Antibiotic uptake in P.

aeruginosa, and its

consequences on the primary metabolome
Garca-Rivera M., Fetz V., Brnstrup M.

Biology Seminar
1st Jun 2016
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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Introduction
Drug resistant bacteria[1]
Acquired resitance has become a major health issue
Health care extra costs of EUR 1.5 billion each year
In the EU, 25 000 deaths per year

Different resistance mechanisms[2]

Alterations in target proteins/enzyme linked to a biosynthetic pathway
Alterations to the drug structure
Alterations that affect permeability (efflux pumps or porins)

E. coli

P. aeruginosa

ESBL1 producing bacteria

Multi-drug resistant bacteria

Extended-spectrum -lactamase
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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Introduction
Drug permeability across Gram-negative bacteria cell wall[3,4]
Lipo polysacharides limited permeation of hydrophobic drugs
Hydrophilic small molecules permeable via non-specific porins
Lack of appropiate direct assays permeability barrier not well understood

Antibiotic uptake quantification methods[3]

Radioactive labelled compounds

(standard)
-lactamase activity (1977)
MS-based promising methods

Single cell imaging with fluorescent

molecules
Single channel ion current
(artificial planar bilayer)

Bottleneck for optimization of effective, new antibiotics

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

flow

Introduction
Drug antibacterial activity[2]
Uptake by bacteria
Mode of action
Bacterostatic or bactericidal
MIC2 and MBC3
Time- vs concentration-dependence

Time

Uptake
Mode
of
action
MIC

Effect of antibiotics on bacterial metabolism[5,6]

Antibiotic stress Alteration in metabolic pool
Unique metaboilic responses according to different antibiotics
Untargeted metabolomics for deciphering mode of action[7]

2
3

Minimum inhibitory concentration

Minimum bactericidal concentration
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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Aims

Establish a reliable, robust and direct MS-based procedure

for uptake quantification (high throughput)

Profiling metabolites for P. aeruginosa upon treatment with

selected antibiotics (LC- and GC-MS)

Validation by comparing antibiotics with known and unknown

mode of action

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Proposed timeline

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Previous work

Translocation4 project
(France)

LC-MS quantification
methods (CBIO)

Uptake quantification
proposed procedure

Six -lactam antibiotics proposed

MIC determined
Two E. coli strains, porin expresion mutants

Mobile phases and buffers composition

Limits of detection and quantification
Calibration curves in two MS devices
MRM5 methods

Different E. coli strain, different antibiotic nature

Large lab-scale volumes (50 mL)
Proposed protocol for cell fractionation

(Heumanns thesis)
4
5

New Drugs for Bad Bugs (ND4BB) / Innovative Medicines Initiative (IMI)
Multiple-reaction
monitoring
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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Uptake quantification
-lactam antibiotics
Meropenem
C17H25N3O5S,
383,5 g/mol
Company:
Sequoia

Ceftazidim
C22H22N6O7S2 5
H2O
637 g/mol
Company: Sigma

Imipenem
C12H17N3O4S
299,35 g/mol
Company:
Sequoia

Cefepime
C19H24N6O5S2
480.56 g/mol

Ertapenem
C22H25N3O7S
475,5 g/mol
Company:
Sequoia

Ticarcillin
C15H16N2O6S2
384.429
g/mol
Company:
Sigma

OmpC porin expresion mutants[8]

Control permeability of <600 kDa hydrophilic compounds
Porin alteration in clinical isolates: omp35, omp36, omp37
Reduced porin expression less -lactam susceptibility
E. coli BZB1107 and W3110, transformed with AmpR pTrc99A plasmid

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Uptake quantification
-lactam antibiotics within the cell

a)

b)

Source: http://watcut.uwaterloo.ca/webnotes/Pharmacology/microbesBacterialCellWall.html
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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Uptake quantification
General high-throughput workflow
Cell growth in LB medium + amp/carb up to (OD 600 = 0.6). Centrifugation and wash. Resuspension
with 0.9% NaCl in 1/10 of the initial volume

600 L bacteria (OD600 = 6.0) + 50 L antibiotic and -lactamase inhibitors

Incubation time = 10 min

400 L transferred to 550L of sucrose

Sucrose-cushion centrifugation 15 min, supernatant discarded

400 L of solvent for cell disruption

200 L of liquid to be vaporized

5
6

Resuspension in 50 L of MS-buffer

Source: http://www.agilent.com/en-us/products/automation-solutions/automated-liquid-handling/bravo-automated-liquid-handling-platform
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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Uptake quantification
Disruption solvent comparison
100000

10000

Antibiotic concentration (ng/mL)

1000

Ticarcillin
Cefepime
Ertapenem
Ceftazidim
Meropene
m

100

10

1
ACN

ACN + 0.1% FA
MeOH
Disruption Solvent

MeOH + ACN

MeOH + Ace

High standard deviation due to the robot? Or due to the assay itself?

Uptake quantification
Manual vs Robot-assisted liquid handling
120

Manual: apparent lower

deviation

100
80
MS concentration (ng/mL)

60
Manual
Robot

40

ANOVA: 0 min not

significant

20
0
0 2 5 8 10 12 15
Incubation time (min)
Disruption solvent: ACN

a) Is the sucrose cushion long enough for the uptake to take place?
b) No uptake takes place and just remaining antibiotic is measured?

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Uptake quantification
Short sucrose-cushion centrifugation time
100
90

Long centrifugation: 15 min, 2 250xg

80
70
60

Short centrifugation: 1 min, 10 000xg

0 min

50
MS concentration (ng/mL)
40

10 min

30
20
10

800

0
Long

Short

Disruption solvent: ACN

700
600
Ms concentration (ng/mL)
500
400
300
200
100
0
ACN

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

0.9% NaCl + ACN

MeOH

MeOH + ACN

Uptake quantification
Negative control (fast test)

Living cells

Cell debris

No cells

350
300
250
200
150
100
50
0

MS Concentration (ng/mL)

100%

Same
initial
OD600

Same
initial
OD600

8%

26%

Just liquid
Disruption solvent: ACN + 0.1% formic acid

However, same pellet volume is not a guarantee

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Uptake quantification
Antibiotic mass balance
0)

1)

2)

-lactamase
inhibitors:
-Tazobactam
-Clavulanate
uptake
fraction

4)

3)

sucrose-cushion
loss

5)

disruption
fraction

With robot:

V: volume (L), C: concentration (g/mL), m: mass (mg)

: amount of bacteria (mg or number of cells)
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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

6)

Uptake quantification
Packed pellet volume
Packed cell volume measurement (tube with a

scale)
Losses in pellet volume due to sucrose cushion
Before and after incubation
After sucrose cushion

Sample description

Pellet volume
recovery

400 mL control and sucrose cushion in Eppendorf

tube.
Pellet resuspended in 400 mL (1 min, 10,000xg)

81 %

400 mL of incubated bacteria and sucrose cushion

in Eppendorf tube. Pellet resuspended in 400 mL (1
min, 10,000xg)

81%

Sucrose in TPP, add 400 mL of control

(centrifuged 15 min, 2250xg)

81%

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Up to 1 mL

Up to 5 L

TPP PCV tube

Uptake quantification
Overall uptake fraction
=1
=0

Total uptake and total

release
a) No uptake
b) No release
c) Both

0.0250

Based only on antibiotic

0.0200

quantification
Losses in pellet volume

1.8%

0.0150

uf

due to sucrose cushion

0 min
10 min

0.0100
0.0050
0.0000
ACN 0.9% NaCl + ACN MeOH
Disruption solvent

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

MeOH + ACN

Uptake quantification
Dry weight
1.4

Culture in LB medium
Centrifugation and

resuspension in 0.9% NaCl

Adjusted OD600
Dried overnight at 80C

1.2
1
Dry weight (mg)
0.8

E
m
pt
y

0.6
0.4
0.2
0
3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8
OD600

Only able to correlate initial OD600 (before incubation)

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Uptake quantification
Control corrected uptake
1400

MS concentration (ng/mL)

1200

Negative control:

1000
800

Emp
ty

600
400

Dead cells = cell debris

omp
35

200
0

0 min 15 min
Incubation time

80
70

Antibiotic uptake (ng/mgdryweigth)

Other authors:
Report mgantibiotic /mgdry weight[9]
Control at 0C
Show steady-state ~15-20 min
[10]

60
50
Empty

40

omp35
omp36

30

omp37

20
10
0
0 min
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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

15 min
Incubation time

Uptake quantification
No-uptake scenario
0)

1)

2)

-lactamase
inhibitors:
-Tazobactam
-Clavulanate
uptake
fraction

3)

liquid fraction
sucrose-cushion
loss

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Uptake quantification
No-uptake scenario

Packing
factor
Eppendorf

<<

Packing
factor TPP

500
450

For a TPP tube:

MS concentration (ng/mL)

400
350

349.59

300
250
200

154.83

150

(absurd)
Expected MS concentration : 166 ng/mL

100
50
0

Eppendorf
Format

Expected MS concentration : 83 ng/mL

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

TPP

Outlook
Polishing the method for a precise uptake quantification
Losses might be correlated by an internal
(after/before sucrose cushion or cell disruption)

standard

Disruption fraction estimated from optimal conditions (MeOH +

sonification + ultrasonic bath)
Possibility to measure the inactive form of -lactams
Determination of bacterial mass at the end, not only at the
beginng of the process
Hands on with metabolics procedures and data analysis

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

References
1.
2.

EDDC & EMEA (2009). The bacterial challenge: time to react. Technical Report.
Anderson, Rosaleen, et al. Antibacterial agents: chemistry, mode of action, mechanisms of resistance
and clinical applications. John Wiley & Sons, 2012.
3. Winterhalter, M., Ceccarelli, M., Physical methods to quantify small antibiotic molecules uptake into
Gram-negative bacteria, European Journal of Pharmaceutics and Biopharmaceutics, Volume 95, Part
A, September 2015, Pages 63-67, ISSN 0939-6411
4. Davis, T.D., C.J. Gerry, and D.S. Tan, General platform for systematic quantitative evaluation of smallmolecule permeability in bacteria. ACS Chem Biol, 2014. 9(11): p. 2535-44.
5. Dorries, K., R. Schlueter, and M. Lalk, Impact of antibiotics with various target sites on the
metabolome of Staphylococcus aureus. Antimicrob Agents Chemother, 2014. 58(12): p. 7151-63.
6. Allen, J., et al., Discrimination of modes of action of antifungal substances by use of metabolic
footprinting. Appl Environ Microbiol, 2004. 70(10): p. 6157-65.
7. Vincent IM, Ehmann DE, Mills SD, Perros M, Barrett MP. Untargeted Metabolomics To Ascertain
Antibiotic Modes of Action. Antimicrobial Agents and Chemotherapy. (2016) 60(4):2281-2291.
8. Pags, Jean-Marie, Chlo E. James, and Mathias Winterhalter. "The porin and the permeating
antibiotic: a selective diffusion barrier in Gram-negative bacteria." Nature Reviews Microbiology 6.12
(2008): 893-903
9. K J Williams and L J Piddock. Accumulation of rifampicin by Escherichia coli and Staphylococcus
aureus. J. Antimicrob. Chemother. (1998) 42 (5): 597-603
10. Cai, H., et al., Development of a liquid chromatography/mass spectrometry-based drug accumulation
assay in Pseudomonas aeruginosa. Anal Biochem, 2009. 385(2): p. 321-5.

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Extra
Washing steps
MEROPENEM MS concentration (ng/mL)
Wash step after
sucrose cushion

No wash step

Living cells

Wash step before

and after sucrose
cushion

Wash step before

sucrose cushion

A1

282.0542

B1

1.7304

C1

0.6691

D1

0.5431

A1

277.7709

B1

1.5922

C1

0.7310

D1

0.4350

A2

339.4214

B2

2.9545

C2

1.0990

D2

0.8677

A2

335.5864

B2

3.0770

C2

1.0897

D2

0.7823

A3

323.5455

B3

2.8641

C3

0.6542

D3

0.4777

A3

324.2513

B3

3.0498

C3

0.8577

D3

0.4762

Average

313.7716

2.5447

0.8501

0.5970

StdDev

26.9859

0.6897

0.2024

0.1820

%RelDev

9%

27%

24%

30%

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Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

Extra
Washing steps
100
Antibiotic uptake (ngantibiotic/mgbacteria)

8.48

10

48.46

46.66

8.98

3.63
No wash
1.56
1

0.1

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0.20

0.28

Antibiotic uptake in P. aeruginosa, and its consequences on the primary metabolome

0.12

1.38

Wash