Table 1 Efflux characteristics, MIC and relative polysaccharide content for reference strains versus CF clinical isolates of P. aeruginosa.

From: Mechanisms of intrinsic resistance and acquired susceptibility of Pseudomonas aeruginosa isolated from cystic fibrosis patients to temocillin, a revived antibiotic

Reference strains and clinical isolates (patient’s identification code; country; date of collection)

Description (phenotype; length of MexA/B protein; AA deletions and affected MexA/B domains)

Efflux characteristics

MIC (mg/L)

EPS (β-1, 4/β-1, 3 glycosidic bonds)

mexA alterations

mexB alterations

Vmax (units/s)

TMO

TMO +PAβN

TMO+VNL

PAO1

Wild type MexA (383 AA) MexB (1046 AA)

—

—

−0.480

512

128

768

1

PT629 a

PAO1 MexAB-OprM overproducer

—

—

−0.720

1024

256

1536

 

PAO1mexABa

PAO1 ΔmexAB

—

—

−0.112

2

2

2

 

3724 (AL1; France; 14/12/2006)

-MexA (383 AA).

-Truncated MexB (878 AA): S879_Q1046del deletion of 168 AA affecting 5 TMs (TM8-TM12).

-Uncommonb synonymous mutations: C546G

-Prevalentb synonymous mutations: A345G G993A

-Uncommon synonymous mutations: C486T

-Prevalent synonymous mutations: A495G C1308T C1692T T2280C T2730C G3117A

-Nonsense mutation: C2636A

−0.178

2

2

4

1.2

144 (PCF79; Belgium; 20/09/2010)

-Truncated MexA (124 AA): A125_G383del deletion of 259 AA affecting the α-hairpin, second, third and MP domains.

-MexB (1046 AA)

-Prevalent synonymous mutation: G993A

-Nonsense mutation, Δ 13 nt (367–379)

Prevalent synonymous mutations: A495G T2280C T2730C G3117A

−0.016

2

2

4

1.1

W024 (DM; UK; 2009)

-Truncated MexA (69 AA): V70_G383del deletion of 314 AA in α-hairpin, second, third and MP domains.

-MexB (1046 AA)

Nonsense mutation, Δ 1 nt (164)

No mutations

−0.117

4

2

6

1.4

W049 (DB; UK; 2009)

-Isogenic to W024

-Truncated MexA (69 AA): V70_G383del deletion of 314 AA in α-hairpin, second, third and MP domains.

-MexB (1046 AA)

Nonsense mutation: Δ1 nt (164)

No mutations

−0.117

4

2

6

2.2

143-1 (143; Germany; 12/07/2012)

-Nonstop mutation in mexA (stalled ribosome, proteolysis; recycled ribosome)13

-MexB (1046AA)

-Prevalent synonymous mutation: G993A

-Nonstop mutation: Δ 2 nt (837–838)

-Uncommon synonymous mutations: C2412T

-Prevalent synonymous mutations:A495G T2226C T2280C T2730C G3117A

−0.019

4

4

8

0.5

AG3 (JP; UK; 10/05/2006)

-Shortened MexA (372 AA): G337_E347del deletion of 11 AA in the MP domain.

-MexB (1046AA)

-Prevalent synonymous mutations: A345G G993A

-Deletion: Δ 33 nt (1011–1043)

Prevalent synonymous mutations: C474T C492T A495G T2280C T2730C G3117A

−0.004

8

4

12

1.4

129 (RCF62; Belgium; 09/09/2010)

-MexA (383 AA).

-MexB (1045 AA): N254del deletion of asparagine 254 in the DN subdomain and missense mutation (T557N) in TM7.

-Uncommon synonymous mutations: C633T G702A C780T

-Prevalent synonymous mutations: T333C A345G G447A A639G T655C T729C C732T A738G C789G G792A T1002C T1095C

-Uncommon synonymous mutations: C909T G1362A

-Prevalent synonymous mutations: A495G T2730C G3117A

-Conservative missense mutation: C1670A

-Deletion: Δ3 nt (760–762)

−0.053

8

2

12

1.1

135-1 (135; Germany; 10/07/2012)

-MexA (383 AA): A206S missense mutation (third domain).

-MexB truncated (719 AA): M720_Q1046del deletion of 327 AA in DC, PN1, PC2 and TM8-TM12 and 300 AA substitutions (I186V; M421_719) in TM5-7, DN, PC1, and PC2.

-Uncommon synonymous mutations: G438T G558C

-Prevalent synonymous mutations: T333C A345G G447A A639G T655C T729C C732T A738G C789G G792A C951G T1002C T1095C

-Radical missense mutation: G616T

-Uncommon synonymous mutations: G348A C1101T C1389A

-Prevalent synonymous mutations: C315T A351G A495G C498G G591A C600G C537T A1938G T2067C A2079G T2280C T2730C

-Conservative missense mutation: A556G

-Insertion and deletion leading to premature stop codon: G1261_C1262insG + Δ 2 nt (1947–1948)

−0.100

8

8

12

3.8

126 (VIF68; Belgium; 09/09/2010)

-SCV isolate.

-MexA (383 AA): aberrant signal peptide (S19L).

-MexB (1046 AA): missense mutations (I186V, Q773P) in DN and DC subdomains at the OprM-docking domain of MexB

-Uncommon synonymous mutations: C582T C912T

-Prevalent synonymous mutations: T333C A345G G447A T655C T729C C789G G792A C951G T1002C T1095C

-Radical missense mutation: C56T

-Uncommon synonymous mutations:C1314A G2004A C2014T

-Prevalent synonymous mutations: C315T A351G A495G C498G C537T G591A C600G A804C G1587T T1749C A1938G T2067C A2079G T2226C T2280C T2730C T2826C A2892G C2955T

-Conservative missense mutation: A556G

-Radical missense mutation: A2318C

−0.007

8

2

12

3.3

279 (RRM59; Belgium; 28/10/2010)

-MexA (383 AA).

-Aberrant MexB (1027 AA): K151_T169del deletion of 19 amino acids in the periplasmic pore domain (subdomain PN2)

No mutations

-Prevalent synonymous mutations: A351G C1308T T2280C T2730C G3117A

-Deletions: Δ57 nt (455–511)

−0.016

16

4

24

2

BM1 (AD; UK; 13/09/2006)

- LES isolate.

-Truncated MexA (119 AA): Y120_G383del deletion of 264 AA in α-hairpin, second, third and MP domains.

-MexB (1046 AA)

-Prevalent synonymous mutation: A345G

-Nonsense mutation: C360G

-Uncommon synonymous mutation: T2015G

-Prevalent synonymous mutations: A495G, C1692T, T2280C, T2730C, G3117A

−0.025

16

16

16

3.7

AJ3 (ML; UK; 22/05/2006)

- LES isolate.

-Truncated MexA (119 AA): Y120_G383del deletion of 264 AA in α-hairpin, second, third and MP domains.

-MexB (1046 AA)

-Prevalent synonymous mutation: A345G

-Nonsense mutation: C360G

-Uncommon synonymous mutation: T2015G

-Prevalent synonymous mutations: A495G, C1692T, T2280C, T2730C, G3117A

−0.025

16

16

32

1.4

CF15 (CT; UK; 2007)

-LES isolate.

-Truncated MexA (119AA): Y120_G383del deletion of 264 AA in α-hairpin, second, third and MP domains.

-MexB (1046 AA)

-Prevalent synonymous mutation: A345G

-Nonsense mutation: C360G

-Uncommon synonymous mutation: T2015G

-Prevalent synonymous mutations: A495G, C1692T, T2280C, T2730C, G3117A

−0.025

16

16

24

3.2

CF53 (DP; UK; 2007)

-LES isolate.

-Truncated MexA (119 AA): Y120_G383del deletion of 264 AA in α-hairpin, second, third and MP domains).

-MexB (1046 AA)

-Prevalent synonymous mutation: A345G

-Nonsense mutation: C360G

-Uncommon synonymous mutation: T2015G

-Prevalent synonymous mutations: A495G, C1692T, T2280C, T2730C, G3117A

−0.025

16

16

32

1.6

CF19 (LS; UK; 2007)

-LES isolate.

-Truncated MexA (119 AA): Y120_G383del deletion of 264 AA in α-hairpin, second, third and MP domains).

-MexB (1046 AA)

-Prevalent synonymous mutation: A345G

-Nonsense mutation: C360G

-Uncommon synonymous mutation: T2015G

-Prevalent synonymous mutations: A495G, C1692T, T2280C, T2730C, G3117A

−0.025

16

16

24

1.9

CF16 (RC; UK; 2007)

-LES isolate.

-MexA (383 AA).

-Truncated MexB (30 AA): P31_Q1046del only TM1 is encoded.

Prevalent synonymous mutation: A345G

-Uncommon synonymous mutation: T2015G

-Prevalent synonymous mutations: A495G, C1692T, T2280C, T2730C, G3117A

-Nonsense mutation: Δ 154 nt (85–239)

−0.120

32

8

48

2.7

BV1 (DC; UK; 11/10/2006)

-LES isolate.

-MexA (383 AA).

-Truncated MexB (672 AA): E673_Q1046del deletion of 374 AA in DC, PN1, PC2 and TM8-TM12, and 154 AA substitutions (F519_G672) in TM6-7, PC1 subdomains.

Prevalent synonymous mutation: A345G

-Uncommon synonymous mutation: T2015G

-Prevalent synonymous mutations: A495G, C1692T, T2280C, T2730C, G3117A

-Nonsense mutation: Δ 8 nt (1555–1562)

−0.080

32

8

64

3.6

191-4 (191; Germany; 03/09/2012)

-Truncated MexA (27 AA): E28_G383del deletion of 356 AA.

-MexB (1046 AA).

-Prevalent synonymous mutation: G993A

-Nonsense mutation: G82T

Prevalent synonymous mutations: A495G T2280C T2730C G3117A

−0.014

64

32

128

4.1

207 (207; Germany; 2012)

-Truncated MexA (27 AA): E28_G383del deletion of 356 AA.

-MexB (1046 AA).

-Prevalent synonymous mutation: G993A

-Nonsense mutation: G82T

Prevalent synonymous mutations: A495G T2280C T2730C G3117A

−0.014

64

32

64

2.6

109 (ENM88; Belgium; 03/09/2010)

-MexA (383 AA).

-15 base pairs (5 AA residues PAIAP [P36_P40]) minisatellite repeat in MexB (1051 AA), toxic or malfunctioning proteins.

-Uncommon synonymous mutation: G117A C468T

-Prevalent synonymous mutations: T333C A345G G447A T729C T1002C

-Uncommon synonymous mutations: G1452A C1920T

-Prevalent synonymous mutations: C315T A351G A495G T642C A804C C1308T T1749C A1938G T2067C A2079G T2226C T2280C

-15 nt minisatellite repeat: 118–132 c

−0.008

64

toxic

64

2.5

618 (FJ1; France; 01/01/1996)

-Truncated MexA (297 AA): V298_G383del, deletion of 86 AA in MP domain.

-MexB (1046 AA)

-Prevalent synonymous mutation: A345G

-Nonsense mutation: Δ1 nt (C869)

Prevalent synonymous mutations: A495G C1308T C1851T T2067C T2226C T2280C T2730C T2826C A2892G C2955T G3117A

−0.114

128

toxic

128

2.9

3179 (MP1; France; 03/02/2004)

-SCV isolate.

-MexA (383 AA).

-Truncated MexB (719 AA): M720_Q1046del deletion of 327 AA in TM8-TM12, DC and PN1/PC2 subdomains.

Prevalent synonymous mutation: G993A

-Prevalent synonymous mutations: A495G C1308T C1851T T2067C T2226C T2280C T2730C T2826C A2892G C2955T G3117A

-Nonsense mutation: Δ1 nt (G2147)

−0.117

128

128

192

3.1

180-3 (180; Germany; 30/07/2012)

-MexA (383 AA): W332R missense mutation in MP domain;

-MexB (1046 AA)

-Prevalent synonymous mutations: A345G T729C T1002C

-Radical missense mutation: T994C

-Uncommon synonymous mutation: G1608A

-Prevalent synonymous mutations: C492T A495G T2280C T2730C G3117A

−0.185

128

32

128

1.2

3319 (BV1; France; 03/08/2004)

-MexA (383 AA);

-MexB (1046 AA): Y182C, A707S, I963V, S1041E and V1042A, missense mutations in PC2, DN, TM11, and cytoplasmic C-terminal domain.

-Uncommon synonymous mutations: G51T C147G C291T G399A C423T G426A C591T C651T C687T C705T C777A

-Prevalent synonymous mutations: T333C A345G G447A T655C T729C C951G T1002C

-Uncommon synonymous mutations: C129T G165A T423G T519C G1080A C1152T T1332C C1383T C1476G T1563C C1581T C1614T A1713G G1748C T1758G G1836C C1980T G2001T C2070A C2134T A2139G C2148T C2223T G2319A T2457C G2469C C2604A T2907C T3033C

-Prevalent synonymous mutations: A351G A495G C498G C537T T642C A804C G1587T T1749C T2067C T2226C T2280C T2730C A2892G C2955T

-Radical missense mutation: G2119T

-Conservative missense mutations: A545G; A2887G; G3120T T3121G C3122A C3123G T3125C

−0.150

256

32

384

1.8

208-3 (208; Germany; 09/08/12)

-Isogenic to 135-1.

-MexA (383 AA): missense mutation A206S (third domain).

-MexB (1045 AA): missense mutation I186V (DN subdomain), and 30 AA replaced by 29 new AA (R620_R650 in PC1 subdomain).

-Uncommon synonymous mutations: G438T G558C

-Prevalent synonymous mutations: T333C A345G G447A A639G T655C T729C C732T A738G C789G G792A C951G T1002C T1095C

-Radical missense mutation: G616T

-Uncommon synonymous mutations: G348A C1101T

-Prevalent synonymous mutations: C315T A351G A495G C498G C537T G591A C600G A1938G T2067C A2079G T2280C T2730C

-Conservative missense mutation: A556G

-Deletions: Δ 1 nt (T1854) + Δ 2 nt (1947–1948)

−0.320

256

256

256

4.4

208-2 (208; Germany; 09/08/12)

-Isogenic to 135-1.

-MexA (383 AA): missense mutation A206S (third domain).

-MexB (1045 AA): missense mutation I186V (DN subdomain), and 19 AA replaced by 18 new AA (L631_R650 in PC1 subdomain)

-Uncommon synonymous mutations: G438T G558C

-Prevalent synonymous mutations: T333C A345G G447A A639G T655C T729C C732T A738G C789G G792A C951G T1002C T1095C

-Radical missense mutation: G616T

-Uncommon synonymous mutations: G348A C1101T

-Prevalent synonymous mutations: C315T A351G A495G C498G C537T G591A C600G A1938G T2067C A2079G T2280C T2730C

-Conservative missense mutation: A556G

-Deletions: Δ 1 nt (T1889) + Δ 2 nt (1947–1948)

−0.253

512

256

512

4.6

128 (DAF69; Belgium; 09/10/2010)

-Isogenic to AG3.

-MexA (383 AA).

-MexB (1046 AA): L376V, missense mutation in TM3.

Prevalent synonymous mutations: A345G G993A

-Prevalent synonymous mutations: C474T C492T A495G T2280C T2730C G3117A

-Conservative missense mutation: C1126G

−0.576

1024

1024

1024

4.9

129-6 (129; Germany; 11/07/2012)

-Isogenic to AG3.

-Synonymous mutations in MexA (383 AA) and MexB (1046 AA)

Prevalent synonymous mutations: A345G G993A

Prevalent synonymous mutations: C474T C492T A495G T2280C T2730C G3117A

−0.672

1024

256

1536

2.1

4289 (JV1; France; 13/08/2007)

-Synonymous mutations in MexA (383 AA) and MexB (1046 AA)

Prevalent synonymous mutations: G993A

Prevalent synonymous mutations: A495G T2280C T2730C G3117A

−0.528

1024

512

1536

1.5

  1. aSee ref. 6 for references.
  2. bUncommon synonymous mutations: rare mutations found in isolates belonging to only one clone or one single patient; prevalent synonymous mutations: identical mutations found in different isolates belonging to different clones.
  3. cThe presence of minisatellites could be consecutive to bacterial exposure to genotoxic compounds42,43; the resulting proteins are malfunctioning.
  4. The table shows (a) the mutations detected in mexA and mexB and the changes in the corresponding proteins, the Vmax for NPN efflux (see Fig. 2, panels c-d), (b) temocillin MICs in control conditions, or in the presence of 20 mg/L PAβN as efflux pump inhibitor and 2 mM vanillate (VNL) as OpdK substrate, and (c) the culture content in exopolysaccharides relative to PAO1 (see Fig. 3). Abbreviations used: TM, transmembrane α-helix; MP, membrane-proximal domain; PN1/PN2 (periplasmic, N-terminal), PC1/PC2 (periplasmic, C-terminal), DN (docking, N-terminal) and DC (docking, C-terminal) are six periplasmic subdomains that build the pore and docking domains of MexB, respectively (see Fig. S1 for more details); nt, nucleotide; AA, amino acids; SCV, small colony variant; LES, Liverpool Epidemic Strain; EPS, extracellular polymeric saccharides. Molecular graphics for mexA and mexB mutants are represented in the supplementary data (Table S2a–e) together with the position of the different protein domains.
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