Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

Summary

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

Abstract

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings.

To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces.

For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

Introduction

In the US alone, 1.7 million individuals develop a hospital-acquired infection (HAI) annually, with one in every 17 of these infections resulting in death¹. In addition, it is estimated that the treatment costs for HAIs range from $28 billion to $45 billion annually^1,2. These HAIs are predominated by methicillin-resistant Staphylococcus aureus (MRSA)^3,4 and Pseudomonas aeruginosa⁴, which are commonly isolated from chronic wound infections and usually require extensive treatment and time ....

Protocol

To present the direct co-culture and direct exposure methods, we use magnesium oxide nanoparticles (nMgO) as a model material to demonstrate bacterial interactions. To present the direct culture and focused-contact exposure methods, we use an Mg alloy with nanostructured surfaces as examples.

1. Sterilization of nanomaterials

NOTE: All the nanomaterials must be sterilized or disinfected prior to microbial culture. The methods that can be used include heat, pressure, radiation, and disinfectants, but the tolerance of the materials for each method must be identified prior to the in vitro exp....

Results

The identification of the antibacterial activity of magnesium oxide nanoparticles and nanostructured surfaces has been presented using four in vitro methods that are applicable across different material types and microbial species.

Method A and method B examine bacterial activities when exposed to nanoparticles at a lag phase (method A) and log phase (method B) for a duration of 24 h or longer. Method A provides results regarding the MIC and MBC, while method B determines the inhibito.......

Discussion

We have presented four in vitro methods (A-D) to characterize the antibacterial activities of nanoparticles and nanostructured surfaces. While each of these methods quantifies bacterial growth and viability over time in response to nanomaterials, some variation exists in the methods used to measure the initial bacterial seeding density, growth, and viability over time. Three of these methods, the direct co-culture method (A)¹⁷, the direct culture method (C)¹⁴, and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tube	Milipore Sigma	Z336777
80 L NTRL Certified Convection Drying Oven	MTI Corporation	BPG-7082	https://www.mtixtl.com/BPG-7082.aspx
(hydroxymethyl) aminomethane buffer pH 8.5; Tris buffer	Sigma-Aldrich	42457
AnaSpec THIOFLAVIN T ULTRAPURE GRADE	Fisher Scientific	50-850-291
Electron-multiplying charge-coupled device digital camera	Hamamatsu	C9100-13
Falcon 15 mL conical tubes	Fisher Scientific	14-959-49B
Gluteraldehyde	Sigma-Aldrich	G5882
Hemocytometer	Brightline, Hausser Scientific	1492
Inductively coupled plasma - optical emission spectrometry (ICP-OES)	PerkinElmer	8000
Inverse microscope	Nikon	Eclipse Ti-S
Luria Bertani Broth	Sigma Life Science	L3022
Luria Bertani Broth + agar	Sigma Life Science	L2897
MacroTube 5.0	Benchmark Scientific	C1005-T5-ST
Magnesium oxide nanoparticles	US Research Nanomaterials, Inc	Stock #: US3310   M	MgO, 99+%, 20 nm
MS Semi-Micro Balance	Mettler Toledo	MS105D
Nitrocellulose paper	Fisherbrand	09-801A
Non-tissue treated 12-well polystyrene plate	Falcon Corning Brand	351143
Non-tissue treated 48-well polystyrene plate	Falcon Corning Brand	351178
Non-tissue treated 96-well polystyrene plate	Falcon Corning Brand	351172
Petri dish 100 mm	VWR	470210-568
Petri dish, 15 mm	Fisherbrand	FB0875713A
pH meter	VWR	SP70P
Scanning electron microscopy (SEM)	TESCAN	Vega3 SBH
Sonicator	VWR	97043-936
Table top centrifuge	Fisher Scientific	accuSpin Micro 17
Table top centrifuge	Eppendorf	Centrifuge 5430
Tryptic Soy Agar	MP	1010617
Tryptic Soy Broth	Sigma-Aldrich	22092-500G
UV-Vis spectrophotometer	Tecan	Infinite 200 PRO	https://lifesciences.tecan.com/plate_readers/infinite_200_pro
VWR Benchmark Incu-shaker 10L	VWR	N/A
X-ray power defraction	Panalytical	N/A	PANalytical Empyrean Series 2