We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.
The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.
Implant failure is often a consequence of microbial infections that occur in spite of antimicrobial prophylaxis and aseptic working conditions. This problem is causing very high healthcare costs and is distressing among patients1. Important bacteria such as Staphylococcus aureus are currently considered to be very dangerous pathogens in nosocomial infections associated with catheters and other medical implants and are the main contaminants of medical instruments2. Therefore, the development of novel antimicrobial strategies is urgently needed for both daily and medical uses.
Antimicrobial agents include antibiotics3, quaternary ammonium compounds4, metal ions/oxides5, and antimicrobial peptides (AMPs)6. Antibiotics are gradually becoming less efficient due to bacterial resistance7, which is on the rise due to antibiotic overuse8. Quaternary ammonium compounds are only very efficient for a short-term use because of microbial resistance9. Metal ions/oxides have long been utilized as very effective antimicrobial agents and are used in many common commercial products including bandages, water filters, paints, etc.10,11,12. However, it has been demonstrated that these types of compounds can be toxic to some types of mammalian cells13.
AMPs show excellent antimicrobial and immunomodulatory properties14,15, and bacteria seem to find it very difficult to develop a resistance against them16. However, the process to produce pure AMPs is expensive; therefore, a large-scale production is not viable. Thus, strategies to counter the problems in producing AMPs have been developed (e.g., small molecular antibacterial peptoid mimics17, peptoids18, α-peptides19 and β-peptides20). Methacrylate-ended polypeptides and polypeptoids have been synthesized for antimicrobial and antifouling coatings21.
The development of new antimicrobial agents such as advanced materials in pure or hybrid form, able to prevent and treat multidrug-resistant infections, is increasingly necessary. A broad range of new advanced materials for many bioengineering fields such as tissue and bioprocess engineering have been developed with improved chemical and physical properties in the last decades through several methods: plasma-polymerization grafting onto a hydrophobic substrate22,23,24, tailoring of crosslinking density25,26, polymerization in solution27,28,29,30, porogen dissolution31,32, and by the incorporation of nanomaterials such as graphene oxide (GO)33,34,35,36 and carbon nanofibers (CNFs)37.
The study of the antimicrobial capacity of these new materials could exponentially increase their potential bioengineering applicability and has, therefore, become essential. We present an easy-to-follow protocol to quantify the antimicrobial activity of such new advanced materials. Here, after the sample preparation, two complementary methods are followed: the first is based on the agar disk diffusion test38 (diffusion method) and the second is based on the ISO 22196:2007 norm39 to measure the antimicrobial activity on material surfaces (contact method).
1. Sample Preparation
2. Recommended Microorganisms
NOTE: We recommend the use of 3 different microorganisms to study the antimicrobial capacity of the tested material against a wide range of microorganisms.
3. Agar Disk Diffusion Test (Diffusion Method)
NOTE: When a liquid diffusion of antimicrobial compounds might be the main antimicrobial mechanism of advanced materials, the diffusion method can provide very useful information about the antimicrobial capacity of these materials. The material disk located at the center of the agar plate can form a transparent ring zone (halo) where a growth inhibition of microorganisms occurs after 24 h of culture (see Figure 1).
4. Measurement of Antimicrobial Activity on Material Surfaces (Contact Method)
NOTE: When surface contact might be the main antimicrobial mechanism of some advanced materials, the contact method can provide very useful information about the antimicrobial capacity of these materials. In this method, the microorganisms are placed directly onto the material surface and their growth inhibition can be determined after a certain amount of time.
5. Antimicrobial Results Analysis
Figure 1: Measurements for the normalized width of the antimicrobial "halo". This panel shows the diameter of the inhibition zone (diz) and the disk diameter (d). Please click here to view a larger version of this figure.
This protocol was employed, as an example, to test the antimicrobial capacity of 4 materials with different chemical natures against the 3 recommended microorganisms: Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of the agar disk diffusion tests (diffusion method) exhibited non-antimicrobial activity for the first material (M1) as it occured in the control disk (C, image not shown) and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials M2, M3 and M4 (see Figure 2).
Figure 2: Antimicrobial diffusion method results. This panel shows the antimicrobial diffusion method for the 4 material (M1, M2, M3, and M4) disks (10 mm diameter x 1 mm thickness) against S. aureus and E. coli after 24 h of incubation. Please click here to view a larger version of this figure.
Figure 3 shows the different normalized widths of the antimicrobial "halo" (nwhalo) for the different example materials M1, M2, M3, and M4 against Gram-positive and Gram-negative bacteria calculated with equation (1). However, none of the 4 materials were able to inhibit the growth of the yeast Candida albicans (images not shown).
Figure 3: Antimicrobial diffusion "halo" results. This panel shows the normalized "halo" (nwhalo) for each material (M1, M2, M3, and M4) disk (10 mm diameter x 1 mm thickness) against S. aureus and E. coli after 24 h of incubation. The differences are statistically significant (p <0.01). However, sample M1 exhibited no antimicrobial activity. Please click here to view a larger version of this figure.
The results of the contact method also exhibited non-antimicrobial activity for the first material (M1) as it occurred in the control disk (C) and increasing antibacterial activity against Grampositive and Gram-negative bacteria for the other 3 materials (see Figure 4).
Figure 4: Antimicrobial contact method results. This panel shows the respective 90 mm plates of the 4 material (M1, M2, M3, and M4) surface antimicrobial activity assay according to the ISO 22196:2007 after 24 h of incubation for S. aureus and E. coli (dilution factor of 10-4). C is the viable bacteria recovered from the control disk after 24 h of incubation. Please click here to view a larger version of this figure.
The loss of viability (%) was determined by equation (2) and (3) as indicated in this protocol (see Figure 5).
Figure 5: Loss of viability by contact method. This panel shows the loss of viability (%) for M1, M2, M3, and M4 against S. aureus and E. coli on the material surfaces. Sample M1 exhibited no antimicrobial activity. Please click here to view a larger version of this figure.
However, none of the 4 materials were able to inhibit the growth of the yeast Candida albicans by the contact method either (images not shown). Therefore, 3 of these 4 advanced materials showed positive antimicrobial results against Gram-positive and Gram-negative bacteria and thus could be very useful for many bioengineering applications with high antibacterial activity requirements. However, none of the 4 materials were able to inhibit the yeast growth.
The antimicrobial activity of new advanced materials can be analyzed by this easy-to-follow protocol consisting of 2 complementary procedures based on 2 existing methods: the agar disk diffusion test38 and the antimicrobial activity measured on material surfaces according to the ISO 22196:2007 norm39.
In this research field, many of the antimicrobial tests reported in the literature are highly assay-dependent. Therefore, it is very important to have detailed and consistent protocols in place across laboratories. This article is a step in that direction. Furthermore, it could be very helpful for many researchers who are less experienced in this field and require in-depth, step-by-step procedures to follow for accurate results.
This protocol can be used with many types of materials cut into disk shapes of 10-mm diameter. Brittle materials can be swollen in a suitable solvent for 1 h to render the cutting process easier. Thus, hydrophilic materials such as alginates can be hydrated in autoclaved distilled water. Other solvents, such as ethanol, ketone, and dichloromethane, can be employed to swell hydrophobic materials for 1 h before cutting them. However, some materials such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) do not need to be swollen and they can be cut directly. After that, it is very important to dry the sample material disks in a vacuum oven and sterilize each specimen with ethanol and UV radiation for 1 h to avoid any contamination risk.
This protocol recommends TSA and TSB as culture media and the use of pure cultures of 3 microorganisms to reach a broad range of microorganisms: the Gram-positive bacteria Staphylococcus aureus, the Gram-negative bacteria Escherichia coli, and the yeast Candida albicans. However, alternative culture media and other microorganisms in need of different incubation conditions could also be used with this protocol. Sometimes, only 1 microorganism is tested to have an initial idea of the antimicrobial activity of a new material.
The materials showing strong antimicrobial activity against the recommended 3 different types of microorganisms should also be tested against antibiotic-resistant pathogens such as methicillin-resistant Staphylococcus epidermidis (MRSE), which have been successfully utilized with this protocol. Other important drug-resistant microorganisms which are causing much concern are the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), and the Gram-negative Pseudomonas aeruginosa40,41.
Biofilm inhibition and the antimicrobial activity of materials against other types of microorganisms such as viruses and parasites cannot be tested with this protocol. However, this protocol provides a very useful starting point for an antimicrobial study of a new advanced material.
In the antimicrobial agar disk diffusion test, a critical step occurs when the sample disk has to be placed in the center of the plate because some materials fold as soon as they get in contact with the agar media. In this case, it is recommended to use a sterile pair of tweezers to carefully unfold the sample. On the other hand, in the contact method, it is critical to wash the control and sample disks very well with PBS by pipetting them four times followed by a vigorous vortexing and sonication in order to ensure that no viable microorganisms remain adhered to the material surface.
This video protocol can be utilized in many bioengineering applications, such as bioprocess engineering, tissue engineering, controlled drug delivery, packaging materials, wastewater treatment, and agriculture, which use biomaterials with a highly desirable antimicrobial capacity.
The results obtained with this protocol are qualitative (the images) and quantitative (the normalized width of the antibacterial "halo" and the loss of viability) with a good analysis of its reproducibility (mean ± standard deviation). When comparing different materials, these mean values obtained with the diffusion and contact method results analysis must be analyzed by one-way ANOVA, followed by Turkey's post hoc analysis, in order to study if they are, statistically, significantly different (p <0.01).
The authors have nothing to disclose.
The authors would like to acknowledge the Universidad Católica de Valencia San Vicente Mártir for the financial support for this work through the 2017-231-001UCV and 2018-231-001UCV grants.
Cylindrical punch | 10 mm diameter | ||
Petri dishes | soria genlab | P101 | 90 mm diameter, sterile |
Tryptic soy agar (TSA) | Liofilchem | 610052 | Dehydrated medium 500 g (powder) |
Tryptic soy broth (TSB) | Liofilchem | 610053 | Dehydrated medium 500 g (powder) |
Sterile cotton swab | EUTOTUBO | 300200 | |
Centrifuge tubes | VIDRA FOC, SA | 429900 | 50 mL, sterile |
Ethanol | VWR | 83813360 | Absolute ethanol |
Sterile 48-wells plate | COSTAR | 3548 | Flat bottom with lid, tissue culture treated, non-pyrogenic, polystyrene |
A pair of tweezers | BRAUN | 24612036 | Toothless |
Sterile phosphate buffered saline (PBS). | VWR | E404-100TAPBS | |
Vaccum oven with a connected vacuum pump | JP Selecta, SA | 5900620 | |
Laminar flow hood | TELSTAR Technologies, SL | TELSTAR AH-100 | 12.0 W lamp of UV-C radiation |
Class II Biological safety cabinet | LABOGENE | MARS 1200 | |
Incubator | ASTEC CO, LTD | SCA-165DR | |
Vortex mixer | Biosan | V-1 Plus | |
Spectrophotometer | Macherey-Nagel, Germany | Nanocolor UV/VIS II | |
Bunsen burner | JP Selecta, SA | 7001539 | |
Alcohol burner | VIDRA FOC, SA | 1658/20 | In case sterilisation is necessary to be performed inside class II biological safety cabinet |
Orbital shaker | sartorius stedim | 8864845 | |
Sonicator | SELECTA | 3000617 | 50/60 Hz |
Digital calliper | ACHA | 17-260 | 0-150 mm |
Serological pipette | Fisherbrand | 13-678-11 | 25 mL, sterile |
Serological pipette | VWR | 612-4950 | 5 mL, sterile |
Serological pipette | VWR | 612-5541 | 10 mL, sterile |
Micropipette | GILSON | FA10005P | Pipetman L P200L, plastic 20-200 µL |
Micropipette | GILSON | F123602 | Pipetman P1000, 200-1000 µL |
Micropipette | GILSON | FA10016 | Pipetman L P12X300L, 20-300 µL |
Micropipette tips | LABBOX | TIBP-200-960 | 2-200 µL |
Micropipette tips | LABBOX | TIBP-1K0-480 | 100-1000 µL |
Pre-sterilized tube | INSULAB | 301402 | 10 mL |
Photo camera | Canon EOS 5D | Any camera with high resolution can also be utilized | |
Gram-positive bacteria Staphylococcus aureus | strain V329 | Cucarella et al. J Bacteriol 183 (9), 2888–2896 (2001) | |
Gram-negative bacteria Escherichia coli | Colección Española de Cultivos Tipo CECT | CECT 101 | |
Yeast Candida albicans | Colección Española de Cultivos Tipo CECT | CECT 1394 | |
Microcentrifuge tubes | DASLAB | 175508 | 1,5 mL |
Autoclave | JP Selecta, SA | 4002136 | |
Spectrophotometer-cuvettes | UVAT Bio CB | F-0902-02 | 4,5 mL |
Drigalski spatula | LABBOX | SPRP-L05-1K0 | Sterile, disposable |
glass balls (2 mm diameter) | Hecht Karl | 1401/2 | Autoclavable, alternative device to the Drigalski spatula |
Autoclave bags | DELTALAB | 200318 | To sterilize microbiological residues or contaminated material |
Electronic pipette filling device | JetPip | JET BIOFIL | |
Laboratory bottle with ISO thread, graduated, borosilicate 3.3 | LABBOX | SBG3-100-010 | 100 mL, for autoclaving culture media |
Laboratory bottle with ISO thread, graduated, borosilicate 3.3 | LABBOX | SBG3-250-010 | 250 mL, for autoclaving culture media |
Laboratory bottle with ISO thread, graduated, borosilicate 3.3 | LABBOX | SBG3-500-010 | 500 mL, for autoclaving culture media |
Laboratory bottle with ISO thread, graduated, borosilicate 3.3 | LABBOX | SBG3-1K0-010 | 1000 mL, for autoclaving culture media |
Latex gloves | DENIA | 2278000000 | |
Indicator tape for sterilization | LABBOX | STAP-A55-001 | Self-adhesive tape with impregnated paper turning to colour when exposed to sterilization process. |
Universal test tube rack | LABBOX | MTSP-001-001 | To hold centrifuge tubes |
Microcentrifuge tube rack | VWR | 211-0210 | To hold microcentrifuge tubes |
Sterile loop | ACEFE S.A. | 100140055 | 10 µL of capacity for microbial culture |
Material M1 | Universidad Católica de Valencia San Vicente Mártir (UCV) | Material type 1 | |
Material M2 | Universidad Católica de Valencia San Vicente Mártir (UCV) | Material type 2 | |
Material M3 | Universidad Católica de Valencia San Vicente Mártir (UCV) | Material type3 | |
Material M4 | Universidad Católica de Valencia San Vicente Mártir (UCV) | Material type 4 | |
Material C | Universidad Católica de Valencia San Vicente Mártir (UCV) | Control material |