Supplementary information for

Non-antibiotic triclosan induces multiple antibiotic resistance through genetic mutation

Ji Lua, Min Jina, Son Hoang Nguyenb, Likai Maoa, Jie Lia, Lachlan J. M. Coinb, Zhiguo Yuana,

Jianhua Guoa, *

a Advanced Water Management Centre (AWMC), The University of Queensland, St Lucia,

Brisbane, QLD, 4072, Australia

b Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, QLD,

4072 Australia

* Corresponding author.E-mail address: j.guo@awmc.uq.edu.au (J. Guo).

This file includes:

Supplementary Figure 1

Supplementary Tables 1 to 9

Supplementary References

1

Supplementary Figure

Fig. S1 (A) Total gene regulation after 0.2 mg/L TCS dosage for 8 h in wild-type E. coli K-

12, and 0.2T-AMX, 0.2T-CHL and 0.2T-TET mutants. The blue columns represent up-

regulated genes while orange represents down-regulated genes. (B) Venn diagram of

differentially gene expression (|log2| ≥ 2) level of control (orange), 0.2T-AMX (green), 0.2T-

CHL (blue) and 0.2T-TET (red) strains when exposed to 0.2 mg/L TCS for 8h.

2

Supplementary Tables

Table S1 MIC90 of various antibiotics for E. coli K-12 and antibiotic concentrations in LB agar to determine antibiotic resistance, together with

the highest MIC90 detected in TCS-induced mutants and the comparison to the EUCAST breakpoint concentration for clinical resistance in

Enterobacteriaceae.

Antibiotic Abbreviation Category MIC90 in untreated strains (mg/L)

Screening concentration (mg/L)

Highest MIC90

in mutants (mg/L)

EUCAST breakpoint (mg/L)

amoxicillin AMX beta-lactam 8.0 32.0 64.0 8ampicillin AMP beta-lactam 22.5 90.0 180.0 8cephalexin LEX beta-lactam 16.0 64.0 128.0 -chloramphenicol CHL chloramphenicol 16.0 32.0 96.0 8kanamycin KAN aminoglycoside 16.0 31.3 40.0 -levofloxacin LVX fluoroquinolone 0.1-0.2 0.3 1.0 1norfloxacin NOR fluoroquinolone 0.5 1.0 3.0 1tetracycline TET tetracycline 20 40.0 80.0 -

Table S2 Colony enumeration data antibiotic resistance (AMX, LEX, CHL) after 30 days exposure to TCS

TCS (mg/L)

total bacterial

count (×108

cfu/plate)

AMX mutant count

mutation

frequency

(×10-9)

LEX mutant count

mutation

frequency

(×10-9)

CHL mutant count

mutation

frequency

(×10-9)

0 8.6±0.0 1.0±0.6 1.2±0.7 0.3±0.3 0.4±0.4 0.3±0.3 0.4±0.4

0.02 8.4±0.1 2.0±1.0 2.4±1.2 0.3±0.3 0.4±0.4 0.3±0.3 0.4±0.4

3

0.2 8.4±0.3 97.7±12.7 116.1±14.8** 1.0±0.6 1.2±0.7 15.7±2.6 18.6±3.1**

2 5.1±0.1 0.7±0.7 1.3±1.3 0.3±0.3 0.7±0.7 0.3±0.3 0.7±0.7

** means highly significant different after Independent-samples t-test (p < 0.01).

Table S2 (continued) Colony enumeration data for antibiotic resistance (LVX, NOR, TET) after 30 days exposure to TCS

TCS (mg/L)

total bacterial

count (×108

cfu/plate)

LVX mutant count

mutation

frequency

(×10-9)

NOR mutant count

mutation

frequency

(×10-9)

TET mutant count

mutation

frequency

(×10-9)

0 85.6±0.4 1.3±0.9 1.6±1.0 0.3±0.3 0.4±0.4 1.3±0.9 1.6±1.0

0.02 84.2±0.7 2.7±1.5 3.2±1.7 0.7±0.7 0.8±0.8 1.3±0.3 1.6±0.4

0.2 84.1±3.3 2.7±1.2 3.2±1.4 0.7±0.7 0.8±0.8 32.3±5.7 38.4±6.8**

2 50.7±0.8 0.7±0.3 1.3±0.7 0.3±0.3 0.7±0.7 0.3±0.3 0.7±0.7

4

** means highly significant different after Independent-samples t-test (p < 0.01)

5

Table S3 Illumina DNA sequencing identified genetic mutations in six antibiotic-resistant

strains selected by TCS at sub-MIC (0.2 mg/L) for 30 days.

Gene Mutation Annotation 0.2T-AMX

0.2T-CHL

0.2T-TET

aaeB T→G V311G (GTC→GGC)

acrR A→C L65R (CTG→CGG)

citC C→T A346T (GCT→ACT)

fabI C→G A197G (GCC→GGC)

fabI C→A F203L (TTC→TTA)

fabI A→T T194S (ACT→TCT)

frdD +T coding (345/360 nt)

insB-1 +A coding (267/270 nt)

insL-1 Δ1 bp coding (1031/1050 nt)

marR T→G T72P (ACC→CCC)

rhsC Δ1 bp coding (3334/3375 nt)

rhsC G→A T1036T (ACG→ACA)

rpoD T→C D445G (GAT→GGT)

soxR G→T R20S (CGC→AGC)

Table S4 Functions and possible impacts of gene mutations.

Gene Function Possible impact of mutation

aaeB p-hydroxybenzoic acid efflux pump subunit AaeB, putative membrane subunit

Mutation in aaeB results in hypersensitivity to hydroxylated, aromatic carboxylic acid (Van Dyk et al., 2004).

acrR A negative DNA-binding transcriptional regulator for the multidrug efflux pump AcrAB.

Mutations of acrR gene were reported to contribute to the increased expression of acrA and acrB genes (Wang et al., 2001).

citC Citrate lyase synthetase, activates citrate lyase by acetylation.

Unknown

fabI Enoyl-[acyl-carrier-protein] reductase [NADH] FabI

Missense mutation in the fabI gene could prevent TCS inhibition by altering the structure of the FabI protein, which reduce TCS targeting efficacy (Sivaraman et al., 2003).

frdD Fumarate reductase subunit D Since the promoter of ampC ß-lactamase gene 6

in E. coli K-12 overlap in the frdD gene region (Grundstrom and Jaurin, 1982), and some mutations in ampC promoter were shown to enhance the promoter strength, the mutation in frdD region may have caused significant up regulation of ampC gene expression (Corvec et al., 2002; Tracz et al., 2005).

insB-1 The mutated InsA-B'-InsB were suggested to result in an overall reduction of transposition of IS1 and its defective copy in a cell, allowing stable existence of the element in its bacterial host (Matsutani 1997).

Unknown

Insl-1 insL-1 encodes for polypeptide IS186/IS421 transposase. IS186 is an insertion sequence element that appears three times in a typical E. coli genome.

Unknown

marR MarR is the repressor of the marRAB operon which is involved in the activation of both antibiotic resistance and oxidative stress genes

Mutations in marR gene are involved in multidrug resistance, including ciprofloxacin, doxycycline and chloramphenicol (Sulavik et al., 1995).

rhsC Encodes hydrophilic proteins with repetitive sequence elements and divergent C-termini

Unknown

rpoD RNA polymerase sigma 70 (sigma D) factor RpoD.

The mutation in rpoD gene can cause resistance to TCS by changing gene expression levels (Gantzhorn et al., 2015).

soxR SoxR protein can be activated by univalent oxidation in its iron–sulphur cluster, in turn, activated SoxR targets soxS gene and increases the transcription of soxS.

Mutation in soxR gene is able to increase the expression of soxS, which can induce the expression of more than 100 genes (Koutsolioutsou et al., 2005).

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Table S5 Transcriptional changes in genes related to stress-induced mutagenesis in E. coli after 8 h of 0.2 mg/L TCS treatment

Class Gene COG annotationLog2 fold change of FPKM*

0.2T-AMX

0.2T-CHL

0.2T-TET control

Regulator arcA DNA-binding response regulator, OmpR family, contains REC and winged-helix (wHTH) domain - - - -1.0

argR Arginine repressor - - - -1.0

glnK Nitrogen regulatory protein PII 1.0# - -2.4# 1.4#

hdfR DNA-binding transcriptional regulator, LysR family - - 1.2 -

infA Translation initiation factor IF-1 - - - -1.2

lexA SOS-response transcriptional repressor LexA (RecA-mediated autopeptidase) 1.2 1.0 1.5 -

nusA Transcription antitermination factor NusA, contains S1 and KH domains - - 1.2# -

phoU Phosphate uptake regulator 1.2 1.0 1.9 -

rpoS DNA-directed RNA polymerase, sigma subunit (sigma70/sigma32) -1.4 - -1.5# -1.8

DNA repair recA DNA recombination and repair protein; RecA/RadA recombinase 1.1 - 1.2# -

radA DNA repair protein; Predicted ATP-dependent serine protease - - 1.2# -

recN DNA repair ATPase RecN 1.2 1.4 2.0 -

umu Nucleotidyltransferase/DNA polymerase involved in DNA - - -1.0# 1.1#

8

C repair

yfjY DNA repair protein RadC, contains a helix-hairpin-helix DNA-binding motif -2.1# -1.3# -1.0# 1.2#

aidB DNA alkylation damage repair protein; Acyl-CoA dehydrogenase related to the alkylation response protein AidB - 1.8 - -

Electron transfer

cyoA Heme/copper-type cytochrome/quinol oxidase, subunit 2 -1.7 -1.3 - -2.1

cyoD Heme/copper-type cytochrome/quinol oxidase, subunit 4 -1.0# - - -1.5#

hscB DnaJ-domain-containing proteins 1 - - - -1.0

mdh Malate/lactate dehydrogenase - - - -1.2

nuoK NADH:ubiquinone oxidoreductase subunit 11 or 4L (chain K) - - - -1.1#

nuoL NADH:ubiquinone oxidoreductase subunit 5 (chain L)/Multisubunit Na+/H+ antiporter, MnhA subunit - - - -1.0

pgi Glucose-6-phosphate isomerase - - 2.9 -

sdhB Succinate dehydrogenase/fumarate reductase, Fe-S protein subunit - - - -1.4

sdhD Succinate dehydrogenase/fumarate reductase, cytochrome b subunit -1.4# -1.1# - -1.7#

ubiA 4-hydroxybenzoate polyprenyltransferase - - - -1.0

ubiD 3-polyprenyl-4-hydroxybenzoate decarboxylase - - - -1.0

Metabolism argD Acetylornithine/succinyldiaminopimelate/putrescine

aminotransferase -1.1 -1.2 -1.4 -

cysD 3'-phosphoadenosine 5'-phosphosulfate sulfotransferase (PAPS reductase)/FAD synthetase or related enzyme

2.5 - 4.2 -

9

pgl 6-phosphogluconolactonase, cycloisomerase 2 family 1.5 - 2.1 -

speB Arginase family enzyme 1.0 - 1.4 -

Cellular processes

acrA Multidrug efflux pump subunit AcrA - 2.3 2.7 -

acrB Multidrug efflux pump subunit AcrB - 2.4 2.7 -

pstC ABC-type phosphate transport system, permease component - 1.0 1.6# -

srlA Phosphotransferase system sorbitol-specific component IIC 4.7 2.4 3.1# 2.9

znuA ABC-type Zn2+ transport system, periplasmic component/surface adhesin -1.2 - -1.2 -2.0

znuC ABC-type Mn2+/Zn2+ transport system, ATPase component -1.5 - -1.6 -1.6

Unknown functions

yicC Uncharacterized conserved protein YicC, UPF0701 family 1.5 - - -

yjcC Environmental sensor c-di-GMP phosphodiesterase, contains periplasmic CSS-motif sensor and cytoplasmic EAL domain - - -1.1# 2.0

yifE Uncharacterized conserved protein YifE, UPF0438 family - - - -1.0

yqge Putative transcriptional regulator, AlgH/UPF0301 family - - 1.2# -

yqiC Uncharacterized conserved protein YqiC, BMFP domain 1.0 - - -

*: Comparing with those in the untreated E. coli K-12.#: |log2| (Fold Change of FPKM) ≥1 with false discovery rate (FDR) > 0.001.

-: |log2| (Fold Change of FPKM) <1.

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Table S6 Transcriptional changes in genes related to efflux systems in E. coli after 8 h of 0.2 mg/L TCS treatment.

Efflux system

COG annotation

GeneLog2 fold change of FPKM*

0.2T-AMX

0.2T-CHL

0.2T-TET control

AcrAB-TolC

Multidrug efflux pump subunit AcrA acrA - 2.3 2.7 -

Multidrug efflux pump subunit AcrA acrE - - - 2.4

Multidrug efflux pump subunit AcrA mdtA - - - -

Multidrug efflux pump subunit AcrA mdtE - 1.2 - 1.5

Multidrug efflux pump subunit AcrA yhiI - - - -

Multidrug efflux pump subunit AcrB acrB - 2.4 2.7 -

Multidrug efflux pump subunit AcrB acrD - - - -

Multidrug efflux pump subunit AcrB acrF - - - 1.1

Multidrug efflux pump subunit AcrB mdtB - - - 1.1

Multidrug efflux pump subunit AcrB mdtC - - - 1.2

acrAB operon transcriptional repressor acrS -1.5# -2.8# -1.3# -

DNA-binding transcriptional regulator, AcrR family acrR - - - -

DNA-binding transcriptional regulator, MarR family marR - - 5 -

11

DNA-binding transcriptional regulator, MarR family mprA - - - -1

DNA-binding transcriptional regulator, MarR family slyA - - - -1

Outer membrane protein TolC tolC - - 2.8 -

Part of marRAB operon marB - - 4.2 -1.2#

Transcriptional regulator GlxA family marA - - 5.3 -

DNA-binding response regulator, NarL/FixJ family, contains REC and HTH domains sdiA 1.7 1.0# 2.2 -

EmrE Multidrug transporter EmrE and related cation transporters emrE -1.3 - -1.4 -1.5

Multidrug transporter EmrE and related cation transporters mdtI - - -1.0# -

Multidrug transporter EmrE and related cation transporters mdtJ - - -1.2# -

Multidrug transporter EmrE and related cation transporters sugE - - - -1.8#

EmrAB-TolC

EmrAB multidrug efflux system emrA - - - -

EmrAB multidrug efflux system emrB - - - -

EmrKY-TolC

Multidrug resistance efflux pump, multidrug resistance protein K emrK - -1.7# -3.7# 2.1#

MFS family permease, multidrug resistance protein Y emrY -1 -1.5# -1.9# 2.1#

NAD(P)-dependent dehydrogenase, short-chain alcohol dehydrogenase family yciK - - 1.7 -

Others ABC-type multidrug transport system, ATPase and mdlA - - 1.3# -

12

permease component

ABC-type multidrug transport system, ATPase and permease component mdlB - - 1.6# -

ABC-type multidrug transport system, ATPase component rbbA - - - -

ABC-type multidrug transport system, ATPase component yadG - 1.2 5.4 -

ABC-type multidrug transport system, permease component yadH - - 4.6 -

ABC-type uncharacterized transport system, permease and ATPase components yddA -1.0# -1.5# -1.6# 2.4

MFS family permease hsrA - - - -

MFS family permease mdtH -1.1 - -2.2 -

MFS family;Predicted arabinose efflux permease emrD -1.1 - -1 -

MFS family;Predicted arabinose efflux permease mdfA - - - -

MFS family;Predicted arabinose efflux permease mdtM -1.9 - - -1.5

Na+-driven multidrug efflux pump mdtK 1.3 - - -

Uncharacterized membrane protein DedA, SNARE-associated domain yghB - - - -1.3

*: Comparing with those in the untreated E. coli K-12.#: |log2| (Fold Change of FPKM) ≥1 with false discovery rate (FDR) > 0.001.

-: |log2| (Fold Change of FPKM) <1.

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Table S7 Transcriptional changes in genes related to antibiotics resistance enzymes, bacterial membrane permeability and membrane potential in E. coli after 8 h of 0.2 mg/L TCS treatment

Gene COG annotationLog2 fold change of FPKM*

0.2T-AMX 0.2T-CHL 0.2T-TET control

ampC CubicO group peptidase, beta-lactamase class C family 5.4 - 1.3 -1.7#

ampE Membrane protein required for beta-lactamase induction - - 1.4

gloB Glyoxylase or a related metal-dependent hydrolase, beta-lactamase superfamily II - - 3.2 -

yfeW CubicO group peptidase, beta-lactamase class C family 1.8 - 1.3 2.0#

mrdA Cell division protein FtsI/penicillin-binding protein 2 - - 1.0# -

yjcSAlkyl sulfatase BDS1 and related hydrolases,

metallo-beta-lactamase superfamily- -1.3# -1.2# 2.1

nfsA Nitroreductase - 1.7 4.6 -1.0

nfsB Nitroreductase - 1.7 6.7 -

ompA Opacity protein and related surface antigens 1.7 - 1.3# -

ompC Outer membrane protein (porin) 1.0# 1.5 1.6# -

ompF Outer membrane protein (porin) 4.1 - 1.0# 3.6

ompG - -1.0# - 2.0#

ompL - -1.0# 2.7 2.7#

ompN Outer membrane protein (porin) - -1.0# -1.0# 2.3#

ompR Response regulator in two-component regulatory system with EnvZ;affecting transcription of ompC and ompF - - 1.6 -

14

ompT Outer membrane protease - - - 2.5

ompW Outer membrane protein W - - - -

ompX Opacity protein and related surface antigens - - 1.9# -2.1

phoE Outer membrane protein (porin) -1.3 -1.8 -1.2 1.0

gadA Glutamate or tyrosine decarboxylase or a related PLP-dependent protein -2.3 3.6 1.8# -

gadB Glutamate or tyrosine decarboxylase or a related PLP-dependent protein -2.4 3.9 2.0 -

gadW Glutamate or tyrosine decarboxylase or a related PLP-dependent protein 2.1 1.0 - 1.2

*: Comparing with those in the untreated E. coli K-12.

#: |log2| (Fold Change of FPKM) ≥1 with false discovery rate (FDR) > 0.001.

-: |log2| (Fold Change of FPKM) <1.

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Table S8 Transcriptional changes in genes related to cellular antioxidants in E. coli after 8 h of 0.2 mg/L TCS treatment

Gene COG annotationLog2 fold change of FPKM*

0.2T-AMX 0.2T-CHL 0.2T-TET control

sodA Superoxide dismutase 1.6 2.5 4.4 -sodC Cu/Zn superoxide dismutase - - 1.0# -ahp

C Alkyl hydroperoxide reductase subunit AhpC (peroxiredoxin) - - 1.0# -

ahpF Alkyl hydroperoxide reductase subunit AhpF - - 1.0# -

ygiW Hydrogen peroxide and cadmium resistance periplasmic protein;predicted periplasmic protein YdeI with OB-fold, BOF family -1.7 - -1.5 -1.6

soxS AraC-type DNA-binding domain and AraC-containing proteins;superoxide response regulon transcriptional activator - 3.1 1.4# -1.4#

yhcN cadmium and peroxide resistance protein; stress-induced - 1.7 2.7 -2.0

gorGlutathione oxidoreductase;pyruvate/2-oxoglutarate dehydrogenase

complex, dihydrolipoamide dehydrogenase (E3) component or related enzyme

1.2 - 1.0# 1.4

trxB Thioredoxin reductase - - 1.0# -

yggX oxidative damage protective factor for iron-sulfur proteins; Fe-S cluster biosynthesis and repair protein YggX 1.0# 1.1 2.6 -

oxyR oxidative and nitrosative stress transcriptional regulator; DNA-binding transcriptional regulator, LysR family - - 1.0# -

yajL oxidative-stress-resistance chaperone; Putative intracellular protease/amidase - - 1.8# -

*: Comparing with those in the untreated E. coli K-12.

16

#: |log2| (Fold Change of FPKM) ≥1 with false discovery rate (FDR) > 0.001.-: |log2| (Fold Change of FPKM) <1.

Table S9 Transcriptional changes in mutated-genes and downstream genes related to antibiotic resistance in E. coli after 8 h of 0.2 mg/L TCS treatment

Gene COG annotationLog2 fold change of FPKM*

0.2T-AMX

0.2T-CHL

0.2T-TET control

aaeB p-hydroxybenzoic acid efflux system component - - - 1.2

acrR DNA-binding transcriptional regulator, AcrR family - - - -

acrS acrAB operon transcriptional repressor -1.5# -2.8# -1.3# -

acrA Multidrug efflux pump subunit AcrA - 2.3 2.7 -

acrB Multidrug efflux pump subunit AcrB - 2.4 2.7 -

citC Citrate lyase synthetas) - - - -

fabI Enoyl-[acyl-carrier-protein] reductase (NADH) - - - -

frdD Fumarate reductase subunit D 1.5 1.1 1.8 -

ampC CubicO group peptidase, beta-lactamase class C family 5.4 - 1.3 -1.7#

insB-1 Transposase and inactivated derivatives, IS1 family -1.8 -1.3 1.4 1.0

Insl IS4 transposase - - - -

marR DNA-binding transcriptional regulator, MarR family - - 5.0 -1.0#

marA Transcriptional regulator GlxA family

marB Part of marRAB operon - - 4.2 -1.2#

fis DNA-binding protein Fis (factor for inversion stimulation) - 1.0 1.2# -

sdiA DNA-binding response regulator, NarL/FixJ family, contains REC and 1.7 1.0# 2.2 -17

HTH domains

tolC Outer membrane protein TolC - - 2.8 -

rhsC Uncharacterized conserved protein RhaS, contains 28 RHS repeats - - - -

rpoD DNA-directed RNA polymerase, sigma subunit (sigma70/sigma32) - - - -

soxR Thioredoxin reductase - 2.4 - -

soxS AraC-type DNA-binding domain and AraC-containing proteins, superoxide response regulon transcriptional activator - 3.1 1.4# -1.4#

tsx Nucleoside-specific outer membrane channel protein Tsx 1.7 1.0 2.0 -

udp Uridine phosphorylase 1.0 1.1 2.4 -

ygaQ - -1.6# -3.0# 2.5

rpmG Ribosomal protein L33, resistance to mitomycin C 1.1 - 1.0# -

*: Comparing with those in the untreated E. coli K-12.#: |log2| (Fold Change of FPKM) ≥1 with false discovery rate (FDR) > 0.001.

-: |log2| (Fold Change of FPKM) <1.

Orange marks represent the mutated genes detected by Illumina DNA sequencing.

18

Supplementary References

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Koutsolioutsou A., Pena-Llopis S., Demple B., Constitutive soxR mutations contribute to multiple-antibiotic resistance in clinical Escherichia coli isolates. Antimicrob. Agents Chemother. 49, 2005, 2746-2752, https://doi.org/ 10.1128/aac.49.7.2746-2752.2005 .

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Tracz D.M., Boyd D.A., Bryden L., Hizon R., Giercke S., Van Caeseele P., et al., Increase in ampC promoter strength due to mutations and deletion of the attenuator in a clinical isolate of cefoxitin-resistant Escherichia coli as determined by RT–PCR. J. Antimicrob. Chemother. 55, 2005, 768-772, https://doi.org/ 10.1093/jac/dki074 .

Van Dyk T.K., Templeton L.J., Cantera K.A., Sharpe P.L., Sariaslani F.S., Characterization of the Escherichia coli aaeAB efflux pump: A metabolic relief valve? J. Bacteriol. 186, 2004, 7196-7204, https://doi.org/ 10.1128/jb.186.21.7196-7204.2004 .

Wang H., Dzink-Fox J.L., Chen M., Levy S.B., Genetic characterization of highly fluoroquinolone-resistant clinical Escherichia coli strains from China: Role of acrR mutations. Antimicrob. Agents Chemother. 45, 2001, 1515-1521, https://doi.org/ 10.1128/aac.45.5.1515-1521.2001 .

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