**2. Results**

*2.1. Activity of PANI and P3ABA Suspensions against E. coli and S. aureus*

To examine the activity of PANI and P3ABA, cell viability assays were performed on *E. coli* 25922 *lux* and *S. aureus* 6538 challenged with 0.5% (*w*/*v*) suspensions. Activity was determined in rich media, LB broth, for *E. coli* and *S. aureus,* as well as a minimal media, minimal A salts with succinate as the carbon source, for *E. coli*. At 0.5 h, 1 h, 2 h, and 4 h time points the treated cells were enumerated using the drop count method [64].

*E. coli* and *S. aureus* treated with PANI suspension were present at similar cell numbers at the earlier, 0.5 h and 1 h, time points (Figure 2A). For the later, 2 h and 4 h, time points *E. coli* was knocked down by 1 to 2 logs (measured as the difference between inoculum and the median number of viable cells remaining), while *S. aureus* cell numbers decreased by only ~0.5 log (Figure 2A). The overall difference in sensitivity between *E. coli* and *S. aureus* to 0.5% PANI suspension was statistically significant (linear regression analysis, intercepts are different, *p* value: less than 0.05).

**Figure 2.** *Cont.*

**Figure 2.** Sensitivity of *E. coli* 25922 *lux* and *S. aureus* 6538 to PANI and poly(3-aminobenzoic acid) (P3ABA) suspensions. Cell viability assays of ~10<sup>6</sup> CFU/mL *E. coli* and *S. aureus* treated with 0.5% PANI suspension (**A**) and 0.5% P3ABA suspension (**B**) in Lennox broth (LB), with each symbol representing the median of three technical replicates and the bar representing the median of each biological replicate. The data in A and B is replotted together in (**C**) where each point represents the median of biological replicates and the error bars are the range Viable cell counts (colony forming units (CFU)/mL) were obtained for each strain at 0.5 h, 1 h, 2 h, and 4 h time points. The limit of detection is 50 CFU/mL.

Both *E. coli* and *S. aureus* were more susceptible to P3ABA suspension when compared to PANI suspension (Figure 2). P3ABA suspension reduced *E. coli* viable cell numbers to below the limit of detection within 2 h, while *S. aureus* was knocked down by ~2 log followinga4h exposure (Figure 2B; i.e., the difference between inoculum and the median number of viable cells remaining was about 2 logs, or 100-fold). As observed for PANI suspension, P3ABA suspension was more active against *E. coli* as compared to *S. aureus* (Figure 2B,C). The difference in susceptibility between *E. coli* and *S. aureus* to 0.5% P3ABA suspension was statistically significant (linear regression analysis, intercepts are different, *p* value: less than 0.05). These results confirm that PANI and P3ABA in suspension are active against the model Gram-negative and Gram-positive organisms tested and support investigation of their surface activity.

The antimicrobial activity of 0.5% PANI and P3ABA suspensions in rich and minimal media was determined against *E. coli* 25922 *lux*. PANI suspension mediated a greater reduction in the viable cell count in minimal media when compared to rich media (Figure 3A). *E. coli* in minimal media reached the limit of detection (~4 log reduction) after a 2 h challenge, while a decrease of ~1–2 log was observed in rich media after 4 h (Figure 3A). The greater activity of PANI suspensions against *E. coli* in minimal media when compared to LB broth was statistically significant (linear regression analysis; intercepts are different, *p* value: less than 0.05).

**Figure 3.** *Cont.*

**Figure 3.** Sensitivity of *E. coli* 25922 *lux* to PANI and P3ABA suspensions in rich and minimal media. Cell viability assays of ~10<sup>6</sup> CFU/mL *E. coli* treated with 0.5% PANI suspension (**A**) and 0.5% P3ABA suspension (**B**) in Lennox broth (LB) and minimal A salts, with each symbol representing the median of three technical replicates and the bar representing the median of each biological replicate. The data in A and B is replotted together in (**C**) where each point represents the median of biological replicates and the error bars are the range. Viable cell counts (CFU/mL) were obtained for experimental sample at 0.5 h, 1 h, 2 h, and 4 h time points. The limit of detection is 50 CFU/mL.

P3ABA suspension was more active against *E. coli* in rich media as compared to minimal media (Figure 3B). *E. coli* in LB broth was knocked down ~3 log after 1 h and reached the limit of detection by 2 h (Figure 3B). In comparison, the levels of viable *E. coli* in minimal media were stable at the 0.5 h and 1 h time points (Figure 3B). After 4 h of treatment, P3ABA knocked down *E. coli* in minimal media by ~2 log (Figure 3B). The difference in activity of P3ABA against *E. coli* in LB broth and *E. coli* in minimal media was statistically significant (linear regression analysis, slopes are different, *p* value: less than 0.05). These results confirm that PANI and P3ABA in suspension are active against *E. coli* 25922 in rich and minimal media, and support the investigation of their surface activity.

### *2.2. Activity of Absorbent Surfaces Containing PANI and P3ABA against E. coli and S. aureus*

Following the demonstration of activity for PANI and P3ABA against *E. coli* and *S. aureus*, we investigated the antibacterial activity of absorbent surfaces containing PANI and P3ABA to simulate surfaces that absorb water droplets, such as fabrics [65]. LB agar as used as a model of an absorbent surface. Agar containing 1% and 2% concentrations of PANI and P3ABA in agar were tested along with a higher concentration of PANI (8% PANI), because PANI was less active than P3ABA in suspension (Figure 2).

To investigate antimicrobial activity, *E. coli* 25922 *lux* or *S. aureus* 6538 were inoculated onto the experimental agar, with cells being rescued at various time points in fresh media. Survival of the PANI or P3ABA in agar challenge was based on growth of rescued cells. A *lux*-tagged version of *E. coli* 25922 was used in this work as bioluminescence is a practical alternative to enumeration by plate counts [66–68] (Appendix A, Figures A1 and A2). Growth of *E. coli* 25922 *lux* was determined by measuring bioluminescence using the VICTOR X Multilabel Plate Reader. Growth of *S. aureus* 6538 was determined by examining optical density at 600 nm (OD600) using the μQuant™ Microplate Spectrophotometer as a *lux*-tagged *S. aureus* 6538 strain was not available for testing.

Agar containing 8% PANI mediated a decrease in bioluminescence levels of *E. coli* 25922 *lux* to background levels after 1 h of treatment, while 2% PANI agar required 2 h to reduce surface bacterial load (Figure 4A). The limit of detection for this assay is ~20 CFU (Figure A3). Agar containing 1% PANI did not exhibit antimicrobial activity against *E. coli* (Figure 4A). P3ABA agar had greater surface antimicrobial activity than PANI agar (Figure 4). Agar containing 1% P3ABA and 2% P3ABA reduced bioluminescence levels from *E. coli* 25922 *lux* to background levels within 1 h and 15 min, respectively (Figure 4B).

**Figure 4.** Sensitivity of *E. coli* 25922 *lux* to PANI and P3ABA in agar. (**A**) ~10<sup>4</sup> CFU of *E. coli* was exposed to 8% PANI, 2% PANI, and 1% PANI incorporated into LB agar for 0.25 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h. (**B**) ~10<sup>4</sup> CFU of *E. coli* was exposed to 2% P3ABA and 1% P3ABA incorporated into LB agar for 0.25 h, 0.5 h, 1 h, and 2 h. Following treatment, the cells were rescued by washing the agar surface with LB broth and transferred to a 96 well plate. Each point represents the median of three technical replicates and each bar represents the median of each biological replicate. The data from A and B is combined for comparison in (**C**). The rescued cells were incubated at 37 ◦C for 16 h and light release was measured. The vertical axis shows the bioluminescence measurements (relative light units per second, RLU s<sup>−</sup>1) from the recovered cells with each data point representing an independent experiment and the line representing the median. Background luminescence readings are ~10 RLU s<sup>−</sup>1.

PANI in agar was less active against *S. aureus* 6538 than *E. coli* 25922 *lux*, reflecting the trend observed with suspension testing (Figures 2A, 4A, and 5A). Agar containing 2% and 8% PANI reduced *S. aureus* surface load to background levels within 8 h and 4 h, respectively (Figure 5A). The limit of detection for this assay is ~20 CFU (Figure A4). As observed for *E. coli*, 1% PANI in agar was inactive against *S. aureus* within the time constraints (8 h) of the experiment (Figures 4A and 5A). The viability of *S. aureus* cells following treatment with 8% PANI in agar for 2 h, 4 h, and 8 h, and following treatment with 2% PANI in agar for 8 h was significantly different from that of untreated cells (Friedman test, *p* value: less than 0.05, Dunn's multiple comparison test).

**Figure 5.** Sensitivity of *S. aureus* 6538 to PANI in agar. (**A**) ~10<sup>4</sup> CFU of *S. aureus* was exposed to 8% PANI, 2% PANI, and 1% PANI incorporated into LB agar for 0.25 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h. (**B**) ~10<sup>4</sup> CFU of *S. aureus* was exposed to 2% P3ABA and 1% P3ABA incorporated into LB agar for 0.25 h, 0.5 h, 1 h, 2 h, and 4 h. Following treatment, the cells were rescued by washing the agar surface with LB broth and transferred to a 96 well plate. Each point represents the median of three technical replicates and each bar represents the median of each biological replicate. The data from A and B is combined for comparison in (**C**). The rescued cells were incubated at 37 ◦C for 16 h and optical density at 600 nm (OD600) was measured. The vertical axis shows the OD600 measurements from the recovered cells with each data point representing an independent experiment and the line representing the median. OD600 readings above 0.05 are considered as growth.

Agar containing 2% P3ABA effectively decontaminated *S. aureus* on a surface following a 15 min treatment (Figure 5B), which was consistent with the activity against *E. coli* (Figure 5A). 1% P3ABA in agar was less active than the higher concentration, with the former requiring a 4 h exposure to reduce bacterial load (Figure 5B). The activity of 2% P3ABA in agar over 15 min and 1% P3ABA in agar over 4 h was statistically significant (Friedman test, *p* value: less than 0.05, Dunn's multiple comparison test).

The activity of PANI and P3ABA presented in Figures 4 and 5 demonstrates their efficacy against *E. coli* and *S. aureus* when incorporated into an absorbent surface.

### *2.3. Activity of Non-Absorbent Surfaces Containing PANI and P3ABA against E. coli and S. aureus*

The activity of PANI and P3ABA as surface antimicrobials at non-absorbent surfaces to simulate surfaces that do not absorb water, such as walls and door handles, was investigated using SEBS films containing 5% PANI or 3% P3ABA [69]. The concentrations of the additive in these films are within the range typically used for incorporation into surfaces (0.1–5%) [69] and reflect the greater activity of P3ABA against *E. coli* and *S. aureus* compared to PANI as demonstrated in suspension (Figure 2) and in agar (Figures 4 and 5). The activity of PANI and P3ABA films was examined in a 'micro-surface testing assay' (MSTA), in which 10 μL of inoculum in LB broth is sandwiched between two pieces of film and recovered at particular time points in fresh LB broth in a 96 well plate [28,70]. Cell viability was determined by measuring the bioluminescence for *E. coli* 25922 *lux* and OD600for *S. aureus* 6538.

The MSTA for testing 5% PANI and 3% P3ABA films against bacteria was optimised using *E. coli* 25922 *lux*. Following either 2 h or 24 h challenges on the films, bacteria were rescued by washing with LB broth and incubated in a 96-well plate for 16 h, after which bioluminescence was measured. The bacteria present in the remaining recovery broth were enumerated using plate counts to verify the ability of bioluminescence levels to infer cell number.

PANI and P3ABA films gave no reduction in bacterial viability for *E. coli* 25922 *lux* for after a 2 h challenge, which was indicated by both plate counts and bioluminescence readings (Figure 6A,B). Films containing P3ABA were more active than their PANI counterparts after 24 h exposure, with the former reducing the plate counts and bioluminescence levels by ~2 log relative to the untreated cells (Figure 6A,B). The activity of 3% P3ABA films against *E. coli* after 24 h treatment was statistically significant (2-way RM ANOVA; CFU/mL *p* value: less than 0.05; RLU s<sup>−</sup><sup>1</sup> *p* value: less than 0.05). The similarity in trends seen between the plate counts and bioluminescence measurements from *E. coli* 25922 *lux* that was treated with PANI and P3ABA films confirmed that the bioluminescence-based experimental approach to determining the activity of a non-absorbent surface was appropriate to use for further testing. The results presented show that non-absorbent surfaces containing P3ABA can reduce bacterial load after a 24 h exposure.

**Figure 6.** *Cont.*

**Figure 6.** Sensitivity of *E. coli* 25922 *lux* and *S. aureus* 6538 to PANI and P3ABA films. ~10<sup>4</sup> CFU of *E. coli* (**A**,**B**) or *S. aureus* (**C**) in 10 μL LB broth was sandwiched between two pieces of PANI film, P3ABA film, or control film for 2 h (**A**,**B**) and 24 h (**A**–**C**). The cells were rescued by washing the film samples with LB broth and transferred to a 96 well plate (**B**,**C**). Each point represents the median of three technical replicates and each bar represents the median of each biological replicate. The rescued *E. coli* cells were also enumerated with plate counts (**A**). The cells in the 96 well plate were incubated at 37 ◦C for 16 h and light release (**B**) or OD600 (**C**) was measured. The vertical axes show the viable cell counts (**A**) and bioluminescence measurements (**B**) from the recovered *E. coli* cells, and OD600 measurements (**C**) from the recovered *S. aureus* cells, with each data point representing an independent experiment and the line representing the median. The limit of detection for the plate counts is 50 CFU/mL. Background luminescence readings are 10 RLU s<sup>−</sup>1. OD600 readings above 0.05 are considered as growth.

Following from this, the optimised protocol for determining the activity of 5% PANI and 3% P3ABA films was used against *S. aureus* 6538 with an increase in OD600 above 0.05, indicating the presence of viable cells. *S. aureus* 6538 was treated for only 24 h as PANI and P3ABA films were not active against *E. coli* 25922 *lux* following 2 h treatments (Figure 6A,B) and *S. aureus* was less sensitive than *E. coli* to PANI and P3ABA in suspension (Figure 2). Both 5% PANI in films and 3% P3ABA in films displayed no activity against *S. aureus* 6538 inoculated in LB broth (Figure 6C).

### *2.4. Characterisation of the Action of PANI and P3ABA Films against E. coli and S. aureus*

The activity of 5% PANI and 3% P3ABA films against *E. coli* and *S. aureus*, was poorer than expected. We hypothesised that this might be due to inoculating large numbers of bacteria in rich media. To test this hypothesis, films containing PANI and P3ABA were challenged with a range of concentrations of *E. coli* 25922 *lux* and *S. aureus* 6538. The test organisms were washed in saline to

simulate a low nutrient environment. A 2 h exposure was used, as this contact time is more effective in disrupting transmission pathways. The influence of the presence of organic matter was examined by challenging PANI and P3ABA films with *E. coli* 25922 *lux* washed in LB broth or in 0.85% saline for 2 h.

Bioluminescence from all of the doses of *E. coli* exposed to films containing 5% PANI was reduced to below background levels (Figure 7A). Films containing 3% P3ABA exhibited similar levels of antimicrobial activity (Figure 7A). The antimicrobial activity of PANI and 3PABA films against each inoculum level tested was significantly different from the control film (2-way RM ANOVA, interaction of film type and CFU dose *p* value: less than 0.05). These results indicate that PANI and P3ABA films are active against *E. coli* in low nutrient conditions.

**Figure 7.** Activity of PANI and P3ABA films against a range of CFU doses of *E. coli* 25922 *lux* and *S. aureus* 6538. ~10 CFU–~10<sup>4</sup> CFU of *E. coli* (**A**) or *S. aureus* (**B**) in 10 μL 0.85% saline was sandwiched between two pieces of PANI film, P3ABA film, or control film for 2 h. The cells were rescued by washing the film samples with LB broth and transferred to a 96 well plate. The rescued cells were incubated at 37 ◦C for 16 h and light release (**A**) or OD600 (**B**) was measured. The vertical axes show the bioluminescence measurements (**A**) or OD600 (**B**) from the recovered cells with each data point representing an independent experiment and the line representing the median. Background luminescence readings are 10 RLU s<sup>−</sup>1. OD600 readings above 0.05 are considered as growth.

OD600 values from ~10<sup>4</sup> CFU and ~10<sup>3</sup> CFU doses of *S. aureus* 6538 treated with 5% PANI and 3% P3ABA films were above OD600 of 0.05, the threshold for growth, indicating that the films were not active against these higher CFU doses (Figure 7B). The OD600 from lower doses of *S. aureus* that was exposed to PANI and P3ABA films did not increase above the threshold for growth implying killing of the inoculated cells occurred (Figure 7B). The activity of PANI films against ~10<sup>2</sup> and ~10 CFU, and P3ABA films against ~10 CFU was statistically significant (2-way RM ANOVA, film type *p* value: less than 0.05, CFU dose *p* value: less than 0.05). It can be concluded that PANI and P3ABA films are active against low inocula of *S. aureus* in saline.

The effect of the presence of organic matter on the surface activity of films containing PANI or P3ABA was determined by challenging *E. coli* 25922 *lux* in LB broth and 0.85% saline. Bioluminescence levels from *E. coli* 25922 *lux* recovered from 5% PANI films and 3% P3ABA films when inoculated in 0.85% saline were below background levels, whereas *E. coli* 25922 *lux* inoculated in LB broth released the same amount of light as bacteria that were recovered from control films (Figure 8). This indicates that *E. coli* in saline was much more sensitive to PANI and P3ABA films than *E. coli* in LB broth. It is possible that the constituents of LB broth interfere with the contact killing of *E. coli* on films containing PANI and P3ABA.

**Figure 8.** Activity of PANI and P3ABA films against *E. coli* 25922 *lux* in the presence and absence of organic matter. ~10<sup>4</sup> CFU *of E. coli* in 10 μL LB broth or 10 μL 0.85% saline was sandwiched between two pieces of PANI film, P3ABA film, or control film for 2 h. The cells were rescued by washing the film samples with LB broth and transferred to a 96 well plate. The rescued cells were incubated at 37 ◦C for 16 h and light release was measured. The vertical axis shows the bioluminescence measurements (RLU s<sup>−</sup>1) from the recovered cells with each data point representing an independent experiment and the line representing the median. Background luminescence readings are ~10 RLU s<sup>−</sup>1.
