Textiles and clothing are in continuous contact with microorganisms through the environment and skin. These can cause serious problems such as the material aging and deteriorating, staining and creating unpleasant odours, and can also promote allergic reactions, toxic responses, infections or disease. Therefore, one of the most recent objectives of the textile industry is to control these undesirable effects caused
by microorganisms1.

Generally, microbial growth is generated as a result of perspiration, and the microorganisms multiply and adhere to the tissues and/or materials at a microscopic level by means of biofilms, which increase their chances of survival.

Today there are a large number of fabrics on the market claiming to have antibacterial properties, but in fact, only a small part of the clothes advertised as antibacterial are really so; most of the clothes on the market lack these properties. Hence, the importance of checking the characteristics attributable to the footwear component by means of the methodologies that will be explained later.

Among the professionals who work in industrial sectors, the ones who stand out are, for example, those who work in the HORECA, Food and Health sector. These workers are exposed to a high risk of intoxication and therefore it is essential to have a type of footwear that not only inhibits the growth of different bacteria, including Campylobacter, Legionella, E. Coli, MRSA, Listeria, Salmonella, etc., but also eliminates them one hundred percent.

The CTCR has researched on this subject and has become a pioneering Technology Centre in the application of solutions in the bacterial field to Personal Protective Equipment (PPE).

According to the FAO (Food and Agriculture Organization of the United Nations), food-borne diseases caused by contaminated food are the greatest health hazard at present on an international level. In recent years, infections and intoxications have proliferated due to the reproduction of bacteria in work environments, affecting both the health of workers and customers, and consequently the activity and reputation of companies. Cases such as Campylobacter, Legionella, E. Coli, MRSA, Listeria4
and Salmonella have caused millions of poisonings, countless hospitalisations and a very high cost for medical expenses and loss of working hours… hence its relevance.

Antimicrobial compounds on the market

Currently, a large number of projects are being developed that give rise to compounds that prevent the growth of microorganisms and their incorporation into materials. The treatments applied to textile materials, polymers or others in the industry confer protection against bacterial proliferation. However, the effectiveness of these treatments depends largely on factors such as the area of contact, release and dispersion and their success depends on the ease of industrial application, impregnation and speed.

Some of the most recent development projects of antimicrobial treatments in textiles use various active agents such as silver, quaternary ammonium salts, triclosan, chitosan, dyestuffs and peroxyacids3. Many of the fabrics on the market include silver ions among their fibres, which are non-harmful in contact with the skin and proven to be effective against a wide variety of bacterial species. Others incorporate antimicrobial nanoparticles such as copper oxide or zinc oxide and are commonly used in healthcare and hospital fabrics. Many of the material and textile finishes limit the growth of microorganisms through the use of these biocides and their incorporation is carried out either during the final stages of production with a finishing treatment or by incorporating them into the synthetic fibres during extrusion.

It is important to note that the use of certain antimicrobial additives by industry can have harmful effects such as altering the natural flora of the skin and causing allergic reactions. Therefore, new agents of biological and renewable origin such as chitosan derived from fungi are being researched.

It should be noted that in general one speaks of antibacterial activity, but the material can incorporate different types of these active substances. Bactericides or fungicides kill the microorganism, and bacteriostatic or fungistatic substances inhibit growth. Their mode of action on bacterial cells prevents their multiplication either by damaging their external cytoplasmic membrane, interfering with the functionality of DNA and RNA or reacting with protein groups and enzymes.

International methodologies

Being a growing industry, a large number of standards and methodologies have been developed in recent years. However, there is no consensus as to the most appropriate application method to use, which will depend on factors such as:

– Contact time

– Final results of qualitative or quantitative type

– Type of test material: the same methodologies are not used for absorbent and non-absorbent materials

– Type of antimicrobial agent

– Type of attachment of this agent to the material

Numerous international methods have been published to test the antibacterial activity in textiles. The most important standards include qualitative techniques: AATCC TM147:20115, UNE-EN ISO 20645:20056 and JIS L 1902:20157, which involve the appearance of a halo of inhibition and quantitative methods: AATCC TM100:20198, UNE-EN ISO 20743:20149 and ASTM E2149-1010, which are absorption methods in which a count of microorganisms is made. Agar diffusion methods or qualitative methods are very easy to perform, fast and very useful when several samples are evaluated. Quantitative or sorption methods provide values of antimicrobial activity based on growth reduction, but are more costly in terms of time and material.

Another standard is that which applies to plastic materials and other non-porous surfaces, ISO 22196: 201111, which serves as a guide to quantitatively evaluate the effectiveness of an antibacterial product when applied to material surfaces such as polymers and ceramics.

With regards to the evaluation of antifungal activity, the most important standards for the textile sector are UNE-EN 14119: 200412, which describes the methods for evaluating the action of microscopic fungi on textiles, this being a qualitative technique, and ISO 13629-2:201413, which also evaluates antifungal activity on textiles, but using the plate-count technique, the final result being of a quantitative type. To evaluate the activity of microorganisms in other types of materials such as plastics or polymers, UNE-EN ISO 846:199814 or ASTM G2115, respectively, are used.

The UNE-EN ISO 16187:201416 standard specifies quantitative methods for evaluating antibacterial activity in footwear and its components. Recently, a new specific standard for the footwear and components sector for the evaluation of the quantitative challenge of fungal activity has been published: UNE-EN ISO 20150:202017. Both are used within the footwear sector to evaluate all types of materials in footwear and components, in which antibacterial treatments are used or have been used.

Antibacterial activity is defined in the context under consideration as the effectiveness of a footwear material or finish in preventing or mitigating bacterial growth, i.e. the ability to reduce the number of bacteria or to kill those bacteria. Similarly, antifungal activity is defined as the effectiveness of a material or finish to prevent or mitigate the growth of microfungi, to reduce their number or to eliminate them.

UNE-EN ISO 16187 methodology

Sample preparation

When dealing with the analysis of the antibacterial capacity of the different components and materials of the footwear, the standard refers to the preparation of the sample. The area must be specifically 500mm2 and a minimum thickness of 2mm. The area and weight are important for calculating the initial volume of bacterial suspension, and if the sample size is increased, the volume of bacterial suspension must be increased in proportion since the initial characteristics of the sample influence the way in which the bacteria interact with the surface. At least 6 samples are prepared for each material or component and for each bacterial species tested.

The samples must be obtained directly from the footwear model. In addition, each sample to be tested must have at least 80% of the surface of the component of the material claimed to be antibacterial (instep, lining, sole pad, heel, sole…), which has antibacterial properties.

Microorganisms and inoculum preparation

The species used to make the antibacterial activity assays are:

– Staphylococcus aureus AS 1.89 or ATCC 6538 (Gram positive)

– Klebsiella pneumoniae AS 1.1736 or ATCC 4352. (Gram negative)

The standard allows the use of other bacterial strains as long as the test includes at least one Gram-positive and one Gram-negative bacterium, since their activity is different against antibacterial agents and the difference is a consequence of their cellular structure. Bacteria such as: Mycrococcus lylae, Corybacterium xerosis, Brevibacterium epidermidis, Micrococcus luteus have been used.

These are bacteria classified in Biological Risk Group 2 which carries a moderate risk, they are pathogenic bacteria that can cause human or animal disease but rarely cause harm if appropriate preventive measures are used in the laboratory. Therefore, their handling requires experienced personnel and appropriate safety measures.

Using a sterile seeding loop, a colony is transferred to 20 ml of Nutrient Broth (NB) growth medium and incubated in agitation at 37 ± 2°C for 16 hours. Then the number of bacteria is counted, a specific volume is taken to prepare the final inoculum in NB 1% broth with a final concentration of approximately 2.5×105 CFU/ml.

Procedure

There are different methods that are applied depending on the initial material. For example, Method A should be used only for absorbent materials and Method B should be used only for non-absorbent materials, however, Method C can be applied for each of them or when they are combined.

All the methodologies established by the standard are quantitative and have in common that a determination of the number of viable bacteria is made, before and after contact with the sample under study. The aim is to determine whether the antibacterial active substance has an effect on the growth of the bacteria. All methodologies have an incubation period of 24 ± 1h and the temperature is 37 ± 2ºC.

The main difference lies in how the inoculation protocol is carried out and the incubation conditions, which can be static or with agitation.

Expression of results

The antibacterial performance of the footwear or footwear components is reported separately based on the calculation of the antibacterial activity ratio. It is expressed as a percentage (%) according to the following formula:

R= [(Ct-Tt)/Ct ]*100

Therefore, the antimicrobial activity is determined by comparing the results obtained between the growth value in CFU/ml of the control sample (Ct ) and the growth value of the treated sample (Tt), before and after the determined contact time (24 hours at an optimal temperature for the growth of the selected micro-organism) and subsequent culture.

In case no samples of the same material with no antibacterial capacity are available, the ratio is calculated using the following formula:

R= [(T0-Tt)/T0 ]*100

Where T0 is the number of colonies at initial time, before incubation expressed in CFU/ml.

UNE-EN ISO 20150 methodology

Sample preparation

The sample must be a piece with an area of 500 mm2 and a minimum thickness of 5 mm. Area and weight are important and should be noted. At least 6 samples are prepared for each material or component and for each test strain against which the test is performed.

The samples should be obtained directly from the footwear model. In addition, each sample to be tested must have at least 80% of the surface of the component of the material claimed to be antifungal (upper, lining, sole, heel, sole…).

The samples should be pre-treated or sterilized if high initial contamination is assumed, but it is necessary to ensure that this pre-treatment does not affect the properties.

Microorganisms and preparation of spore suspensions

The strains used for the antifungal activity test depend on the activity claimed, and Candida albicans must be used:

• Candida albicans ATCC 10231

• Aspergillus niger ATCC 6275

• Aspergillus brasiliensis ATCC 16404

Trichophyton mentographytes ATCC 9533

For the preparation of C.albicans inocula, a colony is transferred to 20 ml of malt extract and incubated with agitation at 28±2ºC in overnight culture. The necessary volume of this is added to a fresh buffer solution with wetting agent to reach a yeast concentration of 1 to 5×105 CFU/ml. The preparation procedure of the fungal spore suspension is more complex and includes the formation of spores first on a PDA plate and then scraping them off and introducing them into a tube with 5 ml of salt solution, stirring with glass beads and filtering into gauze. These solutions are centrifuged and washed several times

 to dilute them and adjust them to a concentration of 1 to 5×106 spores/ml.

Procedure

As with the specific standard for antibacterial testing, there are different methods that are applied depending on the starting material. For example, Method A should be used only for absorbent materials and Method B should be used only for non-absorbent materials, however, Method C can be applied for each of them or when they are combined.

The methodology is quantitative and is based, as in the case of antibacterial tests, on the determination of the number of viable fungi before and after contact with the sample under study. The aim is to determine whether the active substance has an effect on the growth of the fungi. All methodologies have an incubation period of 24 ± 2h and the temperature is 28 ± 2°C.

The major difference lies in how the inoculation protocol is carried out and the incubation conditions, which may be static or agitated.

Expression of results 

It uses the same formulas as for the antibacterial test, since the expression of the final value is in the form of percentage (%) inhibition. R is the antifungal activity index.

In short, the evaluation of the antimicrobial activity of materials that make up safety footwear, among others, whether against bacteria or fungi, is important to provide safety to both producers and traders in the face of changes in international markets and the antimicrobial requirements defined by regulations in each country.

References 

1 Teufel, L., Redl, B., 2006. Improved methods for the Investigation of the Interaction between textiles and microorganisms. Lezinger Berichte, 85: 54-60.

2 Gabriela Durán, 2011. Textiles antimicrobianos. UTN, Buenos Aires.

3 Gao, Y., Cranston, R., 2008. Recent Advances in Antimicrobial Treatments of Textiles. Textile Research Journal, 78 (1): 60-72.

4 Pinho, E., Maghalaes, L., Henriques, M., Oliveira, R., 2010. Antimicrobial activity assessment of textiles: standard methods comparison. Ann Microbiol, 61: 493-498.

5 AATCC TM147:2011, Antibacterial activity assessment of textile materials: parallel streak method

6 UNE-EN ISO 20645:2004. Textile fabrics- Determination of antibacterial activity – Agar diffusion plate test.

7 JIS L 1902:2015, Determination of antibacterial activity and efficacy of textile products.

8 AATCC TM100:2019, Assessment of Antibacterial Finishes on Textile Materials

9 UNE-EN ISO 20743:2014, Textiles – Determination of antibacterial activity of textile products.

10 ASTM E2149-10. Standard Test Method for Determining the Antimicrobial Activity of Immobilized Antimicrobial Agents Under Dynamic Contact Conditions.

11 ISO 22196: 2011, Measurement of antibacterial activity on plastics and other non-porous surfaces.

12 UNE-EN 14119: 2004 Testing of textiles – Evaluation of the action of microfungi.

13 ISO 13629-2:2014. Determination of antifungal activity of textile products.

14 UNE-EN ISO 846:1998. Plastics – Evaluation of the action of microorganisms.

15 ASTM G21. Standard Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi

16 UNE-EN ISO 16187:2014. Footwear and footwear components – Test method to assess antibacterial activity

17 UNE-EN ISO 20150:2020. Footwear and footwear components – Quantitative challenge test method to assess antifungal activity.