# Copper and nanostructured anatase rutile and carbon coatings induce adaptive antibiotic resistance – AMB Express

Sep 7, 2022

### Bacterial strains, culture conditions, materials and chemicals

Bacterial strains used in this study are shown in Table 1. E. coli BW25113 (Baba et al. 2006) is a useful strain for genetic studies because it the parent in a series that includes nearly every possible gene knockout. We have measured the baseline responses of efflux phenotypes and gene expression using this strain (Kurenbach et al. 2017). Strains are stored in 15% (v/v) glycerol solution at − 80 °C. They were recovered for use on Lauria Bertani (LB) agar plates (Lennox-L-Broth Base, Invitrogen, Auckland (New Zealand) and agar (Bacteriological Agar No.1, Oxoid, Hampshire (UK)) and then incubated at 37 °C. Plates were replaced at one-week intervals. All antibiotics were purchased from Sigma, Auckland (New Zealand).

Paraformaldehyde used for fixation of cells prior to fluorescence microscopy was purchased from Sigma, Auckland (New Zealand) and stored as powdered stock at 4 °C. 4% (v/v) solution of paraformaldehyde was made up on the day it was to be used and kept at 4 °C.

Stainless steel (25 × 25 mm) coupons were used as an inert control material as recommended by the ISO 27447:2009 (Mills et al. 2012), and commercially pure copper (25 × 25 mm) coupons were selected as a positive control material because they have known antimicrobial properties that are independent of light (Wasa et al. 2021). The NsARC (25 × 25 mm) coupons and control (stainless steel and copper) samples were sterilised by immersion in 70% ethanol and the NsARC test pieces were then aseptically stored in the dark for > 48 h. Gelatine coated slides were made by immersing glass microscopy slides (Mareinfeld-Superior, 76 × 26 mm, approx. 1 mm thick) in 70% ethanol for 1 h, then air drying and dipped in 0.1% (v/v) gelatine solution at 70oC before air drying again. After drying the slides were kept at 4 °C and used within 1 week of drying.

### Determining if NsARC can cause a change in susceptibility of E. coli and S. aureus to antibiotics

The E. coli culture was exposed to the tests and control metal samples using a modified version of ISO 27447:2009 (Wasa et al. 2021). 100 µl of the test organisms containing ~ 1,000,000 cells at the stationary growth phase were placed on 25 × 25 mm NsARC and stainless-steel surfaces and 24 × 24 mm sterile coverslip (Lab Supply, Dunedin (New Zealand)) was place on top, causing the liquid to spread evenly on the surface. The samples were then placed in 60 × 15 mm petri dishes. Replicates were then simultaneously exposed to visible light of 2100 lx (450–650 nm) or kept in the dark for 8 h. Afterwards, the samples were rinsed using phosphate buffered saline (PBS). The number of bacteria in 100 µl of the PBS was about ~ 102 cells. This volume was transferred to the surface of LB plates containing 0, 0.01, 0.02, 0.03, 0.04, 0.05 µg/ml of ciprofloxacin, or 0, 5, 6, 7, 8, 9 µg/ml of chloramphenicol, or 0, 4, 5, 6, 8, 9 µg/ml of kanamycin or 0, 0.5, 0.7, 1, 1.5, 2 µg/ml of tetracycline.

The same procedure as followed using Staphylococcus aureus. Wash off from NsARC was transferred to the surface of LB plates containing 0, 1, 2, 3, 4, 5, 6 µg/ml of erythromycin, or 0, 0.09, 0.1, 0.2, 0.3, 0.4 µg/ml of fusidic acid, or 0, 0.9, 1, 2, 3, 4 µg/ml of kanamycin, or 0, 0.07, 0.08, 0.09, 0.1, 0.2 µg/ml of oxacillin, or 0, 0.09, 0.1, 0.2, 0.3, 0.4 µg/ml of tetracycline or 0, 0.5, 0.8, 1, 1.5, 2 µg/ml of vancomycin. The E. coli and S. aureus wash off from stainless steel was diluted threefold to achieve ~ 102 cells and then was transferred to the surface of LB plates containing various concentration of antibiotics as described above. The minimum inhibitory concentrations of the antibiotics are shown in Additional file 1: Table S21. Plates were incubated at 37 °C for 24 h. All experiments were conducted three times to obtain biological replicates. Three samples of each (test and control) were used for each experiment to obtain technical replicates. The plates were monitored for up to three days. This was compared for the various treatments (material and exposure conditions). There were variations in the values, thus, the cfu/ml counts were normalised to efficiency of plating (EOP) values using the formula.

$${rm EOP} =frac{titre,of,treatments,(text{L}text{B} + text{a}text{n}text{t}text{i}text{b}text{i}text{o}text{t}text{i}text{c}text{s})}{titre,of,control left(LB,only right)}.$$

The EOP values were then used to plot graphs using graphpad prism software.

### Determining if NsARC can induce gene expression

Individual colonies formed by reporter strains E. coli BWtolC and BWsoxS (Jun et al. 2019) were placed into LB broth (Lennox-L-Broth Base, Invitrogen, Auckland (New Zealand)) supplemented with kanamycin and then placed on a shaker platform at 37 °C and grown to exponential phase. 100 µl containing about ~ 107cells was then placed on separate NsARC (test), stainless steel (negative control) and copper (positive control) coupons. Sterile cover slips (24 mm × 24 mm) were used to spread the cultures on the sample surfaces. The samples were placed in petri dishes (60 mm × 15 mm) containing damp filter paper. Replicates were simultaneously exposed to visible light of 2100 lx (450–650 nm), UV light (365 nm), ambient light (650–750 nm) and also kept in the dark for a period of not more than 2 h before washing off with PBS and the cells fixed with paraformaldehyde (Chao and Zhang 2011). 2 µl of the fixed cells were then smeared onto a gelatine-coated glass slide and allowed to dry at room temperature. 6 µl of 50% (v/v) glycerol was then added to the dried smear and a 25 × 25 mm glass cover slip was used to spread it over the dried smear.

### Microscopy and image processing

The fixed cells were examined with an Axio Imager.M1 (Zeiss, Oberkochen, Germany) using 556/20 nm excitation bandpass. Digital images were captured at 100 magnification with an AxioCam MRm camera (Zeiss) in phase contrast and through a 556/20 nm (red) filter set. Approximately 10 NsARC and 10 control samples each were used for this experiment. And about 50 images were captured from each individual sample. A total of 500 images from each treatment was taken for each treatment replicate. Images were then analysed using Fiji ImageJ (Schindelin et al. 2012). 100 separate images for each sample were analysed. Single-cell fluorescence intensity of the individual cells was obtained by acquiring multichannel images of the fluorescence signals and phase contrast signals. Multichannel images were imported into the Fiji ImageJ program. Thresholding command and standard settings were used to separate cells from the background based on the phase contrast. The analyse particles command was then used to add cells to the region of interest manager and the average fluorescence of the individual cells was determined using the “multi-measure” command (Remus-Emsermann et al. 2016). The relative fluorescence is also just an arbitrary unit (au) of measurement.

### Statistical analysis

R was used for statistical analysis (Rosario-Martinez et al. 2015). We used analysis of variance (ANOVA) to analyse the data from the experiment we carried out to determine if exposure of bacteria to NsARC caused a change in susceptibility to antibiotics. A multifactor ANOVA was performed on EOP values to test for effect of the materials (NsARC and control) and antibiotic concentration under two exposure conditions (light and dark). Residual plots were examined to determine if EOP values were normally distributed, which is an assumption for ANOVA (Crawley 2007). The plots were not normally distributed. So, the EOP scores were log transformed to meet the assumption. In each case, we tested for significant difference between materials. The null hypothesis was that there was no difference between the EOP values from the materials at various antibiotic concentrations. We also tested for interaction between materials, antibiotic concentrations and exposure conditions. A Bonferroni’s post hoc test was used to compare the EOP to determine if there is a significant difference between NsARC and the controls. The value for statistical significance was set at P < 0.05. The results of each post hoc are available in supplementary material (Additional file 1: Table S1–S12), however, we were most interested in the differences in EOP between individual treatment combinations as follows: NsARC vs. steel under light and NsARC vs. steel in the dark. Contrast matrices listing the contrast of interest mentioned were drawn up and the test Interactions function in the phia package in R was used to evaluate the contrasts as described in the result section (Rosario-Martinez et al. 2015).

For the experiment to determine if NsARC can induce changes in the expression pattern of genes that can alter susceptibility to antibiotics, a statistical model based on ANOVA was also used. A Tukey’s post hoc test was used to compare the means of the relative fluorescence to determine if there is a difference between NsARC and the controls. The value for statistical significance was set at P < 0.05. The results of each post hoc are available in supplementary material (Additional file 1: Table S13–S20). However, we were most interested in the differences between individual treatment combinations, these include NsARC vs. Positive control (copper), Negative control (stainless steel) vs. Positive control (copper) and NsARC vs. Negative control (stainless steel). These were calculated using contrasts and the results can also be found in the supplementary materials (Additional file 1: Table S13–S20). Violin plots were then made using ggplot2 (Wickham 2016).