Tomislav Ivankovic 1,*, Antonija Rajic 1, Sanja Ercegovic Razic 2, Sabine Rolland du Roscoat 3 and Zenun Skenderi 2
1. Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia; tona31@gmail.com
2. Department of Materials, Fibres and Textile Testing, Faculty of Textile Technology, University of Zagreb,
10000 Zagreb, Croatia; sanja.ercegovic@ttf.unizg.hr (S.E.R.); zenun.skenderi@ttf.unizg.hr (Z.S.)
3. Laboratoire Sols, Solides, Structures et Risques (3SR), UMR 5521, Université Grenoble Alpes, CNRS G-INP,
38000 Grenoble, France; sabine.rolland-du-roscoat@univ-grenoble-alpes.fr
* Correspondence: tomislav.ivankovic@biol.pmf.hr
Abstract: Wool is considered to possibly exhibit antibacterial properties due to the ability of wool
clothing to reduce the build-up of odor, which arises from the microbial activity of skin microbiota.
Indeed, when tested with a widely used agar diffusion plate test method, even wool or other textiles
not treated with any antimicrobial agent can be interpreted to show certain antibacterial effects due
to the lack of growth under the specimen, as instructed in ISO 20645:2004 standard. Therefore, we
analyzed in detail what happens to bacterial cells in contact with untreated wool and cotton fabric
placed on inoculated agar plates by counting viable cells attached to the specimens after 1 and 24 h of
contact. All wool and several cotton samples showed no growth under the specimen. Nevertheless, it
was shown without a doubt that neither textile material kills bacteria or inhibits cell multiplication. A
reasonable explanation is that bacterial cells firmly attach to wool fibers forming a biofilm during
multiplication. When the specimen was lifted off the nutrient agar surface, the cells in the form
of biofilm remained attached to the wool fibers, removing the biomass and resulting in a clear, no
growth zone underneath it. By imaging the textile specimens with X-ray microtomography, we
concluded that the degree of attachment could be dependent on surface topography. The results
indicate that certain textiles, in this case, wool, could exhibit antibacterial properties by removing
excess bacteria that grow on the textile/skin interface when taken off the body.
Keywords: textile; cotton; ISO standards; antimicrobial; agar diffusion
1. Introduction
The possibility that wool has antibacterial properties comes from the ability of wool
clothing to reduce/resist the onset of odor build-up, and odor is primarily considered to
originate from microbial activity [1–3]. Caven et al. [1] suggested three possible explanations for the supposed antibacterial properties of wool. First, the complex wool fiber
composed of epicuticle, lipid monolayer, and the cortex has an antibacterial effect, as
suggested by Johnson et al. [3]. Nowadays, this statement does not seem to be correct,
as several studies have undoubtedly shown that untreated wool itself does not exhibit
bactericidal or bacteriostatic properties [1,4,5]. These studies used the absorption method
(i.e., ISO 20743:2013) and compared the number of bacteria inoculated with liquid nutrient
media onto the fiber specimen and the number of bacteria on the specimen after a certain
period of time, showing that bacteria either remained viable or multiplied on the wool
fibers. In one of our previous studies, the number of bacteria inoculated, according to the
absorption method, on untreated wool fabric increased by four log values after 24 h of
contact [6].
The second explanation would be that wool bonds or adsorbs odorous fumes without
actually inhibiting bacterial growth, for which there is strong evidence [7–9].
The third possibility is that wool fibers’ hydrophobic surface and specific microstructure create a microclimate unfavorable for bacterial growth [1]. This third possibility was
the object of our investigation. To simulate the real-life conditions where wool is in direct
contact with skin covered with normal microbiota, we tested scoured plain weave woolen
fabric by using the standard method ISO 20645:2004 “Textile fabrics—Determination of
antibacterial activity—Agar diffusion plate test.” In this method, the textile specimen is
placed on top of the inoculated agar, and as the antimicrobial agent diffuses into the agar, it
either kills or stops bacterial cells from multiplying, giving a clear zone of “no bacterial
growth” around the specimen. However, according to the method instructions, even the
observation of no growth under the specimen can be classified as a “good effect” (the term
“good effect” is a direct quote from the document).
However, there is a methodological issue with the agar diffusion test; the ISO 20645:2004
is suitable only to test the textiles treated with an antimicrobial agent compared with the
control, untreated specimens [10,11]. However, earlier, we observed that untreated wool
and some other untreated textiles also show “no growth” under the specimen when tested
by the agar diffusion test [6]. Thus, can this be classified as an antibacterial effect?
To try and provide an answer, we further investigated the results of agar diffusion
testing by determining what is happening with the bacteria in contact with textile samples.
This was undertaken by counting the viable cells on the specimens after 1 h and 24 h of
contact of the textile specimen with agar inoculated with bacteria (Figure 1).
Figure 1. Experimental setup for determining the number of viable bacteria attached to the samples during the contact with inoculated agar. Initially, the bacteria are inoculated across the nutrient agar plate, and specimens are placed onto the plate. After the incubation (37 ◦C/24 h), the growth under the specimen was determined visually (1) and under the microscope (2). The textile specimen was transferred to 20 mL of sterile saline solution and vortexed five times for 5-sec bursts (3) to detach the bacterial cells from the specimen. The supernatant was serially diluted up to 10–7; plated and grown colonies were counted after the incubation at 37 ◦C/24 h (4). The number of bacteria was reported as log CFU·cm−2 of the specimen (5).
By doing this, we wanted to unravel the methodological issue of interpreting the “no
growth” under the sample (instructed by ISO 20645:2004 method) and to define whether
non-modified wool and other textiles adsorb the bacteria, kill the bacteria, or stop their
growth, or do not have any effect on bacterial cells at all.
2. Results and Discussion
2.1. Agar Diffusion Test
We assessed the antibacterial efficacy of three types of tested textiles, namely wool
fabric, the cotton of a standard laboratory coat, and cotton of standard sterile compressed
gauze, toward several bacterial species (Table 1). Bacterial species that were used in
the present research were chosen to represent diversity regarding their metabolic and
morphologic characteristics. The Staphylococcus aureus and Klebsiella pneumoniae are respectively Gram-positive and Gram-negative bacteria are listed as test organisms in ISO
20645:2004 standard. In our test battery, we added another Gram-positive bacterium, Bacillus cereus, which produces endospores. Finally, three strains of Gram-negative opportunistic pathogenic bacteria Acinetobacter baumannii were tested as well, an ATCC type strain and two hospital isolates resistant to multiple antibiotics.
Comparing the three materials, the best antibacterial effect was exhibited by wool
fabric (Table 1). Wool showed good antibacterial effect towards all tested bacteria except S. aureus, where it was at the limit of efficacy. Cotton was at the limit of efficacy towards all of the bacteria, again except S. aureus where it had insufficient effect, and two strains of A.
baumannii where it showed good effect. Compressed gauze exhibited either insufficient
effect or limit of efficacy. Comparing different bacteria, the results were similar for each
material (Table 1), suggesting that the specific textile affects all bacteria in the same manner, again exempting S. aureus, which seemed to have the strongest growth under the specimen,regardless of the material. To summarise, out of the six different bacteria we tested, the wool exhibited good antibacterial effect toward five of them, the cotton toward two of them, and compressed gauze towards none (Table 1).
2.2. Comparison to Literature Data
The comparison of our results with literature data was somewhat challenging. A
standardized method for antibacterial testing of textile samples resembling ours is perhaps
the AATCC 147 Standard [12], where bacteria are inoculated on top of the agar plates as
parallel streaks. The textile specimen is placed on the agar surface, and the inhibition zone around the specimen is monitored. Therefore, this method also enables direct contact of bacterial cells with the textile, assessment of growth under the sample, and can be used for textiles without diffusible agents [10]. We were able to find only one study reporting growth under the untreated wool sample; Liu et al. [13] tested capsaicin-coated wool fabric and reported “heavy growth” under control, untreated wool fabric, indicating no antibacterial effect. Another case of a very similar experimental setup was reported in Gomes et al. [14], where cotton modified with chitosan was tested by the JIS L 1902-Halo standard method.
This method is very similar to ISO 20645:2004 and is also essentially a pour plate method;
the difference is that “ISO” demands two layers of agar, the bottom layer clean and the
upper layer inoculated with bacteria, while the “Halo method” demands only one layer
of agar inoculated with bacterial culture. However, Gomes et al. [14] inoculated bacterial
suspension on top of the agar layer, making it identical to our setup. They report no
antibacterial effect on control untreated cotton, but only the “Halo zone”, the inhibition
zone around the specimen, was monitored. There is no mention of bacterial growth under
the specimen, preventing comparison to our results. A modified Kirby—Bauer test, a nonstandardized method for antibacterial testing of textiles, is identical to the experimental
setup we were using. However, we could not find any mention that growth under any
textile, let alone wool, was monitored and reported, only the inhibition zone [15–17].
What is clear from the available literature is that wool itself does not exhibit bactericidal
properties. Using the standard pour plate method, Pollini et al. [18] tested wool treated with silver and reported no antibacterial effect on untreated control samples. The same was reported in a couple of other studies [4,19]. From the aforementioned results, it is clear that the wool itself does not release any antibacterial agent whatsoever. However, as already mentioned, it also does not exhibit antibacterial activity when in contact with bacterial cells, as shown using the standard absorption method [1,4–6].
3.2. Textile Materials
To test the antibacterial properties of untreated textiles, one sample of scoured wool
fabric and two cotton samples were used in the experiment. The plain weave woolen
fabric of linear density (for warp/weft) 15.5 × 2/25 tex, count (warp/weft) of 21/24 cm−1
,and mass per unit area of 140 g/m2, is labeled as (W) and was industrially prepared
(washed, decatised, sheared and dried) and supplied by Varteks Ltd. (Varazdin, Croatia).
For comparison, two samples of cotton material of different degrees of finishing were
also tested. The first cotton sample was a standard laboratory white coat (C) in twill with
an embroidery count (warp/weft) of 18/17 cm−1 and mass per unit area of 220 g/m2
,manufactured by Marija Ltd. (Zagreb, Croatia). The second one was a standard sterile
compressed gauze (G) in canvas embroidery, with a count (warp/weft) of 11/8 cm−1
and mass per unit area of 40 g/m2, manufactured by Lianyungang Ruikang Sanitary
Dressing Company Ltd. (Lianyungang, China). For a positive control, a textile with known antibacterial properties, Aquacell® wound dressing that is incorporated with ionic
silver (Convatec Inc., Berkshire, UK). The textiles were cut into 20 × 20 mm squares and
used in the experiment without prior sterilization, as noted in ISO 20645:2004 standard
method. However, disinfected nitrile hand gloves were used during the cutting to minimize
contamination of samples from the skin bacteria.
3.3. Antibacterial Testing
The procedure of antibacterial testing (Figure 1) was based on a method described in
ISO 20645:2004—“Textile fabrics—Determination of antibacterial activity—Agar diffusion
plate test” (reference ISO 2004). Suspensions of concentration 105 and 108 Colony Forming Units (CFU) per mL of sterile 0.3% saline were made for each tested bacterium. Such solutions were made by dispersing bacterial biomass in sterile saline up to 0.5 McFarland units (corresponding to ~108 cells per mL). Next, the solution was serially diluted to obtain 105 CFU mL−1 suspension. The CFU’s were checked by plating prior to each experimental batch. To grow the “bacterial lawn”, bacterial suspension was spread across the Tryptic soy agar (Biolife, Italy) plate using a sterile cotton swab. After plate inoculation, two textile specimens (20 × 20 mm) were placed on the agar surface using sterile tweezers. The agar plates were then incubated for 24 h at 37 ◦C. The results were interpreted according to ISO 20645:2004 standard by examining colony growth under the textile sample, visually and under the microscope (Olympus Japan, CX21) at 40× magnification (Figure 1).
3.4. Determining the Number of Bacteria Attached to Textile Samples
To determine if and in what amount the bacteria remain attached to textiles during the
antibacterial testing (as previously described), the samples were gently removed (Figure 1) and immersed in 20 mL of sterile saline (in 50 mL Falcon-type tubes). The tubes were
shaken-out on a vortex shaker according to ISO 20743:2013—“Textiles—Determination
of the antibacterial activity of textile products”, for 5 × 5 s cycles. Shaking detaches the
bacteria from the fabric, and the cells remain free-floating in the saline suspension. A
total of 1 mL of suspension was serially diluted up to 10−7, and 0.1 mL was inoculated on
TSA plates. After the incubation (24 h/37 ◦C) the grown colonies were counted, and the
bacterial numbers were reported as CFU per cm2 of textile material. As a control, clean
textile specimens were used, meaning they were not previously incubated on agar plates.
Several bacterial colonies usually grew as the specimens were not sterile, but total counts
were less than 10 CFU·mL−1.
3.5. X-ray Microtomography
X-ray microtomography was performed to visualize the inner structure of the three
types of textiles at the micron scale. The 3D images of the textiles were obtained on a
laboratory tomograph manufactured by RX Solutions (Annecy, France) equipped with a
Hamamatsu X-ray source (Hamamatsu City, Japan) and a Varian flat panel detector (Varian Medical Systems, Salt Lake City, UT, USA). Each sample was irradiated with an X-ray beam (generated with a 100 kV 100 µA electron beam on a tungsten target) for 2400 angular projections equally spaced over 360◦
. The 2D radiographs were converted into a 3D dataset using a filtered back projection algorithm, and 3D views were obtained using the 3D viewer of Fiji software (v 1.52f). The chosen pixel size was set to 10 µm. This pixel size was chosen as it allows the visualization of the fibers and the fiber bundle that constitute the textile and a representative volume of the textile as many periods of the structure can be seen.
3.6. Statistical Analysis
All the experiments were undertaken in triplicate. The growth under the textile
specimen was determined qualitatively by visual inspection. The numbers of bacteria
attached to textile samples were quantitatively compared and analyzed using Statistica®
software (StatSoft, Tulsa, OK, USA). Ordinary Student’s t-test was used, and statistical
significance was set at p < 0.05.
4. Conclusions
The wool fabric showed antibacterial efficacy towards several bacterial species if interpreted according to the agar diffusion test as “no growth” under the textile sample. On the other hand, experiments monitoring the number of viable cells after 24 h of bacteria/textile contact showed that neither the wool sample nor two different cotton samples exhibited any bactericidal or bacteriostatic activity in terms of inactivating or killing bacterial cells.
Instead, bacterial cells readily multiplied on the textile samples during 24 h of incubation
on nutrient agar plates. The explanation would thus be that bacteria strongly adsorb to
wool while actively multiplying, developing a firmly attached biofilm. When the wool
sample was lifted off, the bacterial biofilm remained attached to wool fibers, removing the
biomass from the surface of the nutrient agar, resulting in a clear “no growth” zone under
the sample.
Since similar observations were present in experiments with cotton but not with
compress gauze, it would seem that the surface topography and structure of the textile
plays an important role in the antibacterial efficacy of the textiles which are unmodified
with some antibacterial agent. The results indicate that certain textiles, in our case woven
wool fabric, could exhibit antibacterial properties by removing excess bacteria that grow on the textile/skin interface when taken off the body.
A deeper analysis of biofilm formed on the textile fibers, visualized by scanning electronic microscopy, and quantified as it develops, would also be desirable. Along with
further tests with different non-modified textile materials and the same materials of differing structure and porosity, the question of why some textiles seem to show antibacterial efficacy without showing any bactericidal activity could be resolved.
Author Contributions: T.I. and S.E.R., conceptualization, experimental design, writing original draft preparation, review and editing.; A.R., methodology and experimental setup.; S.R.d.R., X-ray Microtomography analysis and editing.; Z.S., project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.
Funding: This work has been fully supported by the Croatian Science Foundation under the project(IP-2016-06-5278). The X-ray Microtomography imaging was enabled with the support of “COGITO” Hubert-Curien Partnership (Campus France) program no. 42815QE. The 3SR is part of LabEx Tec 21—ANR-11-LABX-0030 and of Institut Carnot PolyNat (ANR16-CARN-0025).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
