Electronic Supplementary Material (ESI) for Environmental Science: Water Research & Technology. This journal is © The Royal Society of Chemistry 2020 Electronic Supplementary Information Sonophotocatalysis mediated morphological transition modulates virulence and antibiotic resistance in Salmonella Typhimurium A. P. Habeeb Rahman a, b, Ananyo Jyoti Misra a, b, Swagatika Panda c, Bhaskar Das d, Prachi Bhol a, Priti Sundar Mohanty a, b, Ashok J. Tamhankar b, e, Amrita Mishra b, Cecilia Stålsby Lundborg e, Suraj K. Tripathy a, b * a School of Chemical Technology, Kalinga Institute of Industrial Technology, Bhubaneswar, India b School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, India c ICMR-Regional Medical Research Center, Bhubaneswar, India dDepartment of Biotechnology and Medical Engineering, National Institute of Technology, Raurkela, India eDepartment of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden * Corresponding author: S. K. Tripathy (E-mail: [email protected], Tel: +91-674-2725466) Synthesis of Fe-ZnO NPs: Supplementary Scheme. 1 Flowchart of Fe-ZnO NPs synthesis 8 7 6 5 L m / 4 U F C 0 1 3 g o l STm 2 STm SPI SB300 1 STm GFP SEH 0 0 20 40 60 80 100 120 Time (min) Fig. S1. Efficacy of SPC disinfection against different mutants of S. enterica. STm- antibiotic resistant mutant, STm SPI- Salmonella pathogenicity island induced STm, SB300- virulent strain of wild type S. Typhimurium, STm GFP- Green fluorescence protein tagged mutant, SEH- Hydrogen peroxide resistant mutant of S. Enteritidis. (Bacterial loading ≈ 107 CFU/mL, Fe-ZnO NPs loading = 200 mg/L, Temperature=35±2°C). Error bar indicates the standard deviation of replicates (n=3). Fig. S2. Time dependant analysis of resazurin metabolic assay of STm sampled at different time points of SPC disinfection. Each reaction was performed in triplicate. (Bacterial loading ≈ 107 CFU/mL, Fe-ZnO NPs loading = 200 mg/L, Temperature = 35±2°C). 2.5 1.7 Avg Diameter (14d) Avg Diameter (7d) ) 1.6 2.0 m c ( r e t e m a i d g v 1.5 A ) m c 1.5 ( r 1.4 e t 7 8 9 10 11 12 13 14 e Incubation (days) m a i 1.0 d g v A 0.5 0.0 0 40 60 80 Treatment time (min) Fig. S3. Average colony diameter of STm sampled at different time intervals of SPC disinfection. Diameter was measured after 7 and 14 days of incubation at 37°C. Inset represents the change in diameter of untreated STm. (Bacterial loading ≈ 107 CFU/mL, Fe-ZnO NPs loading = 200 mg/L, Temperature = 35±2°C). Error bar indicates the standard deviation of replicates (n=3). Fig. S4. (a) and (b) shows fluorescent images of GFP tagged untreated and treated (recovered after sublethal SPC - +45 min) STm captured during bacterial motion in the prepared slides, depicting the (a)absence and (b) presence of spheroplast like cells respectively. 0.0300 0.0008 0.0007 0.0006 0.0225 0.0005 n 0.0004 n o o i i s a s v n i a % 0.0003 v 0.0150 n i 0.0002 % 0.0001 0.0075 0.0000 UT T Invasion time = 50 min 0.0000 UT T UT/Cell T/Cell Invasion time = 50 min Fig. S5. Invasion assay of untreated (UT) and treated (T) STm after sublethal SPC (sampled after +30 min of SPC) in HCT116 colon epithelial cells at MOI=10. Inset represents the difference in total invasion by UT and T STm. Error bar indicates the standard deviation of replicates (n=3). 0.015 0.00040 0.012 0.00032 0.009 0.00024 n n o i s o a i v n i s % a 0.00016 v n i 0.006 % 0.00008 0.003 0.00000 UT T Invasion time = 50 min 0.000 UT T UT/Cell T/Cell Invasion time = 50 min Fig. S6. Invasion assay of untreated (UT) and SPC treated SPI induced (T) STm (sampled at +45 min of SPC) in HCT116 colon epithelial cells at MOI=10. Data represents the survived bacterial cells during invasion. Inset represents the difference in total invasion by SPI induced UT and T STm. Total invasion time = 50 min. Error bar indicates the standard deviation of replicates (n=3). 140 Membrane Protein 120 DNA Average 100 ) m 80 m ( I O 60 Z 40 20 0 t .1 .1 .1 .1 .1 n 5 1 1 1 1 U C C C C L P P S P P S S S Treatment Fig. S7. ZOI of antibiotics clustered according to their target (membrane, protein and DNA) after 45min treatment of SPC 5.1 (Sonophotocatalysis 5th cycle), and 1st cycle of SPC (Sonophotocatalysis), SC (Sonocatalysis), PC (Photocatalysis), SPL (Sonophotolysis) compared with untreated STm. ZOI is measured excluding the diameter of antibiotic disc = 6mm. Fig. S8. Antibiotic resistance/susceptibility profiling of STm recovered after SPC 5.2 (Sonophotocatalysis 5th cycle), and 1st cycle of SPC (Sonophotocatalysis), PC (Photocatalysis), SC (Sonocatalysis) compared with untreated STm. 80 Membrane Protein DNA 70 Average 60 50 ) m c ( 40 I O Z 30 20 10 0 .2 .2 .2 .2 nt 5 1 1 1 U C C C C P P S P S S Treatment Fig. S9. ZOI of antibiotics clustered according to their target (Membrane, Protein and DNA) of STm recovered after SPC 5.2 (Sonophotocatalysis 5th cycle), and 1st cycle of SPC (Sonophotocatalysis), PC (Photocatalysis), SC (Sonocatalysis) compared with untreated STm. ZOI is measured excluding the diameter of antibiotic disc = 6mm. Table. S1. Change in fatty acid composition of sublethal SPC treated and untreated STm Sl No Fatty acid Untreated Sublethal SPC treated STm 1 12:0 3.85 3.85 2 11:0 3OH 0.43 0.37 3 13:0 0.32 0.29 4 14:0 iso 0.13 0.25 5 14:0 5.03 5.07 6 15:1 iso G 0.41 0.45 7 15:0 anteiso 3.27 2.86 8 15:1 w5c 0.62 0.30 9 14:0 2OH 0.77 0.67 10 16:0 N alcohol 0.48 0.32 11 16:0 iso 0.29 0.30 12 16:0 anteiso 0.35 0.33 13 16:1 w5c 0.14 0.19 14 16:0 24.34 24.13 15 15:0 2OH 0.66 0.57 16 17:1 anteiso A 0.61 0.77 17 17:0 anteiso 2.25 1.93 18 17:0 cyclo 6.38 6.29 19 17:0 0.97 1.01 20 18:3 w6c (6,9,12) 0.66 0.61 21 18:0 1.64 1.36 22 19:0 cyclo w8c 4.32 5.02 23 19:0 0.46 0.43 24 20:2 w6,9c 0.92 0.47 25 20:1 w7c 0.26 0 . Table. S2. List of antibiotics used in the current study and its major target sites. Sl No Antibiotic Class or Subgroup Target site 1 Imipenem B-lactam/ Carbapenem Cell wall 2 Ceftriaxone Lactams/cephalosporin Cell wall 3 Cefuroxime Lactams/cephalosporin Cell wall 4 Doripenem Lactams/Thienamycins Cell wall 5 Meropenem Lactams/Thienamycins Cell wall 6 Ampicillin Lactams/Penicillins Cell wall 7 Ertapenem Lactams/Thienamycins Cell wall 8 Aztreonam Lactams/Monobactams Cell wall 9 Cephotaxime Lactams/cephalosporin Cell wall 10 Polymyxin B Polymyxin/polypeptide Cell wall 11 Penicillin G Penicillins/B-lactam Cell wall 12 Colistin Cyclic peptides Cell wall 13 Vancomycin Glyco/cyclic peptides Cell wall 14 Methicilin Carboxylic acids/ dipeptides Cell wall 15 Cefepime Lactam/4th gen cephalosporins Cell wall 16 Cefixime/Clauvulanic Acid Cephalosporins/alpha amino acids Cell wall 17 Ceftazidime Lactams/cephalosporin Cell wall 18 Chloramphenicol Amphenicol/ Nitrobenzene Protein 19 Nalidixic acid Diazanaphthalenes Protein 20 Erythromycin Aminoglycosides Protein 21 Gentamicin Aminoglycosides Protein 22 Amikacin Aminoglycosides Protein 23 Tetracyclin Tetracyclins Protein 24 Linezolid Oxazinanes/ Phenylmorpholines Protein 25 Streptomycin Aminocyclitol glycosides Protein 26 Kanamycin Aminocyclitol glycosides Protein 27 Clindamycin Lincosamide Protein 28 Tobramycin Aminocyclitol glycosides Protein 29 Azithromycin Macrolides/Aminoglycosides Protein 30 Clarithromycin Macrolides/Aminoglycosides Protein 31 Ciprofloxacin Quinolines DNA 32 Co-trimoxazole Sulfonamides DNA 33 Norfloxacin Quinolines DNA 34 Rifampicin Macrolactams DNA 35 Sparfloxacin Quinoline carboxylic acids DNA 36 Moxifloxacin Quinoline carboxylic acids DNA 37 Gatifloxacin Quinoline carboxylic acids DNA 38 Gemifloxacin Quinoline carboxylic acids DNA 39 Levofloxacin Quinoline carboxylic acids DNA.