**Appendix A.**

*Appendix A.1. Validation of Utilisation of E. coli 25922 lux as a Proxy of E. coli 25922 for Investigation of PANI and P3ABA as Surface Antimicrobial Agents*

Appendix A.1.1. PANI Has Similar Activity against *E. coli* 25922 and *E. coli* 25922 *lux* While P3ABA Is Less Active against the Latter

For *E. coli* 25922, a *lux*-tagged version was used as the released bioluminescence can be detected and serves as a marker of cell viability [28,92,93]. Utilisation of a bioluminescently-tagged strain is a practical alternative to enumeration by plate counts for future testing of these potential surface additives against slow growing bacteria, such as *Mycobacterium tuberculosis* [63]. *E. coli* 25922 *lux* has a chromosomal insertion of the bacterial luciferase (*lux*) operon (*luxCDABE*) into the 16S locus [93]. A cell expressing the *lux* operon (*luxCDABE*) will be in an altered state compared to the non-tagged version as cellular energy is diverted in order to generate the luminescence [28]. The bioluminescence reaction consumes reduced flavin mononucleotide (FMNH2) and a long chain fatty aldehyde, and tetradecanoic

acid is diverted from the fatty acid biosynthesis pathway to regenerate the aldehyde substrate [97]. Therefore, it is possible that the *lux*-tagged version of *E. coli* 25922 may have different sensitivities to PANI and P3ABA compared to the non-bioluminescent version.

Following from this, the activity of PANI and P3ABA in LB broth was determined against *E. coli* 25922 and *lux*-tagged *E. coli* 25922. The measure of activity used was the standard minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) [55]. For PANI in suspension, *E. coli* 25922 and the *lux*-tagged version had similar sensitivities (Figure A1). P3ABA had a similar MIC against *E. coli* 25922 and *E. coli* 25922 *lux* (Figure A2); however, the MBC of P3ABA against *E. coli* 25922 (0.125%) was lower than that for *E. coli* 25922 *lux* (1–4%), which was statistically significant (Mann-Whitney test, *p* value: less than 0.05). The difference in activity of P3ABA observed against *E. coli* 25922 and *E. coli* 25922 *lux* may be reflective of the metabolic burden of light production. The *lux*-tagged *E. coli* 25922 may be less susceptible to the bactericidal action of P3ABA over a 24 h treatment time. Overall, these results support the use of *lux*-tagged *E. coli* for testing of PANI and P3ABA.

**Figure A1.** Activity of PANI against *E. coli* 25922 and *E. coli* 25922 *lux*. The MIC (circles) and MBC (squares) of PANI against *E. coli* 25922 (EC) and *E. coli* 25922 *lux* (EC *lux*) in LB broth. Data obtained from *E. coli* 25922 is represented by filled data points while data obtained from *E. coli* 25922 *lux* is represented by unfilled data points.

**Figure A2.** Activity of P3ABA against *E. coli* 25922 and *E. coli* 25922 *lux.* The MIC (circles) and MBC (squares) of PANI against *E. coli* 25922 (EC) and *E. coli* 25922 *lux* (EC *lux*) in LB broth. Data obtained from *E. coli* 25922 is represented by filled data points while data obtained from *E. coli* 25922 *lux* is represented by unfilled data points. Statistical significance is represented by \* (Mann-Whitney test, *p* value: less than 0.05).

Appendix A.1.2. The Limit of Detection for *E. coli* 25922 *lux* and *S. aureus* 6538 Recovered in a 96 Well Plate Is 100 CFU/mL

Examination of the activity of PANI and P3ABA in surfaces involved recovery of challenged cells in a 96 well plate, which facilitated high-throughput testing of many concentrations and treatment times against one inoculum [95]. Therefore, it was necessary to determine the limit of detection of *E. coli* 25922 *lux* and *S. aureus* 6538 growing in this manner to enable interpretation of surface testing results. The limit of detection was examined by determining the lowest number of cells added to 180 μL of LB broth in a 96 well plate that can grow to detectable levels [95]. The limit of detection is presented as CFU/mL to relate back to the initial inoculum concentration. The absolute number of cells present in the 180 μL volume in the wells would be roughly 5-fold lower than the CFU/mL value.

After a 16 h incubation of *E. coli* 25922 *lux*, bioluminescence 2 log or more above background levels was detected in the wells inoculated with ~10<sup>9</sup> to ~10<sup>3</sup> CFU/mL indicating bacterial growth (Figure A3). For the well inoculated with ~10<sup>2</sup> CFU/mL, the median bioluminescence level was 2 log above background levels; although, two points were only slightly above background levels (Figure A3). Therefore, the limit of detection of *E. coli* 25922 *lux* in a 96 well plate was 100 CFU/mL, which would correspond to ~20 CFU in the well.

After a 16 h incubation of *S. aureus* 6538, growth was detected in wells inoculated with ~10<sup>9</sup> to ~10<sup>2</sup> CFU/mL (with growth defined as OD600 above 0.05) (Figure A4). The median OD600 value from starting inoculum of ~10 CFU/mL was above the threshold for growth; however, two points were below background levels (Figure A4). Therefore, the limit of detection of *S. aureus* 6538 in 96 well plate was 100 CFU/mL, which would correspond to ~20 CFU in the well.

**Figure A3.** The limit of detection of *E. coli* 25922 *lux* when grown in a 96 well plate. *E. coli* 25922 *lux* was serially diluted in LB broth from ~10<sup>9</sup> CFU/mL to ~1 CFU/mL in a 96 well plate and incubated at 37 ◦C for 16 h. Light release (RLU s<sup>−</sup>1) was measured from each dilution and detectable light levels above 10 RLU s<sup>−</sup><sup>1</sup> indicates growth. The data presented are from four independent experiments.

**Figure A4.** The limit of detection of *S. aureus* 6538 when grown in a 96 well plate. *S. aureus* 6538 was serially diluted in LB broth from ~10<sup>9</sup> CFU/mL to ~1 CFU/mL in a 96 well plate and incubated at 37 ◦C for 16 h. OD600 was measured from each dilution and OD600 readings above 0.05 are considered as growth. The data presented are from four independent experiments.
