of smaller size will have highest antibacterial activity against H446. The rationale for the hypothesis was the availability of larger surface for smaller size ZnO NPs to interact with the bacteria.

Materials and methods

Nanoparticles and characterization

ZnO nanomaterials were obtained from BYK (ALTANA, Wesel, Germany) as liquid suspensions with reported average sizes of 20 nm (NANOBYK 3820) and 60 nm (NANOBYK 3860). Total elemental composition of the NPs was confirmed by microwave-assisted acid digestion (EPA 3051B) and subsequent analysis of ICP-OES (Thermo Fisher iCAP 6000 Series). Benzisothiazolinone (BIT, 77 mg L−1) and glycol ethers (≤0.2%) were present in the undiluted stock suspension (40% ZnO by mass). The presence of a glycol ether stabilizing agent was confirmed with FTIR using a Bruker Vertex 80 (Bruker Corporation Billerica, Massachusetts). Prior to IR analysis, the samples were dried in a 40 °C oven to remove water, the dried films were analyzed using a single-bounce diamond ATR cell. The final spectra presented were averaged from 256 individual scans. The hydrodynamic diameter (HDD) and zeta potential of the pristine material were determined using a ZetaSizer Nano Series (Malvern, Worcestershire, UK) after a 10:1 dilution in Milli-Q water (18.2 mΩ). HDD values were recorded using an average of three runs, each run consisting of 12 measurements. Primary particle size and morphology were investigated with a JEOL JEM-2010F scanning transmission electron microscope (STEM) equipped with a High-angle annular dark field detector (HAAFD; Peabody, MA). Finally, zinc speciation in pristine material was determined by X-Ray photoelectron spectroscopy (XPS) using a PHI Quantera II (Physical Electronics, Chanhassen, MN) and X-Ray absorbance fine structure (XAFS) spectroscopy.

Antimicrobial assay

Haemophilus influenzae strains Rd. (wild type), H446 (Chop− mutant), and H543 (glpQ− mutant) were used in the study (Fan et al. 2001; Lysenko et al. 2000). A single isolated colony of the bacteria was inoculated in 4 mL of brain heart infusion (BHI) broth supplemented with 2 μg mL −1 nicotinamide adenine dinucleotide and 2.0% (v/v) hemin. The broth was incubated at 37 °C for 210 min to ensure the bacteria is in exponential growth phase. Cells were centrifuged at 10,000 rpm for 1 min and washed twice with sterile distilled water. It was important to carry out the study without any protein from the culture media since proteins and other organic molecules present in the media could interact with the NPs and form a dynamic nanoparticle-protein corona (Maiorano et al. 2010). Cell pellet was resuspended in sterile-distilled water. Four milliliters of reaction system was set up in sterile glass test tubes with appropriate concentration of NPs and 0.5-mL culture. Reaction tubes were incubated at room temperature with no shaking. Ten microliters of aliquot was taken at 0, 15, 30, and 60 min. Serial dilutions and plating on nutrient agar were performed to determine the colony forming units (CFU) present in the media post incubation. Percent anti-bacterial activity was calculated by comparing the log number of CFU present in the media after the incubation, as compared to the log number of CFU at time zero. At each time point, three samples were taken, and each sample was plated and duplicated. Data analysis was performed based on the average values, and the standard deviation for the results did not exceed 10% for any result. A plot of log CFU/ml vs. time was generated in Microsoft Excel and the slope of the curve (death rate, kd obtained (Surwade et al. 2018). It is worth noting that the values are negative as a death rate, and lower values indicate higher death rates.

Transmission electron microscopy (TEM)

Bacterial cells micrographs were prepared by collecting a 2 μl sample after 30 min of incubation with NPs at room temperature with no shaking and placed on copper grids (Formvar/Carbon Square Mesh, Cu, 200 Mesh). The sample was allowed to air dry in laminar hood and observed under TEM using the protocol described by Singh et al. (2013)

Results

Particle characterization

FTIR spectra collected for the two NPs showed a very similar absorption spectra, indicating the same capping agent and dispersant was used for the 20 and 60 nm NPs (Supplemental Fig. 1). Based on the spectra collected from the samples and the Material Safety Data sheet provided, a glycol ether was identified as the stabilizing agent and the spectra were very similar to polyethylene glycol, a common NP suspension stabilizing agent. The speciation of the ZnO NPs was confirmed based upon the XAFS spectra collected. The two ZnO NPs obtained from BYK shared identical spectral features in the XANES region (Supplemental Fig. 2). STEM images of the ZnO NPs from BYK were elongated hexagonal crystals (Fig. 1). A non-significant amount of crystal twinning was present in each of the size fractions. Based on the elongate crystal shape, determining an average single particle size is difficult. For the ZnO NPs used in the current study, the average hexagonal diameter and length were determined for each size. Using the diameter (d) and length (l), the aspect ratio (d l−1) for each reported size was calculated and the results are presented in Fig. 2. The range of values measured for the length and diameter varied greatly as evidenced by the reported standard deviations. The measured crystal length and diameter for 20 nm NPs were significantly smaller than the 60 nm NPs. The aspect ratio calculated for the ZnO NPs exhibited a trend with an increasing aspect with increasing size (Fig. 2). Based on a Tukey ad-hoc means comparison, the difference between the 20 and 60-nm aspect ratio was significant. The specific surface area (SSA) of the 20 and 60 nm particles were determined using the data obtained from the SEM images and assuming a ZnO bulk density of 5.61 g cm−3. The SSA was calculated by determining the mean particle surface area and volume using an elongated hexagonal prism. The calculated SSA for the 20 and 60 nm ZnO particles was 54 ± 2 and 18 ± 1 m2 g−1. In addition to determining the NP size by microscopy, the hydrodynamic diameter (HDD) of the NPs were measured. The diameter reported for the HDD assumes that the NPs being measured are spherical in shape. While this is not the case for the ZnO NPs used in the current study, it does provide important information on the extent of interparticle aggregation that occurs in solution, and the approximate size of the aggregates (Supplemental Fig. 3). The reported HDD for the ZnO NPs at a native water pH (7.47 and 7.95) were 255 and 352 nm for the 20 and 60 nm NPs, respectively. The dashed line in the percent under range plot indicates that greater than 50% of the intensity measured for each size was less than the maximum intensity of the size distribution curve, indicating a high degree of skewness towards smaller values. The HDD data does suggest that all the BYK ZnO NPs are aggregated in suspension. Finally, the surface charge of the particles was measured as a function of pH (Supplemental Fig. 4). Both the 20 and 60 nm ZnO particles have a weak negative zeta potential between pH 6.5 and 7.5. All studies were conducted at near neutral pH where dissolution of ZnO would be minimal (Jiang et al. 2015; Wanetal. 2017).

Fig. 1.

Fig. 1

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STEM micrographs of ZnO nanoparticles. a 20 nm, b 60 nm

Fig. 2.

Fig. 2

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Diameter, length, and aspect ratio of the 20 nM and 60 nM zinc oxide nanoparticles used in the current study

Antibacterial activity

Comparison of death rate (kd) obtained from experiments performed under ideal experimental conditions allows one to compare the antibacterial activities of NPs against different mutants of H. influenzae. Antibacterial activity of 20 nm NPs against the three strains tested shows that the wild type strain (Rd) was the most sensitive at all three concentrations tested (Fig. 3a). However, the sensitivity of strains H543 and H446 was concentration-dependent. Between the two mutants, strain H543 was more sensitive at 1 and 3 ppm concentration whereas H446 was sensitive at 5 ppm concentration (Fig. 3a). Antibacterial activity of 60 nm NPs shows different patterns with wild type strain being the least sensitive of all the three strains. H446 was the most sensitive at 1 ppm concentration whereas H543 was the most sensitive at 3 and 5 ppm (Fig. 3b). Comparison of antibacterial activity at same concentrations for two different NPs shows that 20 nm NPs exhibit significantly higher activity, particularly against wild type strain. TEM images of the strains incubated with 1 ppm of NPs for 30 min shows that 20 nm ZnO NPs are less aggregated as opposed to 60 nm NPs (Fig. 4). Twenty nanometers of ZnO NPs are present in high concentrations around the strain in decreasing order for strains H446, H543, and Rd. (wild type). Sixty nanometers of nanoparticle aggregates are also seen to be covering the cell surface area in same decreasing order.

Fig. 3.

Fig. 3

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Influence of various concentrations of 20 nm (a) and 60 nm (b) zinc oxide nanoparticles on the death rate (kd) of wild type (Rd) and two mutants of H. influenza (H446 and H543)

Fig. 4.

Fig. 4

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TEM images of wild type strain (a) control (b) incubated with 1 ppm 20 nm ZnO NPs (c) incubated with 1 ppm 60 nm ZnO NPs; H446 strain (d) control (e) incubated with 1 ppm 20 nm ZnONPs (f) incubated with 1 ppm 60 nm ZnO NPs; H543 strain (g) Control (h) incubated with 1 ppm 20 nm ZnO NPs (i) incubated with 1 ppm 60 nm ZnO NPs

Discussion

Numerous studies have documented the significance of NP particle size and concentration for determining the antibacterial activity of ZnO NPs. In general, smaller ZnO NPs and elevated concentrations are correlated with higher antibacterial activity (Nair et al. 2009; Zhang et al. 2007; Raghupathi et al. 2011; Jones et al. 2008). Results of the current study support similar phenomenon for ZnO NPs against H. influenza (Fig. 3). Wild type H. influenza (Rd) were more sensitive to 20 nM NPs compared to 60 nM NPs. The strain also showed increasing sensitivity with increasing NP concentration for both the particle sizes tested.

Antimicrobial activity of the NPs and the TEM images indicates the surface charge present on the outer membrane of the organism impacts the antimicrobial activity of ZnO NPs. Presence of ChoP on the outer membrane increases the net positive surface charge. Strain H446 has no ChoP present in its outer membrane and hence has the most negative charge of the three strains tested. As can be seen in Fig. 4, high concentration of NPs were sequestered around the outer membrane. The large concentration of NPs at the membrane surface will reduce the number of NPs that could enter the cell and elicit antibacterial activity. Figure 4 shows that at 1 ppm concentration of 20 nm ZnO NPs, H446 was the most resilient (least sensitive) of the three strains tested. Strain H543 and Rd. have increasing concentration and presence of ChoP in the membrane, and as can be predicted, stain Rd. is most sensitive to 20 nm ZnO NPs. Large degree of aggregation of 60 nM ZnO NPs around H. influenza is evident in the TEM images.

The study illustrates ZnO NPs interact with the outer membrane of the bacteria, and these interaction plays a key role in the mechanism of action. Results show that if the outer membrane has higher negative charge, the toxicity of NPs against the bacteria will be minimal. In contrast, if the membrane has higher positive charge functional groups, the bacteria will be sensitive to the toxicity of metal NPs. We need to caution that in the current study, the ionization of metals from the aggregated NPs in the membrane may not be a significant source of toxicity against H. influenzae. However, same may not be true for other bacteria being tested. Zhang et al. (2007) and Nair et al. (2009) observed that ZnO NPs interact directly with the outer membrane of the bacteria due to electrostatic interactions, form aggregates around the cells, and cause damage to the membrane eventually leading to cell death. Further, Kumar et al. suggested that the cell death following binding of NPs to outer membrane can be attributed to the neutralization of the surface potential of the bacterial membrane resulting in increased surface tension and membrane depolarization (Kumar et al. 2017). The differences in each of the study are the test bacteria being studied and its sensitivity to the ions being released from the NPs.

In conclusion, our results indicate that our hypothesis was false, and increasing negative charge on the outer membrane of bacteria decreased the antibacterial activity of the ZnO NPs. The study also opens the possibility of using ZnO NPs as a therapeutic agent to treat H. influenza infections in clinical settings.

Supplementary Material

Supplementary Material

Funding information

The work was partially funded through National Science Foundation awards numbers CBET No 1748439 and CBET No. 1559792.

Footnotes

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11051-020-4767-z) contains supplementary material, which is available to authorized users.

Conflict of interest The authors declare that they have no conflict of interest.

Contributor Information

Priyanka Surwade, College of the Sciences and Mathematics, West Chester University of Pennsylvania, West Chester, PA 19382, USA.

Todd Luxton, National Risk Management Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45224, USA.

Justin Clar, College of Arts and Science, Elon University, Elon, NC 27244, USA.

Fan Xin, College of the Sciences and Mathematics, West Chester University of Pennsylvania, West Chester, PA 19382, USA.

Vishal Shah, College of the Sciences and Mathematics, West Chester University of Pennsylvania, West Chester, PA 19382, USA.

References

  1. Berekaa MM (2015) Nanotechnology in food industry; advances in food processing, packaging and food safety. Int J Curr Microbiol App Sci 4:345–357 [Google Scholar]
  2. Bisht G, Rayamajhi S (2016) ZnO nanoparticles: a promising anticancer agent. Nanobiomedicine 3:3–9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cavassin ED, de Figueiredo LFP, Otoch JP, Seckler MM, de Oliveira RA, Franco FF et al. (2015) Comparison of methods to detect the in vitro activity of silver nanoparticles (AgNP) against multidrug resistant bacteria. J Nanobiotechnol 13:64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clark S (2013) Evasion of host recognition by phase variation in Haemophilus influenzae. Publicly Accessible Penn Dissertations 623 [Google Scholar]
  5. Clark SE, Snow J, Li J, Zola TA, Weiser JN (2012) Phosphorylcholine allows for evasion of bactericidal antibody by Haemophilus influenzae. PLoS Pathog 8(3):e1002521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Durán N, Durán M, de Jesus MB, Seabra AB, Fávaro WJ, Nakazato G (2016) Silver nanoparticles: a new view on mechanistic aspects on antimicrobial activity. Nanomed: Nanotechnol Biol Med 12:789–799 [DOI] [PubMed] [Google Scholar]
  7. Fan X (2004) The choline incorporation pathways of Haemophilus influenzae Dissertations available from ProQuest AAI3125813 [Google Scholar]
  8. Fan X, Goldfine H, Lysenko E, Weiser JN (2001) The transfer of choline from the host to the bacterial cell surface requires glpQ in Haemophilus influenzae. Molecular Microbiol 41:1029–1036 [DOI] [PubMed] [Google Scholar]
  9. Fan X, Pericone CD, Lysenko E, Goldfine H, Weiser JN (2003) Multiple mechanisms for choline transport and utilization in Haemophilus influenzae. Molecular Microbiol 50:537–548 [DOI] [PubMed] [Google Scholar]
  10. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO (2000) A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Materials Res 52:662–668 [DOI] [PubMed] [Google Scholar]
  11. Jahnke JP, Cornejo JA, Sumner JJ, Schuler AJ, Atanassov P, Ista LK (2016) Conjugated gold nanoparticles as a tool for probing the bacterial cell envelope: the case of Shewanella oneidensis MR-1. Biointerphases 11:011003. [DOI] [PubMed] [Google Scholar]
  12. Jiang C, Aiken GR, Hsu-Kim H (2015) Effects of natural organic matter properties on the dissolution kinetics of zinc oxide nanoparticles. Environ Sci Technol 49:11476–11484 [DOI] [PubMed] [Google Scholar]
  13. Jones N, Ray B, Ranjit KT, Manna AC (2008) Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiol Letters 279:71–76 [DOI] [PubMed] [Google Scholar]
  14. Kuklinska D, Kilian M (1984) Relative proportions of Haemophilus species in the throat of healthy children and adults. European J Clinical Microbiol 3:249–252 [DOI] [PubMed] [Google Scholar]
  15. Kumar R, Umar A, Kumar G, Nalwa HS (2017) Antimicrobial properties of ZnO nanomaterials: a review. Ceram Int 43:3940–3961 [Google Scholar]
  16. Lysenko E, Richards JC, Cox AD, Stewart A, Martin A, Kapoor M, Weiser JN (2000) The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C-reactive protein-mediated killing. Molecular Microbiol 35:234–245 [DOI] [PubMed] [Google Scholar]
  17. Maiorano G, Sabella S, Sorce B, Brunetti V, Malvindi MA, Cingolani R, Pompa PP (2010) Effects of cell culture media on the dynamic formation of protein– nanoparticle complexes and influence on the cellular response. ACS Nano 4:7481–7491 [DOI] [PubMed] [Google Scholar]
  18. McNeil SE (2009) Nanoparticle therapeutics: a personal perspective. Wiley Interdisciplinary Reviews: Nanomedicine Nanobiotechnol 1:264–271 [DOI] [PubMed] [Google Scholar]
  19. Mishra PK, Mishra H, Ekielski A, Talegonkar S, Vaidya B (2017) Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications. Drug Discov Today 22:1825–1834 [DOI] [PubMed] [Google Scholar]
  20. Mukha IP, Eremenko AM, Smirnova NP, Mikhienkova AI, Korchak GI, Gorchev VF, Chunikhin AY (2013) Antimicrobial activity of stable silver nanoparticles of a certain size. Appl Biochem Microbiol 49:199–206 [DOI] [PubMed] [Google Scholar]
  21. Nair S, Sasidharan A, Rani VD, Menon D, Nair S, Manzoor K, Raina S (2009) Role of size scale of ZnO nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells. Journal of Materials Science: Materials Med 20:235. [DOI] [PubMed] [Google Scholar]
  22. Raffi M, Hussain F, Bhatti TM, Akhter JI, Hameed A, Hasan MM (2008) Antibacterial characterization of silver nanoparticles against E. coli ATCC-15224. J Materials Sci Technol 24:192–196 [Google Scholar]
  23. Raghupathi KR, Koodali RT, Manna AC (2011) Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir 27:4020–4028 [DOI] [PubMed] [Google Scholar]
  24. Sahoo SK, Misra R, Parveen S (2017) Nanoparticles: a boon to drug delivery, therapeutics, diagnostics and imaging. In Nanomedicine in Cancer (pp. 73–124). Pan Stanford; [DOI] [PubMed] [Google Scholar]
  25. Singh R, Wagh P, Wadhwani S, Gaidhani S, Kumbhar A, Bellare J, Chopade BA (2013) Synthesis, optimization, and characterization of silver nanoparticles from Acinetobacter calcoaceticus and their enhanced antibacterial activity when combined with antibiotics. Int J Nanomedicine 8:4277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sirelkhatim A, Mahmud S, Seeni A, Kaus NHM, Ann LC, Bakhori SKM, Hasan H, Mohamad D (2015) Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism. Nano-Micro Letters 7:219–242 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Slavin YN, Asnis J, Hafeli UO, Bach H (2017) Metal nanoparticles: understanding the mechanisms behind antibacterial activity. J Nanobiotechnol 15:65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sondi I, Salopek-Sondi B (2004) Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. J Colloid Interface Sci 275:177–182 [DOI] [PubMed] [Google Scholar]
  29. Surwade P, Ghildyal C, Weikel C, Luxton T, Peloquin D, Fan X, Shah V (2018) Augmented antibacterial activity of ampicillin with silver nanoparticles against methicillin-resistant Staphylococcus aureus (MRSA). The J Antibiotics 72:50–53 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wan B, Yan Y, Tang Y, Bai Y, Liu F, Tan W, Huang Q, Feng X (2017) Effects of polyphosphates and orthophosphate on the dissolution and transformation of ZnO nanoparticles. Chemosphere 176:255–265 [DOI] [PubMed] [Google Scholar]
  31. Zhang L, Jiang Y, Ding Y, Povey M, York D (2007) Investigation into the antibacterial behaviour of suspensions of ZnO nano-particles (ZnO nanofluids). J Nanopart Res 9:479–489 [Google Scholar]

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Supplementary Materials

Supplementary Material
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