Using an innovative device with microscopic chambers,
researchers from Johns Hopkins and three other
institutions have gleaned important new information about
how bacteria survive in hostile environments by forming
antibiotic-resistant communities called biofilms. These
biofilms play key roles in cystic fibrosis, urinary tract
infections and other illnesses, and the researchers say
their findings could help in the development of new
treatments and preventive measures.
"There is a perception that single-celled organisms are
asocial, but that is misguided," said Andre Levchenko,
assistant professor of
biomedical engineering in the Whiting School of
Engineering and an affiliate of the Institute for
NanoBioTechnology at Johns Hopkins. "When bacteria are
under stress — which is the story of their lives
— they team up and form this collective called a
biofilm. If you look at naturally occurring biofilms, they
have very complicated architecture. They are like cities
with channels for nutrients to go in and waste to go
out."
With a better understanding of how and why bacteria form
biofilms, researchers may be able to disrupt activity in
the bacterial communities and block the harmful effects on
their human hosts. The findings were detailed in an
article published in the November issue of the journal
Public Library of Science Biology.
In the article, the researchers from Johns Hopkins,
Virginia Tech, University of California, San Diego, and
Lund University in Sweden reported on the observation of
the bacteria E. coli growing in the cramped conditions of
a new microfluidic device. The device, which allows
scientists to use nanoscale volumes of cells in solution,
contains a series of tiny chambers of various shapes and
sizes that keep the bacteria uniformly suspended in a
culture medium.
Levchenko and his colleagues recorded the behavior of
single layers of cells using real-time microscopy.
Computational models validated their experimental results
and could predict the behavior of other bacterial species
under similar pressures. "We were surprised to find that
cells growing in chambers of all sorts of shapes gradually
organized themselves into highly regular structures,"
Levchenko said. "The computational model helped explain why
this was happening and how it might be used by the cells
to increase chances of survival."
The microfluidic device, which was designed and fabricated
in collaboration with Alex Groisman's laboratory at UCSD,
allows the cells to flow freely into and out of the
chambers. Test volumes in the chambers were in the
nanoliter range — allowing visualization of single E.
coli cells. Ann Stevens' laboratory at Virginia Tech
helped to generate new strains of bacteria that permitted
visualization of individual cells grown in a single
layer.
Hojung Cho, a biomedical engineering doctoral student from
Levchenko's lab and lead author of this study, captured on
video the gradual self-organization and eventual
construction of bacterial biofilms over a 24-hour period,
using real-time microscopy techniques. The experiments were
matched to a modeling analysis developed in collaboration
with Cho's colleagues at Lund. Images were analyzed using
tools developed with participation of Bruno Jedynak of the
Johns Hopkins Center for Imaging Science.
Observation using microscopy revealed that the longer the
packed cell population resided in the chambers, the more
ordered the biofilm structure became, Levchenko said. Being
highly packed in a tiny space can be very challenging for
cells, so that any type of a strategy to help colony
survival can be very important, he added.
Levchenko also noted that rod-shaped E. coli that were too
short or too long typically either did not organize well
or did not avoid "stampede"-like blockages toward the
exits. The shape of the confining space also strongly
affected the cell organization in a colony, with highly
disordered groups of cells found at sharp corners but not
in the circular-shaped microchambers.
Understanding how bacteria produce biofilms is important to
researchers developing better ways to combat the diseases
associated with them, Levchenko pointed out. For example,
people who suffer from cystic fibrosis — a genetic
disorder that affects the mucus lining of the lungs —
are susceptible to a species of bacteria that colonizes
the lungs; patients choke on the colony's byproducts.
Chronic urinary tract infections result from bacterial
communities that develop inside human cells. And biofilms
cause problems in tissues where catheters have been
inserted or where sutures have been used.
"You can put a patient on antibiotics, and it may seem that
the infection has disappeared, but in a few months it
reappears, and it is usually in an antibiotic-resistant
form," Levchenko said. To explore possible treatments, he
said, the microfluidic device could be used as a tool to
rapidly and simultaneously screen different types of drugs
for their ability to prevent biofilms.
This research was supported by funding from the National
Science Foundation, National Institutes of Health and
Swedish Research Council.
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