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. 2022 Feb 14;10(2):439.
doi: 10.3390/microorganisms10020439.

Bacterial Biofilm Formation on Nano-Copper Added PLA Suited for 3D Printed Face Masks

Annika Kiel  1 , Bernhard Peter Kaltschmidt  2 , Ehsan Asghari  1 , Andreas Hütten  2 , Barbara Kaltschmidt  1 , Christian Kaltschmidt  1
Affiliations

Bacterial Biofilm Formation on Nano-Copper Added PLA Suited for 3D Printed Face Masks

Annika Kiel et al. Microorganisms. .

Abstract

The COVID-19 Pandemic leads to an increased worldwide demand for personal protection equipment in the medical field, such as face masks. New approaches to satisfy this demand have been developed, and one example is the use of 3D printing face masks. The reusable 3D printed mask may also have a positive effect on the environment due to decreased littering. However, the microbial load on the 3D printed objects is often disregarded. Here we analyze the biofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli on suspected antimicrobial Plactive™ PLA 3D printing filaments and non-antimicrobial Giantarm™ PLA. To characterize the biofilm-forming potential scanning electron microscopy (SEM), Confocal scanning electron microscopy (CLSM) and colony-forming unit assays (CFU) were performed. Attached cells could be observed on all tested 3D printing materials. Gram-negative strains P. aeruginosa and E. coli reveal a strong uniform growth independent of the tested 3D filament (for P. aeruginosa even with stressed induced growth reaction by Plactive™). Only Gram-positive S. aureus shows strong growth reduction on Plactive™. These results suggest that the postulated antimicrobial Plactive™ PLA does not affect Gram-negative bacteria species. These results indicate that reusable masks, while better for our environment, may pose another health risk.

Keywords: 3D printing; E. coli; P. aeruginosa; PLA; S. aureus; antimicrobial; biofilm; face masks; nano-copper; personal protective equipment.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Overview of the development of a 3D-printed face mask. The top image shows pictograms of a typical op mask and three coronaviruses. On the left side the slicer software preview of a 3D printable mask is shown. In the bottom graphic the pictogram of a 3D Printer can be seen. The image on the right shows the finished 3D-printed mask mounted on the face.
Figure 2
Figure 2
Scanning electron microscopy (SEM) images showing the biofilm formation and size measurement of P. aeruginosa. (A) The two top SEM images depict the formation of biofilm on Plactive™ PLA. The two bottom images depict the biofilm formation on Giantarm™ PLA. Slime formation is indicated by an arrow. (B) The two top graphs visualize the size distribution of the bacteria on Plactive™ PLA, and the two bottom graphs visualize the size distribution of the bacteria on Giantarm™ PLA. For all four graphs, the y axis represents the relative percentage and the x axis represents the length or width of the bacteria in microns.
Figure 3
Figure 3
Scanning electron microscopy (SEM) images showing the biofilm formation and size measurement of S. aureus. (A) The two top SEM images depict the formation of biofilm on Plactive™ PLA, and the two bottom images depict the biofilm formation on Giantarm™ PLA. (B) The top graph visualizes the size distribution of the bacteria on Plactive™ PLA, and the bottom graph visualizes the size distribution of the bacteria on Giantarm™ PLA. For all graphs, the y axis represents the relative percentage and the x axis represents the radius of the bacteria in microns.
Figure 4
Figure 4
Scanning electron microscopy (SEM) images showing the biofilm formation and size measurement of E. coli. (A) The two top SEM images depict the formation of biofilm on Plactive™ PLA, and the two bottom images depict the biofilm formation on Giantarm™ PLA. (B) The two top graphs visualize the size distribution of the bacteria on Plactive™ PLA, and the two bottom graphs visualize the size distribution of the bacteria on Giantarm™ PLA. For all four graphs, the y axis represents the relative percentage and the x axis represents the length or width of the bacteria in microns.
Figure 5
Figure 5
Confocal laser scanning microscope (CLSM) images, percentages of live versus dead cells and percentages of covered area for P. aeruginosa, S. aureus, and E. coli. (A) CLSM images of P. aeruginosa biofilm formation on Plactive™ PLA (left) and Giantarm™ PLA (right). (B) Percentages of live and dead cells of P. aeruginosa on Plactive™ PLA (blue) and Giantarm™ PLA (white). (C) Percentages of the bacterial covered area in relation to the uncovered area of P. aeruginosa on Plactive™ PLA (blue) and Giantarm™ PLA (white). (D) CLSM images of S. aureus biofilm formation on Plactive™ PLA (left) and Giantarm PLA (right). (E) Percentages of live and dead cells of S. aureus on Plactive™ PLA (blue) and Giantarm™ PLA (white). (F) Percentages of the bacterial covered area in relation to the uncovered area of S. aureus on Plactive™ PLA (blue) and Giantarm™ PLA (white). Note the strong antimicrobial effect on S. aureus of Plactive™ and Giantarm™. (G) CLSM images of E. coli biofilm formation on Plactive™ PLA (left) and Giantarm™ PLA (right). (H) Percentages of live and dead cells of E. coli on Plactive™ PLA (blue) and Giantarm™ PLA (white). (I) Percentages of the bacterial covered area in relation to the uncovered area of E. coli on Plactive™ PLA (blue) and Giantarm™ PLA (white).
Figure 6
Figure 6
Colony forming unit (CFU) assays of P. aeruginosa, S. aureus, and E. coli. (A) CFU assay revealing the number of CFUs per ml (y-axis) for the growth of P. aeruginosa on Plactive™ PLA (blue) and Giantarm™ PLA (white). (B) CFU assay revealing the number of CFUs per ml (y-axis) for the growth of S. aureus on Plactive™ PLA (blue) and Giantarm™ PLA (white). (C) CFU assay revealing the number of CFUs per ml (y-axis) for the growth of E. coli on Plactive™ PLA (blue) and Giantarm™ PLA (white).
Figure 7
Figure 7
Comparison of the measured and literature size values and mean of the measured size values depicted in a bar graph (A) Table showing the measured values and literature values of the three analyzed bacteria (P. aeruginosa, S. aureus, E. coli) [37]. The second and third columns depict the measured values on Plactive™ PLA and Giantarm™ PLA, respectively. The fourth column shows the literature value. (B) Bar graph depicting the mean of the measured size values in microns for the three different bacteria. The first group belongs to P. aeruginosa, of which the two longer bars show the length of the bacteria on Plactive™ PLA and Giantarm™ PLA, respectively. The two shorter bars visualize the width of the bacteria, the first of the two on Plactive™ PLA and the second on Giantarm™ PLA. The group of two bars in the middle of the graph belongs to S. aureus, where the mean of the diameter is visualized, the left bar showing the value on Plactive™ PLA and the right bar the value on Giantarm™ PLA. The four rightmost bars belong to E. coli, where the two left bars of the group depict the mean of the length of the bacteria on Plactive™ PLA and Giantarm™ PLA, respectively. The two right bars show the mean of the width of the bacteria on Plactive™ PLA and Giantarm™ PLA.
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