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Journal of Microbiological Methods 93 (2013) 80–84
Contents lists available at SciVerse ScienceDirect
Journal of Microbiological Methods
journal homepage: www.elsevier.com/locate/jmicmeth
Antibiotic-induced modifications of the stiffness of bacterial membranes
Giovanni Longo a,⁎, Laura Marques Rio b, Andrej Trampuz b, Giovanni Dietler a, Alain Bizzini c, Sandor Kasas a
a
b
c
Laboratory of Physics of Living Matter, EPFL, CH-1015 Lausanne, Switzerland
Infectious Diseases Service, Department of Medicine, University Hospital Lausanne (CHUV), CH-1011 Lausanne, Switzerland
Institute of Microbiology, University Hospital Lausanne (CHUV), CH-1011 Lausanne, Switzerland
a r t i c l e
i n f o
Article history:
Received 5 December 2012
Received in revised form 31 January 2013
Accepted 31 January 2013
Available online 22 February 2013
Keywords:
AFM
Antibiotics
E. coli
Force volume
Mechanical properties
Nucleoids
a b s t r a c t
In the latest years the importance of high resolution analysis of the microbial cell surface has been increasingly
recognized. Indeed, in order to better understand bacterial physiology and achieve rapid diagnostic and treatment
techniques, a thorough investigation of the surface modifications induced on bacteria by different environmental
conditions or drugs is essential.
Several instruments are nowadays available to observe at high resolution specific properties of microscopic samples.
Among these, AFM can routinely study single cells in physiological conditions, measuring the mechanical properties
of their membrane at a nanometric scale (force volume). Such analyses, coupled with high resolution investigation
of their morphological properties, are increasingly used to characterize the state of single cells.
In this work we exploit such technique to characterize bacterial systems. We have performed an analysis of
the mechanical properties of bacteria (Escherichia coli) exposed to different conditions. Such measurements
were performed on living bacteria, by changing in real-time the liquid environment: standard phosphate
buffered saline, antibiotic (ampicillin) in PBS and growth medium. In particular we have focused on the determination of the membrane stiffness modifications induced by these solutions, in particular between stationary and
replicating phases and what is the effect of the antibiotic on the bacterial structure.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Infections due to multidrug-resistant bacteria are a major public
health problem, causing increasing mortality and morbidity worldwide.
One of the most important tasks of the clinical microbiology laboratory
is the identification and characterization of pathogens in samples as
well as the realization of their antibiogram. As a whole, the majority
of the most common characterization techniques are based on the
assumption that the tested organisms represent a homogenous population in relation to their physiological and physical properties (Lay, 2001;
Poppert et al., 2005; Turnidge and Paterson, 2007). For instance, only
few works focus on the determination of the mechanical properties
of single bacterial membranes in order to highlight variations occurring
on single living bacteria (Alsteens et al., 2009; Dufrêne, 2008). In view of
this, modern bacteriology is increasingly demanding a more in-depth
study of bacterial characteristics, abandoning the overall population
analyses and leaning towards the single-cell level (Brehm-Stecher and
Johnson, 2004).
Nowadays, several instruments are available to characterize
specific physiologically-related properties of living microscopic samples.
Abbreviations: PDMS, polydimethylsiloxane; PBS, phosphate buffered saline; LB,
lysogeny broth; APTES, 3-aminopropyltriethoxysilane; FD, force distance; FV, force volume.
⁎ Corresponding author at: EPFL-IPSB-LPMV, BSP/Cubotron 414, CH-1015 Lausanne,
Switzerland. Tel.: +41 21 693 4521; fax: +41 21 693 04 22.
E-mail address: giovanni.longo@epfl.ch (G. Longo).
0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.mimet.2013.01.022
Among these, Atomic Force Microscopy (AFM) (Binnig et al., 1986)
stands out as the instrument of choice to perform high resolution imaging
coupled with a point-by-point characterization of the mechanical and
adhesive properties of micro and nanosized samples. Moreover, such
measurements can be performed seamlessly in physiological conditions
and in presence of different liquid media. This versatility is reflected in
the AFM literature which presents with increasing frequency novel results regarding mechanical properties of single cells, bacteria or even
single molecules (Berquand et al., 2010; Kasas and Dietler, 2008). At a
cellular scale, it has been demonstrated that the mechanical properties
of the membrane can reflect the physiological state of the entire system
and that these properties are clearly altered in the occurrence of pathological conditions (Cross et al., 2007; Girasole et al., 2010; Lekka et al.,
2005). It seems reasonable to extrapolate these observations to bacterial
cells, in order to track a possible correlation between their morphological
and mechanical characteristics and their state or pathogenic potential
(Gaboriaud et al., 2008; Miklossy et al., 2008; Rossetto et al., 2007).
Unfortunately, most of the works currently present in literature
exploit only partially the capabilities of the AFM of characterizing living
specimens in physiological buffer. In fact, most papers focus only on the
morphological modifications induced by antibiotics, imaging in
very-high detail the dehydrated membranes of the bacteria (Cui et al.,
2012; Dubrovin et al., 2012; Hyldgaard et al., 2012). The few groups
that investigate the stiffness of the living bacteria have, up to now,
prepared samples using bacteria exposed to different environmental
conditions and have only compared the average stiffness values of
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G. Longo et al. / Journal of Microbiological Methods 93 (2013) 80–84
the different samples (Formosa et al., 2012; Su et al., 2012). This is
somewhat limiting, since the investigation of mean stiffness values
does not allow characterizing more complex mechanical modifications
induced by the antibiotic on the bacterial membrane. Moreover, since
AFM is principally a single-cell technique, in order to exploit this
strength it should be used to study both the morphology and the mechanical properties of one single living microorganism with a high
spatial resolution and while changing its environmental conditions.
Indeed, in this work we present an AFM-based morphological and mechanical investigation of bacteria (specifically the Gram-negative bacilli
Escherichia coli) during their exposure to different environmental conditions. In particular we will highlight the modifications that arise
when these microorganisms are treated with an antibiotic (ampicillin).
2. Materials and methods
2.1. AFM and liquid cell
The mechanical properties were investigated using a Bioscope I
AFM (Bruker AXS), mounted on top of an inverted microscope. The
microscope is equipped with a custom polydimethylsiloxane (PDMS)
sample holder, capable to maintain the sample under investigation in
a controlled environment. This device is coupled with a liquid changing
system capable of replacing the incubation medium during the measurements, with virtually no effect on the AFM acquisition (Fig. 1) (Kasas et al.,
2010).
All the images were collected using Bruker DNP-10 cantilevers,
choosing the tip with a nominal spring constant of 0.06 N/m. The mechanical investigations have been performed by indenting the AFM
tip into the sample and by measuring the cantilever deflection during
the process. The resulting curve, called force distance (FD) curve, delivers high-resolution information regarding the stiffness and adhesion
of the analyzed area. Importantly, the AFM can perform such measurements in virtually any kind of environment, and in particular in different
liquid media, allowing the observation of biological processes in nearly
physiological conditions.
The images presented in this work were collected in force volume
(FV) mode, a setup in which the AFM tip is periodically indented into
the sample and the deformation of the cantilever is recorded to reconstruct an image formed by a large number of FD curves. All the
presented images contain 32× 32 pixels and, for every pixel, 256 points
per force curve were collected. The cantilever speed for all the
presented images was of 0.1 lines per second, i.e. around 5 min per
image. For each curve, the tip–sample interaction was limited to a deflection maximum of 30 nm (~2 nN).
The FV data files were recorded on several bacteria (more than 50
individual cells were imaged) and analyzed assuming that the cantilever
Fig. 1. The Bruker Bioscope I equipped with the custom PDMS sample holder and coupled
with the liquid changing system.
81
behaves accordingly to the Hook law (i.e. the deflection of the cantilever
is directly proportional to the vertical component of the force applied
onto the tip). In this case the FD curves collected on the sample can be
subtracted from FD curves obtained onto a hard surface. The resulting
curves are called indentation curves, indicating the force needed to
push the tip for a defined length into the sample (i.e. to indent it). The
shape of each indentation curve is then used to calculate the mechanical
properties of the sample (specifically the stiffness) (Cerf et al., 2009;
Polyakov et al., 2011; Rossetto et al., 2007). The data analysis was
performed in a fully automated fashion by using OpenFovea, a free data
processing software that uses the Hertz model to reconstruct stiffness
maps of the entire image (Roduit et al., 2012). For the calculations we assumed that the tip is spherical with an apical radius of 20 nm and that the
sample's Poisson ratio is 0.5. Moreover, some of the FD curves have been
processed using the software in stiffness tomography mode, an innovative feature that allows revealing the mechanical differences that the
AFM tip encounters during its indentation path, delivering cross section
images of the sample's mechanical properties (Roduit et al., 2009).
2.2. Bacteria preparation
The bacteria investigated in this work are E. coli, Gram-negative
rod-like shaped bacteria (typically 500 nm wide and several microns
long). More precisely, we chose to study E. coli strain DH5α because it is
a well-known laboratory strain that is susceptible to ampicillin. Frozen
stocks of E. coli were kept at −80 °C in LB media supplemented with
glycerol. For the experiments, bacteria from stock were streaked on LB
agar media. Then, a few bacterial colonies were collected from the
agar plate and incubated overnight at 37 °C in LB. After incubation,
the bacteria were washed three times for 10 min at 5000 rpm,
resuspended in phosphate buffer saline (PBS, pH 7.4) and deposited
on a 12 mm PDMS substrate for AFM investigation. To ensure a
firm adhesion of the living bacteria on the substrate, the substrates
were preliminarily coated with a linker molecule, (3-aminopropyl)
triethoxysilane (APTES), a highly effective silane coupling agent
used on a wide series of substrates to enhance cell adhesion. First
the clean substrates were exposed for 1.5 min to 0.2% APTES and
rinsed in ultrapure water. Next, the bacteria were left to incubate on
the coated substrates for 15 min before thorough rinsing to remove
loosely adhering cells. The samples were finally placed in 0.5 ml PBS
in the liquid cell holder for AFM investigation. During the investigation,
and without interrupting the measurements, the medium was changed,
Fig. 2. 8 × 8 μm, 32 × 32 pixels, 3D reconstruction of the topography of E. coli bacteria
immobilized on the substrate and imaged in PBS. Inset: force information of the very
same area, showing how the different bacteria appear to have uniform stiffness.
Well-defined stiffer structures can be seen on all the bacteria. (For interpretation of the
references to color in this figure, the reader is referred to the web version of this article.)
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first with 0.5 ml of 4.5 mg/ml ampicillin in PBS and, in a second step,
with 0.5 ml LB.
3. Results and discussion
As said, the experiment was divided in three steps. First, the bacteria
were imaged in PBS, a buffer medium without nutrients. This induces a
stationary state reducing the bacterial movement and the ability to
replicate (Tortora et al., 2009). Secondly, the liquid changer system
was used to introduce ampicillin diluted in PBS. This is an antibiotic of
the β-lactam class that interferes with the formation of peptidoglycan,
which is an essential component of the bacterial cell wall and whose
degradation ultimately causes cell lysis and bacterial death (Rodionov
and Ishiguro, 1995). Finally the liquid medium was changed with LB
to verify if the membrane damage could be recovered by the bacteria.
An 8 × 8 μm, 3D reconstruction of the topography of the specimen,
obtained by performing force volume imaging of the sample immersed
in PBS, is depicted in Fig. 2. Several bacteria can be seen attached to
the substrate. The corresponding stiffness is shown in the inset of
Fig. 2, showing the soft (blue) bacteria attached to a much stiffer
(white = no indentation) substrate. Such more wide images were
collected to confirm that all the bacteria in the area had uniform stiffness values. The uniformity of the results was verified by investigating
minimally 3 areas for each sample and by comparing the results
obtained in 10 different and independent preparations. The total number
of bacteria analyzed in this lower resolution exceeded 50 cells.
In order to increase the obtainable resolution, for each sample
preparation we concentrated on a single E. coli cell, in this way we were
able to increase the number of force curves collected on the bacterial
membrane and the time needed to perform the measurements. We
followed the evolution of the topography and membrane stiffness of
these particular microorganisms while changing the media. The
presented results were consistent in all the tested samples and the
presented results well-represent all the experimental evidence.
Fig. 3A depicts the topography of the chosen bacterium in PBS environment (5× 5 μm, Fig. 3A), while Fig. 3B shows the respective stiffness
Fig. 3. Panel A: 5×5 μm, 32×32 pixel topography image of a single E. coli immobilized on the substrate and imaged in PBS. Panel B: corresponding stiffness image evidencing that the mechanical properties are uniform throughout most of the bacterial membrane, except two well-defined stiffer areas. Panel C: histogram of the stiffness values obtained from several images collected on
the very same area. Two curves are evidenced: in red the membrane contribution and in black the peak of the substrate stiffness. Panel D: 5×5 μm, 32×32 pixel topography image of the very
same E. coli imaged after introduction of ampicillin in PBS. Panel E: corresponding stiffness image, showing a reduction of the overall stiffness of the two stiffer areas and of the substrate.
Panel F: histogram of the stiffness values obtained from several images collected on the very same area in the same conditions. The substrate peak has moved towards lower stiffness values.
Panel G: 5×5 μm, 32×32 pixel topography image of the same E. coli, deflated after introduction of LB. Panel H: corresponding stiffness image, showing the disappearance of one of the two stiff
areas and a reduction in stiffness of the other. Panel I: histogram of the stiffness values obtained from several images collected in the same conditions on the very same area. The membrane peak
indicated a dramatic reduction of the stiffness of the entire membrane. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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G. Longo et al. / Journal of Microbiological Methods 93 (2013) 80–84
values collected for each point. Several subsequent images, collected
over a 4 h period (for instance, Fig. 4A shows the same bacterium in
the same environmental conditions 1 h later), indicate that the membrane stiffness is stable in time, with only small stiffness variations,
probably due to the internal metabolism. To better elaborate and
compare the stiffness information from all the collected images, all
the data was collected in a histogram, shown in Fig. 3C. This allowed
discriminating two well-defined stiffness peaks, one centered at
3 MPa that can be attributed to the typical stiffness of the substrate
and the second at 300 ± 70 kPa, associated with the stiffness of the
E. coli membrane. Indeed, such values are in good agreement with
those obtained by similar studies on the same kind of bacteria,
confirming the attribution (Chen et al., 2009). To better compare
these results with the ones collected in different environmental conditions we have highlighted these two peaks in red (bacteria) and in
black (substrate).
On the bacterial surface, two stiffer areas could be identified (red
and yellow ovals marked B and D in Fig. 4A). These features were
present on all the analyzed bacteria, and their complex structure is
83
evidenced in the higher resolution, 1 × 1 μm, images (Fig. 4B and D,
zooms of the higher and lower structures respectively). A complete
characterization of such structures is still underway, but the most
probable origin of such features is the presence of agglomerates of
stiffer internal cell components (mainly DNA), inside the cytoplasm
(nucleoids) (Frenkiel-Krispin et al., 2004; Longo et al., 2012). To better
characterize such areas, force tomography analyses were performed.
Several force tomography sections were calculated from the data
collected in Fig. 4B (section C corresponding to Fig. 4C) and d (section
E corresponding to Fig. 4E), showing that in these areas not only the
membrane is stiffer than the rest of the bacteria, but that stiffer features
were present also inside the bacterial body.
Upon exposure to ampicillin, while the overall topographical
structure appeared unaffected (Fig. 3D), the force volume images
showed several modifications of the mechanical properties (Fig. 3E).
While the softer areas of the membrane were unchanged, the two
nucleoid areas evidenced a reduction of their stiffness. At the same
time the entire substrate appeared to be coated with softer material,
indicated by the reduction of its overall stiffness. This was confirmed
Fig. 4. Panel A: 5 × 5 μm, 32 × 32 pixel stiffness image of the same E. coli shown in Fig. 3. Two circles (B and D) highlight the two stiffer areas. Panel B: 1 × 1 μm FV zoom-in image of
the nucleoid labeled B in Fig. 4A. A complex structure can be seen. Panel C: Force tomography section obtained over the line marked C in Fig. 4B. Stiffer structures can be seen under
the membrane level. Panel D: 1 × 1 μm FV zoom-in image of the nucleoid labeled D in Fig. 4A. Panel E: Force tomography section obtained over the line marked E in Fig. 4D. (For
interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
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G. Longo et al. / Journal of Microbiological Methods 93 (2013) 80–84
by comparing the histograms of the stiffness values shown in Fig. 3C
and f: the peak associated with the membrane stiffness did not move
significantly, while the peak of the substrate fell to values around
2.3 MPa. The most plausible interpretation of these data is that the
ampicillin, interfering with the formation of peptidoglycan, indirectly
caused the formation of membrane lyses and a consequent leak of
some of the internal soft bacterial content on the substrate. Since
the effect of the ampicillin is maximized when associated with a nutrient
medium, this effect was greatly reduced in PBS environment.
Indeed, replacing the medium with LB caused, in a timespan much
smaller than that of a typical image, much more dramatic changes:
the topography image showed that the bacterium deflated (Fig. 3G)
while the corresponding stiffness image (Fig. 3F) indicated that both
the bacterial membrane and the material present on the substrate
had become much softer. This is in agreement with the interpretation
of the phenomena, indicating that most of the material present inside
the bacteria had probably flowed out, probably due to osmotic pressure,
through the lysis of the membrane (Fig. 3F). Also the two nucleoid
areas underwent major changes: while the smaller one had completely
disappeared (probably the stiffer material had entirely leaked on the
substrate), the other had reached stiffness values similar to those of
the rest of the membrane. These dramatic changes are evidenced in
the histogram shown in Fig. 3I. Both peaks appear to have moved towards lower stiffness values: the membrane peak is centered at values
below 100 kPa while the substrate component is still centered at
2.3 MPa but with a much smaller spread.
4. Conclusions
We have investigated the mechanical properties of the membrane
stiffness of E. coli exposed to different environmental conditions. Such
measurements were performed by force volume investigation delivering,
with an unprecedented high resolution, a map of the mechanical properties of the bacterial membrane. We monitored the stiffness values of a
single bacterial cell when exposed to different environmental conditions,
including the exposure to antibiotics.
Such analyses have evidenced the presence on the bacterial surface
of two stiffer areas. By employing an innovative technique capable of revealing stiffer structures below the surface level, such areas have been
associated with the presence of features inside the bacterial body. It
can be speculated that these areas correspond to nucleoids, i.e. areas
where DNA or other proteins agglomerate.
The AFM measurements in liquid were performed using a custom
liquid cell that allows changing the growth medium during the imaging
with no effect on the measurements. This very stable system has been
exploited in order to change the incubation liquid, introducing antibiotics
and other substances, while following the evolution of the bacterial
topography and stiffness. This new versatile setup opens the way to
an all new set of AFM investigations regarding the response of living
systems to environmental stimuli.
In conclusion we have demonstrated how force volume imaging
can be an extremely powerful tool to monitor the surface stiffness
of living systems when exposed to different growing media. We
have shown that surface modifications occur on E. coli when passing
from a stationary to an active state and we have revealed the effect of
the exposure to ampicillin on the bacterial structure. This procedure
to analyze the mechanical properties of living systems in presence of
different media can lead to novel techniques capable to rapidly determine the effect of external stimuli on key cell properties.
Acknowledgments
This work has been supported by Swiss ISJRP 122941 and FN-CR
32I3-130676 grants.
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