Supplementary Materials for...Cirpo 2x o C Fig. S1. Determination of growth rates and of the...

stke.sciencemag.org/cgi/content/full/12/592/eaax3938/DC1

Supplementary Materials for

Slow growth determines nonheritable antibiotic resistance in Salmonella enterica

Mauricio H. Pontes and Eduardo A. Groisman*

*Corresponding author. Email: [email protected]

Published 30 July 2019, Sci. Signal.12, eaax3938 (2019)

DOI: 10.1126/scisignal.aax3938

This PDF file includes:

Fig. S1. Determination of growth rates and of the emergence of antibiotic-resistant mutants in bacterial cultures. Fig. S2. Effect of Shx treatment on the frequency of persisters. Fig. S3. Effects of ATP depletion and inhibition of protein synthesis on antibiotic tolerance. Fig. S4. Effect of growth inhibition on antibiotic tolerance in Salmonella. Table S1. Microbial strains and plasmids used in this study. Table S2. Oligonucleotides sequences used in this study. References (82, 83)

Fig. S1. Determination of growth rates and of the emergence of antibiotic-resistant

mutants in bacterial cultures. (A) Growth dynamics of wild-type (14028s), ∆12TA

(MP1422), and relA::Tn10 spoT (MP342) Salmonella in LB and MOPS medium containing

2 mM K2HPO4 and 10 µM MgCl2. Error bars represent standard deviations. N = 12

independent biological replicates. Growth curves are representative of typical

experiments. Note log scale of y axis. (B) Number of surviving wild-type (14028s),

∆12TA (MP1422) and relA::Tn10 spoT (MP342) Salmonella following two rounds of 24 h

LBCef

o

Cipro

LB 2x

Cef

o 2

x

Cirpo 2

x

0

2

4

6

8

10

2nd Exposure relAspoT Final

LB

Cefo

Cipro

LB 2x

Cefo 2x

Cirpo 2x

LB

C

efo

Cip

ro

LB

C

efo

Cip

ro

2nd exposure

LBCef

o

CiproLB

2x

Cef

o 2

x

Cirpo 2

x

0

2

4

6

8

10

LB

Cefo

Cipro

LB 2x

Cefo 2x

Cirpo 2x

LB

C

efo

Cip

ro

LB

C

efo

Cip

ro

2nd exposure

wild-type

LBCef

o

Cipro

LB 2x

Cef

o 2

x

Cirpo 2

x

0

2

4

6

8

10

2nd Exposure TA Final

LB

Cefo

Cipro

LB 2x

Cefo 2x

Cirpo 2x

LB

C

efo

Cip

ro

LB

C

efo

Cip

ro

2nd exposure TA

relA spoT L

og

CF

Us

/ml

Lo

g C

FU

s/m

l

Lo

g C

FU

s/m

l

𝛥�12TA

0 3 6 9 12 15 18 21 24

0.1

1

Time (h)

Bacte

rial g

row

th (

OD

600)

LB

wild-type

Δ12TA

relA spoT

1.0

0 3 6 9 12 15 18 21 24

0.1

1

Time (h)

Bacte

rial g

row

th (

OD

600)

Growth MOPS

wild-type

Δ12TA

relA spoT

1.0 A

B

exposure to cefotaxime or ciprofloxacin in LB medium. Error bars represent standard

deviations. N = 6 independent biological replicates. Note log scale of y axis.

Fig. S2. Effect of Shx treatment on the frequency of persisters. Quantification of

surviving wild-type (14028s) Salmonella following 24 h treatment with either cefotaxime

or ciprofloxacin. Shx treatment was carried out for 30 min prior to outgrowth as

described in Materials and Methods. Error bars represent standard deviations. N = 6

independent biological replicates. Note log scale of y axis. Populations are not

statistically significant (two-tailed t-test).

LB

C

efo

Sh

x

+

Cef

oCipro

Shx

+ C

ir

po

0

2

4

6

8

10

CF

Us/ m

l

wild-type Shx 24h

LB

Cefo

Shx + Cefo

Cipro

Shx + Cirpo

wild-type

Lo

g C

FU

s/m

l

cefotaxime

Glucose

Mannose

Gluc +Cefo

Man + Cefo

Gluc + Cipro

Man + Cipro

ciprofloxacin

Glucose

Mannose

Gluc +Cefo

Man + Cefo

Gluc + Cipro

Man + Cipro

no treatmentGlucose

Mannose

Gluc +Cefo

Man + Cefo

Gluc + Cipro

Man + Cipro

Shx + Cefo

Shx + Cirpo

cefotaxime + Shx

ciprofloxacin + Shx

Fig. S3. Effects of ATP depletion and inhibition of protein synthesis on antibiotic

tolerance. (A) Quantification of surviving wild-type Salmonella (14028s) following 24h

exposure to cefotaxime or ciprofloxacin. Bacteria expressed the soluble portion of the

F1Fo ATPase from plasmid pATPase or harbored the empty vector (pVector).

Quantification of surviving (B) wild-type (14028s) or (C) relA::Tn10 spoT (MP342)

Salmonella following cefotaxime or ciprofloxacin exposure in the presence of absence of

chloramphenicol. Error bars represent standard deviations. N = 8 independent

biological replicates. Note log scale of y axis.

pVector pATPase

pVecto

r

pATPas

e

0

2

4

6

8

10

pATPase

Legend

+ Cefotax

+Cipro

Lo

g C

FU

s/m

l

cefotaxime

untreated +Gluco + Trp

Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

+Gluco + Trp

Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

+Gluco + Trp

Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

ciprofloxacin

A B

relA

spoT

relA

spoT +

Cm

0

2

4

6

8

10

relA spoT Cm

LB

LB + Cefo

LB + Cipro

untreated + chlora

relA spoT

cefotaxime


Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

+Gluco + Trp

Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

+Gluco + Trp

Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

ciprofloxacin

Lo

g C

FU

s/m

l

wild

-typ

e

wild

-typ

e+Cm

0

2

4

6

8

10

Cm

LB

LB +Cefo

LB +Cipro

untreated + chlora

Lo

g C

FU

s/m

l

cefotaxime


Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

+Gluco + Trp

Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

+Gluco + Trp

Cefo +Gluco +Trp

Cefo +Man -Trp

Cipro +Gluco +Trp

Cipro +Man -Trp

ciprofloxacin

C

w

ild

-ty

pe

tr

pA

r

elA s

poT

0

2

4

6

8

10

Lo

g C

FU

s/ m

l

trpA relA spoT Cefo Rest

LB

NMM

LB + Cefo

NMM + Cefo

Lo

g C

FU

s/m

l

wild-type trpArelA

spoT

LB

NMM

LB + Cefo

NMM + Cefo

LB

NMM

LB + Cefo

NMM + Cefo

permissive

restrictive

cefotaxime

cefotaxime

B

restricted

wild

-typ

e

relA

spoT

0

2

4

6

8

10

relA spoT NMM

LB

NMM

LB + Cefo

NMM + Cefo

LB

NMM

LB + Cefo

NMM + Cefo

LB

NMM

LB + Cefo

NMM + Cefo

permissive

restrictive

cefotaxime

cefotaximerestricted

wild-type relA spoT

Lo

g C

FU

s/m

l

C

permissive

restrictive

cefotaxime cefotaxime

ciprofloxacin

A

Lo

g C

FU

s/m

l

Glucose

Mannose

Gluc +Cefo

Man + Cefo

Gluc + Cipro

Man + Cipro

Glucose

Mannose

Gluc +Cefo

Man + Cefo

Gluc + Cipro

Man + Cipro

Glucose

Mannose

Gluc +Cefo

Man + Cefo

Gluc + Cipro

Man + Cipro

wild-type manAtrpEDCBA

Glucose

Mannose

Gluc +Cefo

Man + Cefo

Gluc + Cipro

Man + Cipro

restrictive

Glucose

Mannose

Gluc +Cefo

Man + Cefo

Gluc + Cipro

Man + Ciprociprofloxacin restrictive

wild-type trpEDCBA manA0

2

4

6

8

10

Lo

g C

FU

s/ m

l

Mannose+TryptophanMan + Cipro

Fig. S4. Effect of growth inhibition on antibiotic tolerance in Salmonella. (A)

Quantification of surviving wild-type (14028s), manA (MP50), and trpEDCBA::Tn10

(AS301) Salmonella following 24 h treatment with either cefotaxime or ciprofloxacin

under growth-permissive and growth-restrictive conditions. For the manA (MP50)

strain, growth restriction was achieved in MOPS minimal medium containing mannose

as the sole carbon source (see Materials and Methods). For trpEDCBA::Tn10 (AS301)

strain, growth restriction was achieved in MOPS minimal medium lacking tryptophan

(see Materials and Methods). (B) Quantification of surviving wild-type (14028s) and

relA::Tn10 spoT trpA::kan (MP1494) Salmonella following 24 h treatment with either

cefotaxime or ciprofloxacin under growth-permissive and growth-restrictive conditions.

For the relA::Tn10 spoT trpA::kan (MP1494) strain, growth restriction was achieved in

MOPS minimal medium lacking tryptophan (see Materials and Methods). (C)

Quantification of surviving wild-type (14028s) and relA::Tn10 spoT (MP342) Salmonella

following 24 h treatment with cefotaxime under growth-permissive and growth-

restrictive conditions (see Materials and Methods). For the relA::Tn10 spoT (MP342)

strain, growth restriction was achieved in N-minimal medium lacking various amino

acids and containing glycerol as the sole carbon source (see Materials and Methods).

Error bars represent standard deviations. N = 8 independent biological replicates. Note

log scale of y axis.

Table S1. Microbial strains and plasmids used in this study.

Strains Relevant characteristics Identifier Source Escherichia coli

DH5a Host strain used for generation and propagation of plasmid constructs

Yale University CGSC#12384 (82)

EC100D Host strain used for generation and propagation of repR6Kγ plasmid constructs

EC100D Epicentre

Salmonella enterica serovar Typhimurium

14028s wild-type 14028s; ATCC 14028 (73)

AS301 trpEDCBA::Tn10 trpEDCBA (Eduardo A.

Groisman, Yale University. This study; lab stock)

MS7953 phoP::Tn10 phoP::Tn10 (73)

MP342 relA::Tn10 DspoT relA spoT (25)

NR-42835 E11 DtrpA::Kan trpA (79)

MP1494 relA::Tn10 DspoT DtrpA::Kan relA spoT trpA (This study)

MP50 DmanA::Cm manA (This study)

MP1142

DTA1::Cm (NCBI locus tags for genesencoded by TA1 are STM14_3505 and STM14_3506)

TA1, TA6‡, tacAT,STM2904/05 (This study)

MP1143

DTA2::Kan (NCBI locus tags for genesencoded by TA2 are STM14_3562 and STM14_3563)

TA2, parDE‡,STM2954/55 (This study)

MP1144

DTA3::Spm (NCBI locus tags for genesencoded by TA3 are STM14_4401 and STM14_4402)

TA3, TA8‡, tacAT,STM3651/52 (This study)

MP1145

DTA4::Tn10 (NCBI locus tags for genesencoded by TA4 are STM14_4844 and STM14_4845)

TA4, relBE5‡ (This study)

MP1146

DTA5::Gen (NCBI locus tags for genesencoded by TA5 are STM14_4847 and STM14_4848)

TA5, higBA2‡ (This study)

MP1147

DTA6::Apr (NCBI locus tags for genesencoded by TA6 are STM14_5191 and STM14_5192)

TA6, TA9‡,STM4317/18 (This study)

MP1148

DTA7::Cm (NCBI locus tags for genesencoded by TA7 are STM14_5441 and STM14_5442)

TA7, shpAB‡, STM4528/29 (This study)

MP1149

DTA8::Kan (NCBI locus tags for genesencoded by TA8 are STM14_4235 and STM14_4236)

TA8, relBE2‡,yafQ/dinJ,

STM3516/17 (This study)

MP1150

DTA9::Spm (NCBI locus tags for genesencoded by TA9 are STM14_4284 and STM14_4285)

TA9, phD/doc‡,STM3558/59 (This study)

MP1151

DTA10::Tn10 (NCBI locus tags forgenes encoded by TA10 are STM14_1870 and STM14_1871)

TA10, relBE1‡,STM1550/51 (This study)

MP1152

DTA11::Gen (NCBI locus tags for genesencoded by TA11 are STM14_5340 and STM14_5341)

TA11, relBE3‡,(STM4449/50) (This study)

MP1153

DTA12::Apr (NCBI locus tags for genesencoded by TA12 are STM14_4703 and STM14_4704)

TA12, higBA1‡,STM3906/07 (This study)

MP1422 DTA1 DTA2 DTA3 DTA4 DTA5 DTA6 DTA7 DTA8 DTA9 DTA10 DTA11 DTA12 D12TA (This study)

MP1454 DTA3::SpmTA3, TA8‡, tacAT,

STM3651/52 (This study)

MP1455 DTA5::Gen TA5, higBA2‡ (This study)

MP1456 DTA7::CmTA7, shpAB‡, STM4528/29 (This study)

MP1457 DTA9::SpmTA9, phD/doc‡,STM3558/59 (This study)

MP1458 DTA10::Tn10 TA10, relBE1‡,STM1550/51 (This study)

Plasmid Relevant characteristics Identifier Source pSIM6 reppSC101

ts AmpR PCI857-gbexo pSIM6 (74)

pSIM19 Plasmid harboring spectinomycin resistant (SpmR ) marker pSIM19 (74)

pSAM_Gent_Pbad Plasmid harboring gentamycin resistant (GenR ) marker pSAM_Gent_Pbad

(Barbara Kazmierczak,

Yale University)

pMCS-7 Plasmid harboring apramycin resistant (AprR ) marker pMCS-7 (78)

pCP20 reppSC101ts l cI857 FLP AmpR CmR pCP20 (77)

pKD3 repR6Kγ AmpR FRT CmR FRT pKD3 (77)

pKD4 repR6Kγ AmpR FRT KmR FRT pKD4 (77)

pKD4-TetR repR6Kγ AmpR FRT tn10-TetR FRT pKD4-TetR (This study)

pKD4-SpmR repR6Kγ AmpR FRT SpmR FRT pKD4-SpmR (This study)

pKD4-GenR repR6Kγ AmpR FRT GenR FRT pKD4-GenR (This study)

pKD4-AprR repR6Kγ AmpR FRT AprR FRT pKD4-AprR (This study)

pUHE-21 reppMB1 lacIq AmpR vector control pVector; pUHE-21-2-lacIq (83)

pUHE-ATPase reppMB1 lacIq AmpR Plac-atpAGD pATPase (33)

Tn10 - tetracycline resistant marker (tet) Amp - ampicillin resistant marker Kan - kanamycin resistant marker ts - temperature sensitive Cm - chloramphenicol resistant marker ‡ - nomenclature used in (14)Spm - spectinomycin resistant marker

Gen - gentamycin resistant marker Apr - apramycin resistant marker

Table S2. Oligonucleotides sequences used in this study.

Primer Sequence (5'−>3') Purpose Source

W3042 TATGTATTATAATTGTACATATTGTTCATAAACAGGAGGCATATGAATATCCTCCTTA

Generation of DTA1::Cm strain (This study)

W3043 GACCAGCGTCAGGCTCGTATAACGAGTGCTCCGGTGGGAGGTGTAGGCTGGAGCTGCTTC


W3044 CCTCATATGTACGCCTTGA PCR verification of DTA1::Cm strain (This study)

W3045 GGATGACGTTGTTGGCAAT PCR verification of DTA1::Cm strain (This study)

W3038 AGATTTATACTTACAGTGGAGGCTGTTATGGCCAGAACAGTGTAGGCTGGAGCTGCTTC

Generation of DTA2::Kan strain (This study)

W3039 CACACTTGTTTTTTAGATAAAAAGGCTATAACCAATTTACATATGAATATCCTCCTTA


W3040 CATTTCCGGTGTACTCTTAT PCR verification of DTA2::Kan strain (This study)

W3041 CTTGGTAATATTGTTGATT PCR verification of DTA2::Kan strain (This study)

W3034 GTTTGCTATACATGGTGGTTGTGCTATTCTTGTAAAGCAAGTGTAGGCTGGAGCTGCTTC

Generation of DTA3::Sp strain (This study)

W3035 AAATTATCGCTTATAGCGATTTGAACATAACAGTCTTTGCCATATGAATATCCTCCTTA


W3036 CGACGACTTGATGTATACC PCR verification of DTA3::Sp strain (This study)

W3037 TTAACCTTAGACATTCTAT PCR verification of DTA3::Sp strain (This study)

W3145 CCGTATGAAGAGGGCGGGAATGAGGAGGAGTAGCTGGACATATCCACATATGAATATCCTCCTT

Generation of DTA4::Tn10 strain (This study)

W3030 AATTGTTTTCAGGTTGAAAACTTTGTAATGGAACTACATTGGTGTAGGCTGGAGCTGCTT


W3032 ACTGGATAACGTTGCGCT PCR verification of DTA4::Tn10 strain (This study)

W3033 TCGTACTACCTGCGAAACCG PCR verification of DTA4::Tn10 strain (This study)

W3026 TAGATTATTTTCACTACTATAAGCCAATGGCGTATGGAATGTGTAGGCTGGAGCTGCTTC

Generation of DTA5::Gen strain (This study)

W3027 GGTATACCGGCTAGCAATCTACGTTAGCCGGATCATTGCCATATGAATATCCTCCTTA


W3028 GATGCTATACGCCATTGACG PCR verification of DTA5::Gen strain (This study)

W3029 AGTTCCATCAACCCGTTCCT PCR verification of DTA5::Gen strain (This study)

W3022 GTATGCTTAATATCCACAGAAGTACAAGAGGACAACACTGTGTAGGCTGGAGCTGCTTC

Generation of DTA6::Apr strain (This study)

W3023 CGGTCTGGCTCACTGACTATCCACAGCGAATTCCGGCCATCATATGAATATCCTCCTTA


W3024 GACATCTGCGTTACGCGTGT PCR verification of DTA6::Apr strain (This study)

W3025 GTTGTCCGGCTGAAGCGGTA PCR verification of DTA6::Apr strain (This study)

W3018 TAAGATAATTGTATATACATTTTTGTATGTACAATTAGGGCATATGAATATCCTCCTTA


W3019 TTTGTAGGCCGGATAAGGCGCAGCCGCCATCCGGCATCTTGTGTAGGCTGGAGCTGCTTC


W3020 TCCAACAATCCAGGTATAAT PCR verification of DTA7::Cm strain (This study)

W3021 AGCGCTTATCTGGCCTGGCG PCR verification of DTA7::Cm strain (This study)

W3014 TACAATTTGAACTGTATAGAGACACAGTACAGGAGACTAAGTGTAGGCTGGAGCTGCTTC


W3015 ATTTGTTTAATCATAAAACCTGAAAGCACCGGTTAACATATCATATGAATATCCTCCTTA


W3016 GTATGTGTGTCTCAGTTGA PCR verification of DTA8::Kan strain (This study)

W3017 CAGGCAACGTCCTGGTGTGT PCR verification of DTA8::Kan strain (This study)

W3010 TGTACATGTTTGTTGTACAATAAACATGTACAACTGGGGAGTGTAGGCTGGAGCTGCTTC


W3011 TGCCGTAACCCAACCAGGCGGAGCGTCGCCGGGATTAACGTCATATGAATATCCTCCTTA


W3012 ATCTGGGCGGTTAGACTGT PCR verification of DTA9::Sp strain (This study)

W3013 ACGGATAGGTACGACATTA PCR verification of DTA9::Sp strain (This study)

W3006 TAAAGGTGCTATATTTAGTCATCAACCAGGAGGTAAATTGTGTAGGCTGGAGCTGCTTC


W3007 ACATAAAAGAACTGAGCCTGTTTGTCGTATCGTTTGACGCATATGAATATCCTCCTTA


W3008 CACTGACCGGGCTGGAGCT PCR verification of DTA10::Tn10 strain (This study)

W3009 GTGGCATATGTCAAGGTGCC PCR verification of DTA10::Tn10 strain (This study)

W3002 GCTAAACTGCATTACACATTCGATTCTACTGGAGGCATAGTGTAGGCTGGAGCTGCTTC


W3003 GTAGCAGCAACCAGGTAGTGCATCATTTGCGTTGATCGCTCATATGAATATCCTCCTTA


W3004 ATTCGATGTAATGCGAATG PCR verification of DTA11::Gen strain (This study)

W3005 GACCCGGCGCTTATCTGGC PCR verification of DTA11::Gen strain (This study)

W2998 CTCACTCGAGCTCTCTCCAATGAAAAAGAAATTCGATGAGTGTAGGCTGGAGCTGCTTC


W2999 AGCGGCTGCATGAGCCGCTAATTGATGCGTTCTGGAAGATCATATGAATATCCTCCTTA


W3000 AGCACCGCCATCACTTGCAC PCR verification of DTA12::Apr strain (This study)

W3001 CGTTTGAACTCGGCGTCTGC PCR verification of DTA12::Apr strain (This study)

12460 ACTTCATTAACTCAGTGCAAAACTATGCCTGGGGAAGTAACATATGAATATCCTCCTTA

Generation of DmanA::Cm strain (This study)

12461 CGTCAGGATAATACTCTGAAATCACGCGGATCGTTTGCCACGGTGTAGGCTGGAGCTGCTTC

Generation of DmanA::Cm strain (This study)

12462 ACCTCCCATGATCTCCACAT PCR verification of DmanA::Cm strain (This study)

12190 TCATTGCCATACGTAATTC PCR verification of DmanA::Cm strain (This study)

W2990 GCCGCCAAGGATCTGATGGCGCAGGGGATCAATTAAGACCCACTTTCACATT

Cloning of tn10-TetR into pKD4 (This study)

W2991 CCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGCCCTAAGCACTTGTCTCCTG

Cloning of tn10-TetR into pKD4 (This study)

W2992 GCCGCCAAGGATCTGATGGCGCAGGGGATCAACCACAGTACCCAATGATCC

Subccloning of SpR marker from pSIM19 into pKD4

(This study)

W3083 CCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGGGTGAGAGATCTGAATTGC

Subccloning of SpR marker from pSIM19 into pKD4

(This study)

W2994 GCCGCCAAGGATCTGATGGCGCAGGGGATCAAGAGCTCGAATTGACATAAG

Subccloning of GenR marker from pSAM_Gent_Pbad into pKD4

(This study)

W3084 CCGAAGTTCCTATTCTCTAGAAAGTATAGGAAC

Subccloning of GenR marker from pSAM_Gent_Pbad into pKD4

(This study)

W2996 GCCGCCAAGGATCTGATGGCGCAGGGGATCAACTTGACCCGCAGTTGCAA

Subccloning of AprR marker from pMCS-7 into pKD4

(This study)

W3082 CCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGTCGTTCTCCGCTCATGAGC

Subccloning of AprR marker from pMCS-7 into pKD4

(This study)

W3081 CATGGCGATAGCTAGACTG

DNA sequencing of antibiotic markers cloned into pKD4 digested with BglII and AfeI

(This study)

pKD4R AGTGACACAGGAACACTTA

DNA sequencing of antibiotic markers cloned into pKD4 digested with BglII and AfeI

(This study)

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