Journal of Antimicrobial Chemotherapy (2006) 58, 444–448

doi:10.1093/jac/dkl225
Advance Access publication 30 May 2006

Mechanisms of the post-antibiotic effects induced by rifampicin

and gentamicin in Escherichia coli

William Stubbings, Julieanne Bostock, Eileen Ingham and Ian Chopra*

Antimicrobial Research Centre and Research Institute of Molecular and Cellular Biology,
University of Leeds, Leeds LS2 9JT, UK

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Received 7 November 2005; returned 28 February 2006; revised 8 March 2006; accepted 9 May 2006

Objectives: The mechanisms by which antibiotics induce a post-antibiotic effect in susceptible bacteria are
poorly understood. To explore the mechanisms more fully we examined the recovery of macromolecular
synthesis in Escherichia coli during gentamicin- and rifampicin-induced post-antibiotic effects.
Methods: E. coli ATCC 25922 was exposed to rifampicin and to gentamicin at 5· MIC for 60 min to induce
post-antibiotic effects. The antibiotics were then removed from the culture medium by washing the cells.
The rates of DNA, RNA and protein synthesis during the post-antibiotic effect and recovery periods were
subsequently determined by measuring the incorporation of radiolabelled uridine, thymidine and
leucine into trichloroacetic acid precipitable material.
Results: Recovery of E. coli ATCC 25922 from the rifampicin-induced post-antibiotic effect coincided with
the recovery of RNA and protein synthesis. Recovery from the gentamicin-induced post-antibiotic effect
coincided with the recovery of protein synthesis.
Conclusions: These data support the hypothesis that antibiotic molecules retained in the cell mediate the
post-antibiotic effect by suppressing the biochemical activity of their molecular targets.

Keywords: E. coli, PAE, macromolecular synthesis, antibiotic action

Introduction depends upon re-establishment of protein synthesis,3 and recov-

ery from the ciprofloxacin-induced post-antibiotic effect depends
The continued suppression of bacterial growth following limited upon restoration of DNA synthesis.4 To explore post-antibiotic
exposure to an antimicrobial agent was first noted nearly 60 years effect mechanisms more fully we have chosen two further anti-
ago.1 The term post-antibiotic effect has now become the accep- biotics, rifampicin and gentamicin, to examine whether recovery
ted description of this phenomenon which results from prior from the post-antibiotic effect induced by these agents also depends
exposure of organisms to an antibiotic, rather than persistence on recovery of the processes targeted by these antibiotics, namely
of sub-minimal inhibitory concentrations (sub-MICs) of drugs in RNA synthesis for rifampicin and protein synthesis for gentamicin.
the medium.1 Determination of the post-antibiotic effect is now The results reported here are consistent with our earlier hypo-
an important part of preclinical evaluation of new antibiotics thesis that resumption of growth after the post-antibiotic effect
because it is a factor that influences antibiotic dosing intervals.2 period reflects the time taken for antimicrobial agents to disso-
However, relatively little is known about the molecular basis of ciate from their targets and be lost from the cell, releasing
the post-antibiotic effect or the events that lead to restoration of sufficient target molecules for growth to resume.5
normal growth at the end of the post-antibiotic effect.1
Inhibitors of protein and nucleic acid synthesis including
aminoglycosides, tetracycline, macrolides, fluoroquinolones and Materials and methods
rifampicin induce lengthy post-antibiotic effects in Escherichia
coli.1 In the few cases examined, recovery from the post-antibi- Bacterial strain and growth medium
otic effect correlates with re-establishment of the process targeted E. coli ATCC 25922 was obtained from the American Type Culture
by the inducing antibiotic. Thus, recovery from the post-antibiotic Collection (ATCC) and was grown in M9 minimal salts medium6
effect induced by the aminoglycoside tobramycin in E. coli supplemented with thiamine (2 mg/L).
.............................................................................................................................................................................................................................................................................................................................................................................................................................

*Corresponding author. Tel: +44-113-343-5604; Fax: +44-113-343-5638; E-mail: i.chopra@leeds.ac.uk

.............................................................................................................................................................................................................................................................................................................................................................................................................................

444

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 Mechanisms of the post-antibiotic effects induced by rifampicin and gentamicin

(a) 6000 0.25

Uridine incorporation (dpm/OD × 10–3)

4800
0.2

OD (600 nm)
3600

0.15
2400

0.1
1200

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0 0.05
–1 1 3 5 7 9
Time (h)

(b) 600 0.25

Thymidine incorporation (dpm/OD × 10–3)

500
0.2
400

OD (600 nm)
300 0.15

200
0.1
100

0 0.05
–1 1 3 5 7 9
Time (h)

(c) 39 0.25
Leucine incorporation (dpm/OD × 10–6)

31
0.2
OD (600 nm)

23

0.15

15

0.1
7

–1 0.05
–1 1 3 5 7 9
Time (h)

Figure 1. Rates of synthesis (squares) of RNA (uridine) (a), DNA (thymidine) (b) and protein (leucine) (c) in E. coli ATCC 25922 during the post-antibiotic effect
induced by exposure to 5· MIC of rifampicin. Culture OD (diamonds) is displayed during both the post-antibiotic effect and re-growth periods. Control rates of
synthesis (triangles) prior to drug exposure were determined by measuring incorporation of precursors into unexposed control cultures. Values displayed are mean (n =
3) determinations. Error bars represent standard deviations calculated for these readings.

445
 Stubbings et al.

(a) 6 0.5

Uridine incorporation (dpm/OD × 10−6)

5 0.45

4 0.4

OD (600 nm)
3 0.35

2 0.3

1 0.25

0 0.2
−1 0 1 2 3 4 5 6 7
Time (h)
(b) 700 0.45

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650
Thymidine incorporation (dpm/OD × 10−3)

600
550 0.4
500
450

OD (600 nm)
0.35
400
350
300
0.3
250
200
150 0.25
100
50
0 0.2
−1 0 1 2 3 4 5 6 7
Time (h)
(c) 130 0.45
120
110
Leucine incorporation (dpm/OD × 10−6)

0.4
100
90
80
OD (600 nm)
0.35
70
60
50 0.3

40
30
0.25
20
10
0 0.2
−1 1 3 5 7
Time (h)

Figure 2. Rates of synthesis (squares) of RNA (uridine) (a), DNA (thymidine) (b) and protein (leucine) (c) in E. coli ATCC 25922 during the post-antibiotic effect
induced by exposure to 5· MIC of gentamicin. Culture OD (diamonds) is displayed during both the post-antibiotic effect and re-growth periods. Control rates of
synthesis (triangles) prior to exposure were determined by measuring incorporation of precursors into unexposed control cultures. Values displayed are mean (n = 3)
determinations. Error bars represent standard deviations calculated for these readings.

Determination of susceptibility of E. coli ATCC 25922 to

Antibiotics, chemicals and radiochemicals
Antibiotics and chemicals were obtained from standard commercial
sources. The following radiochemicals were purchased from Amer-
sham Life Sciences, Little Chalfont, UK: [Me-3H]thymidine (70
–95 Ci/mmol), [5,6-3H]uridine (31–56 Ci/mmol) and L-[4,5-3H]leu-
cine (120–190 Ci/mol).
antimicrobial agents and induction of the post-antibiotic
effect
MICs were determined by broth microdilution in supplemented M9
minimal salts medium using an inoculum of 104 cells/mL in a final
volume of 70 mL. Microtitre plates (384 wells) containing triplicate

446
 Mechanisms of the post-antibiotic effects induced by rifampicin and gentamicin
2-fold dilution series were incubated for 16 h at 37 C in a Spec-
tramax 384 plus microtitre plate reader (Molecular Devices, Abing-
don, Oxfordshire, UK), running SOFTmax PRO 3.1.1 software.
Optical density readings (600 nm) were taken at 10 min intervals.
Plates were shaken for 30 s before each reading. The MIC was taken
as the lowest concentration of antibiotic that prevented growth in the
triplicate wells. Susceptibility determinations were performed on
three separate occasions and the mean of these values recorded as
the MIC.
Post-antibiotic effects were induced by exposing bacteria (2 · 108
cells/mL) in the early logarithmic phase to 5· MIC of each drug for
60 min, followed by washout and re-suspension of bacteria in fresh
growth medium at 37 C as described previously.7
Incorporation of radiolabelled precursors into
macromolecules
E. coli ATCC 25922 was grown to early logarithmic phase (2 · 10 8 and other bacteria during the post-antibiotic effect and the imme-
cells/mL) in M9 minimal salts media and then exposed to rifampicin
and gentamicin at 5· MIC for 60 min. The cultures were then washed
three times in fresh M9 medium (pre-warmed to 37 C) to remove
antibiotics. Samples were taken before the addition of antibiotics and
periodically during the post-antibiotic effect and recovery periods to
establish by pulse-labelling the rates of macromolecular synthesis
under the various conditions. For pulse-labelling, samples of culture
(450 mL) were added to tubes containing 2 mCi of [Me-3H]thymidine
(DNA synthesis), [5,6-3H]uridine (RNA synthesis) or L-[4,5-3H]-
leucine (protein synthesis) followed by incubation at 37 C in an
orbital waterbath for 5 min at 200 rpm. Ice-cold trichloroacetic
acid (TCA) (10% w/v) was then added to the tubes, which were
left on ice for 30 min. The TCA-precipitable material was collected
on 25 mm GF/C glass microfibre filters which were then washed
twice with 10% w/v TCA (5 mL) and 1% v/v acetic acid (5 mL).
Filters were dried overnight and then placed in vials containing 5 mL
of OptiPhase ‘Hisafe’ 2 liquid scintillation cocktail (Perkin Elmer,
Beaconsfield, Bucks, UK), before counting in a TRI-CARB-2100TR
liquid scintillation counter (Packard Biosciences, Berkshire, Pang-
bourne, UK). Macromolecular synthesis was expressed as disinteg-
rations per minute (dpm) incorporated per OD600 unit.4
Results
The MICs of rifampicin and gentamicin for E. coli ATCC 25922
were 4.0 and 0.5 mg/L, respectively. Measurement of culture
optical density at 600 nm (OD600) following removal of rifampi-
cin and gentamicin revealed that each antibiotic induced a post-
antibiotic effect of 4 h (Figures 1 and 2). Exposure to 5· MIC
of rifampicin for 60 min inhibited the rates of macromolecular
synthesis in each case by over 80% (Figure 1). RNA synthesis
remained at this inhibited level until 5 h, mirroring the stationary
state of the cells (Figure 1a). The rate of RNA synthesis returned
to control levels at 9 h. Protein synthesis followed a similar
pattern of recovery to RNA synthesis since inhibition occurred
throughout the post-antibiotic effect period and recovery began
between 5 and 6 h after removal of rifampicin (Figure 1c). In
contrast, DNA synthesis began to recover immediately after
rifampicin was removed from the external environment and
before the resumption of growth measured by the OD600
(Figure 1b).
Exposure to 5· MIC of gentamicin for 60 min had different
effects on the rates of DNA, RNA and protein synthesis.
RNA synthesis (Figure 2a) was inhibited by only 60% following
447
exposure to gentamicin. It remained inhibited for 1 h and then
began to recover before the onset of growth. DNA synthesis
(Figure 2b) was only inhibited by 30% at the beginning of the
gentamicin-induced post-antibiotic effect. However, 70–80%
inhibition of DNA synthesis was observed in the 1–2 h period
following removal of gentamicin (Figure 2b). DNA synthesis
began to recover between 2–3 h and, like RNA synthesis, before
the onset of growth measured by the OD600. Protein synthesis
(Figure 2c) was inhibited by >95% at the beginning of the gen-
tamicin-induced post-antibiotic effect but its recovery closely
mirrored the resumption of bacterial growth.
Discussion
Data which describe the molecular events that occur in E. coli
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diate recovery period are relatively limited and the effects of
rifampicin and gentamicin have not been reported previously.1
However, in those cases where responses to other antibiotics have
been examined, recovery from the post-antibiotic effect corres-
ponds to re-establishment of the process which is the primary
target of the inducing antibiotic.3,4,8,9
We found that recovery from the post-antibiotic effect induced
by rifampicin in E. coli 25922 correlated with resumption of both
RNA and protein synthesis. This is not surprising in view of the
connection between transcription and translation. Furthermore,
as the maximum half-life of mRNA in E. coli is 20 min,10
mRNA synthesized prior to the addition of rifampicin would
have been degraded by the time of antibiotic wash out (60 min).
Recovery of protein synthesis would therefore have been depend-
ent upon recovery of RNA synthesis, as observed.
Recovery of E. coli from gentamicin exposure was also con-
sistent with recovery of the process inhibited by the inducing
antibiotic. Inhibition of protein synthesis persisted throughout the
post-antibiotic effect and recovered rapidly as the cells emerged
from the post-antibiotic effect. These data are consistent with the
observations of Barmada et al.,3 who showed that recovery of
E. coli from the tobramycin-induced post-antibiotic effect
depended on the recovery of protein synthesis.
Data presented in the present study are consistent with other
published reports that the duration of the post-antibiotic effect is
related to the resumption of the biochemical process(es) inhibited
by the antibiotic that induced it. Furthermore, the observations
reported here support our hypothesis that continued interaction of
remaining antibiotic molecules with their intracellular targets is
responsible for the post-antibiotic effect, and that resumption of
growth after the post-antibiotic effect probably reflects the time
taken for antibiotics to dissociate from their targets and be
removed from the cell.5
Acknowledgements
W. S. was supported by a PhD research scholarship from the
University of Leeds.
Transparency declarations
None to declare.
 Stubbings et al.
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