How are bacterial and fungal colonies on facemasks impacted by mask type, usage and lifestyle?

By Pooja Toshniwal PahariaJul 20 2022Reviewed by Aimee Molineux

In a recent study published in Scientific Reports, researchers identified and quantified fungal and bacterial microbial colonies attached to face masks used in the coronavirus disease 2019 (COVID-19) pandemic, and investigated if the microbes were associated with mask type, usage and lifestyles of individuals in Japan.

Face mask use is one of the most conventional practices for preventing respiratory diseases. The effectiveness of face mask use against SARS-CoV-2 transmission has been extensively investigated; however, data on the probable hygiene issues due to fungal and bacterial growth on the face masks are limited.

About the study

In the present study, researchers identified and quantified bacterial and fungal organisms on face masks used during the COVID-19 pandemic in Japan.

The study was conducted between September and October 2020 and comprised medical students [n=109, 63 men and 46 women (aged 21 to 22 years)] at Kindai University Faculty of Medicine, Japan. The study participants were surveyed on their face mask use (type, duration) and lifestyle habits, and fungi and bacteria were isolated from the masks by the outer side and face-side of the masks pressed onto separate agar plates.

The plates were incubated for five days and 18 hours for fungal and bacterial growth, respectively, and subsequently, microbial colonies were counted on both sides of the masks and compared. For evaluating the impact of the face mask type and usage duration, the microbial colony counts were compared for individuals who wore the masks for a day (three to six hours) and ≥ 2 days, based on the type of face mask (gauze, polyurethane, non-woven).

The potential sex-based differences in fungal and bacterial colony counts were assessed and the team explored if lifestyle and environmental factors (such as transportation) could affect fungal and bacterial counts on the masks. In addition, the impact of natto consumption and gargling on face mask microbial growth was evaluated. Further, the bacterial colonies were classified based on their size and morphologically into (i) small and white, (ii) large and white, (iii) small and yellow, (iv) medium and white, and other colony forms including medium to large and yellow or pink.

The colony sizes were interpreted as small, medium or large if they were <2mm, 2-10mm or >10mm, respectively. Furthermore, the bacterial colonies were determined using Gram stain and 16S ribosomal ribonucleic acid (rRNA) sequencing. Fungal colonies were incubated for an additional two days at room temperature to induce the formation of spores. Subsequently, lactophenol cotton blue (LCB) staining was performed to identify fungi on the face masks macroscopically based on colony morphology and microscopically based on the morphology of the hyphae and spores.

Additionally, principal component analysis (PCA) was performed based on daily facial skincare routine (including face wash, face lotion/sunscreen use, and foundation use) and the fungal and bacterial colonies on masks that were worn for four hours. Furthermore, the team explored the potential effect of foundation use on microbial growth on face masks, for which volunteers were asked to apply foundation on the left side of the face only and wear face masks for four hours.

Results

Bacterial colonies were more numerous, whereas fungal colonies were lesser on the face side compared to the outer side of the face masks. The fungal colonies substantially increased with the duration of mask use. Most (78%) of the study participants wore non-woven face masks, of mostly polyurethane type. And 75% of them disposed of the masks after a single day of use.

On the contrary, 58% of individuals who wore other masks used the same mask for ≥2 days, although they washed their masks in-between multiple uses. Bacterial colonies were observed in 94% and 99% of the samples on the outer side and the face side, respectively, with colony counts on the corresponding sides of 36.0 ± 7.0 and 168.6 ± 24.7.

Bacterial colonies were higher by 13.4-fold on the face-side of the masks with no substantial sex-based differences in the type and duration of mask use, except for significantly lesser bacteria on the face side of masks women wore. Fungal colonies were observed in 95% and 79% of the samples on the outer side and face side of the masks, respectively, with corresponding counts of 6.1±1.9 and 4.6±1.9.

Fungal colonies were less numerous than bacterial colonies on the masks and contrastingly, fungal colonies were higher by 2.4-fold on the outer side of the face mask compared to those on the face side. Repeated mask use for ≥2 days increased fungal colonies on the outer side of the masks, irrespective of the type of face mask used (non-woven or others). Significantly more large white colonies of B. subtilis were observed on both sides of the masks for individuals who consumed natto. 

In the 16S rRNA sequencing analysis, small-sized white microbes were most commonly observed, with >80% incidence and >70% total counts, respectively, comprising mainly S. aureus and/or Staphylococcus epidermidis; followed by the large-sized white microbial colonies comprising B. subtilis. The small-sized yellow microbial colonies were mainly formed by S. aureus.

The medium-sized white colonies comprised B. simplex and B. cereus. Most microbes identified were non-pathogenic for humans; however, several potentially pathogenic microbes such as Staphylococcus aureus, Bacillus cereus, Pseudomonas luteola and S. saprophyticus, Fonsecaea, Cladosporium, Trichophyton and Mucor were found.

Gargling and the method of transportation did not affect the growth of microbes on face masks. A 44% variance was observed in PC1, indicating that women practiced more intensive facial skincare, which may explain the lower bacterial counts on the face masks worn by women. However, foundation use did not impact microbial growth on face masks.

Conclusion

Overall, the study findings demonstrated the bacterial and fungal colonies on the outer side and face side of face masks worn during the COVID-19 pandemic. Based on the findings, immunosuppressed individuals must refrain from repeated usage of face masks and must wear non-woven masks daily for preventing potential microbial infections.