Morgan M. Stanton, Byung-Wook Park, Diana Vilela, Klaas Bente, Damien Faivre, Metin Sitti, and Samuel Sánchez*

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1 Supporting Information Magnetotactic Bacteria Powered Biohybrids Target E. coli Biofilms Morgan M. Stanton, Byung-Wook Park, Diana Vilela, Klaas Bente, Damien Faivre*, Metin Sitti*, and Samuel Sánchez* Supporting video SV1 (AVI): Biohybrid: MSR-1 swimming with MSM (60x magnification). Magnetic field is indicated by arrow. Supporting video SV2 (AVI): Biohybrid swimming in a figure eight guided with external magnetic coils (20x magnification). Magnetic field is indicated by arrow. Supporting video SV3 (AVI): Multiple biohybrids guided by external magnetic coils. Supporting video SV4 (AVI): Time lapse video of 3D confocal images of CFX release from CFX-MSMs. E. coli biofilm surrounding CFX-MSMs absorb the released CFX. Video depicts fluorescence only from CFX. Supporting video SV5 (AVI): Biohybrid guided with permanent magnet to island of E. coli biofilm. Biohybrid Formation Previous attempts had (3-Aminopropyl)triethoxysilane (APTES) located inside and outside the tube, causing ubiquitous attachment of the bacteria in and around the biohybrid (Figure S1). The unspecific attachment resulted in multiple applied forces and no net direction with swimming or no movement at all. Modifications in the protocol of MSM synthesis located APTES only within the tube, so MSR-1 cells attached only inside. During the fabrication of the MSMs (see Materials and Methods), initial mesoporous silica formation inside the cyclopore

membrane began with the addition of tetraethyl orthosilicate (TEOS), hexadecyltrimethylammonium bromide, and triethanolamine at the required temperature.

for cell adhesion. Figure S1. Non-specific MSR-1 adhesion. Amine terminated groups (APTES) were located inside and outside the tube, prompting ubiquitous bacteria adhesion.

coli in 96-well plates (Figure S2A). CFX and increased CFX concentrations were directly linked to a decreased proliferation with 0.03 µg ml -1 CFX beginning to reduce E. coli density.

2 membrane began with the addition of tetraethyl orthosilicate (TEOS), hexadecyltrimethylammonium bromide, and triethanolamine at the required temperature. After formation of the outer layer of mesoporous silica, a mixture of APTES and additional TEOS were added to the reaction to create an inner layer of mesoporous silica with amine chemical moieties for cell adhesion. Figure S1. Non-specific MSR-1 adhesion. Amine terminated groups (APTES) were located inside and outside the tube, prompting ubiquitous bacteria adhesion. CFX Effects on Cell Viablity The effects of CFX concentration were initially examined with planktonic E. coli, where CFX was added in varied concentrations (0 0.3 µg ml -1 ) to E. coli in 96-well plates (Figure S2A). CFX and increased CFX concentrations were directly linked to a decreased proliferation with 0.03 µg ml -1 CFX beginning to reduce E. coli density. A different approach measuring cell velocity was used to examine the effect of CFX on MSR-1. The cell biology of magnetotactic bacteria varies significantly from E. coli and magnetotactic bacteria do not swarm or form biofilms. Quinolone antibiotics were developed to interfere with Gram-positive and Gram-

3 negative bacteria enzyme activity by inhibiting DNA synthesis, which ultimately results in bacteria death 40. A small variety of antibiotics including chloramphenicol and kanamycin have inhibited MRS-1 growth at low concentrations 41, however the effects of CFX on MSR-1 velocity have not been documented. MSR-1 cells were then tested for their ability to function in the presence of CFX since they were required to swim with antibiotic loaded MSMs. Here, we examined the ability of MSR-1 to operate in antibiotics by measuring their velocities in a 0.05 µg ml -1 CFX solution in motility media over 1 hour and compared them to a control group of MSR- 1 in a solution with no CFX (Figure S2B). The average velocity of the MSR-1 cells in the presence of CFX (27 µm s -1 ) was slightly higher than the control group (23 µm s -1 ), but no significant difference was observed, indicating MSR-1 were suited to carry antibiotic tubes without diminishing their swimming performance.

d. (n = 5). (B) MSR-1 average velocity in motility media over time with 0.

4 Figure S2. CFX effects on E. coli and MSR-1. (A) E. coli growth curves in different concentrations of CFX (0 0.3 µg/ml). Error bars indicate the s.d. (n = 5). (B) MSR-1 average velocity in motility media over time with 0.05 µg/ml CFX compared to a control group without CFX. Velocities were collected from free swimming MSR-1. Error bars indicate the s.d. of the mean (n = 10).

5 Figure S3. Examples of magnetic guidance of biohybrids. Tracks of biohybrids magnetically guided to biofilm targets (identified with yellow arrow). Figure S4. Example calibration curve of CFX fluorescence. Fluorescence intensity (FI) of various concentrations of CFX in motility media.

SUPPORTING INFORMATION

SUPPORTING INFORMATION SUPPLEMENTARY FIGURE LEGENDS Fig. S1. Separation of non-dissolved nanoparticles. Tests were conducted on the separation of non-dissolved nanoparticles added in excess to BEGM (A)