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Analytical Challenges of Microbial Biofilms on Medical Devices

Microbial colonization of medical devices is a widespread problem that tests the limits of conventional
analytical methods. Successful analytical endeavors require collaboration between clinicians,
microbiologists, biomedical engineers, and analytical chemists.
Akos Vertes,† Victoria Hitchins,‡ and K. Scott Phillips*,‡
†
The George Washington University, Department of Chemistry
‡
Office of Science and Engineering Laboratories, CDRH, US FDA

*
S Supporting Information
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

between the nonsterile outside environment and the sterile

inside of the patient. Indwelling devices such as urinary
catheters are frequently associated with microorganisms which
Downloaded via 118.101.109.230 on May 8, 2020 at 11:59:09 (UTC).

originate from the skin of the patient or healthcare providers

(Figure 1A). The longer an indwelling device remains in a
patient, the more likely the device interface will be colonized,
often by multiple species. For implanted devices, the risk of
colonization may be due to other causes such as nonsterile
presentation of the device during surgical implantation or
hematogenously (blood borne)-derived bacteria from dental
caries and urinary tract infections. The first line of defense
against colonization of indwelling and implanted devices is the
use of sterile techniques when handling and inserting new
devices. Once in use, colonization of a medical device surface
can be difficult to treat if the bacteria have become resistant to
antibiotics, and in many cases successful treatment of persistent
infection may require surgical removal of the device.
Many devices that have been explanted after issues with
microbial colonization have slimy “biofilm” coatings on them
produced by colonizing microbes.4 These biofilms are self-
assembling multicellular communities that behave differently
from their free floating (planktonic) counterparts.5 This Fea-
ture will introduce a few of the unique features and challenges
of biofilms. In recent years, increased research has led to an
Robert Gates
Robert Gates improved understanding of biofilms on devices, yet much work
remains. Although clinicians are increasingly recognizing bio-
films as a medical threat, at present there is no clinical defini-
T he use of medical devices is one of the fastest growing
areas of medicine and an increasing source of healthcare
associated infections (HAIs). In a recent study, 1.7 million
tion for the term biofilm and the quantitative association be-
tween biofilm and probability of infection is poorly understood.
HAIs occurred in the United States in one year, resulting in 99 Medical devices with biofilm resistant technologies such as drug
000 deaths.1 The costs associated with HAIs were estimated to eluting coatings, bactericidal coatings, and adhesion resistant
range from $28−45 billion per year, and medical device chemistries and nanotopologies are being developed, but it is
associated infections account for upward of 60% of HAIs.2 not always clear how these technologies work in vivo or how
Medical device infections are usually linked to colonization of they affect clinical outcomes.
devices by microbes.3 Microbial colonization involves at least Better analytical instrumentation is needed to detect and
three complex factors: microorganisms, device, and host study device colonization and biofilms in vivo, and clinically
environment (tissues, immune cells, etc.) It is often difficult relevant methods are needed for in vitro assays of antimicrobial
to detect microbial colonization, and in some cases it can go technology. In the sections that follow, we will first discuss
undetected for years, whereas in others it can have life- basic biofilm biology before delving into analytical methods to
threatening urgency. The location of a device in the body detect, quantify, and characterize them.
(Supporting Information, Table S1) can affect the means of
colonization. An indwelling device is one that acts as a “bridge” Published: March 16, 2012

© 2012 American Chemical Society 3858 dx.doi.org/10.1021/ac2029997 | Anal. Chem. 2012, 84, 3858−3866
Analytical Chemistry Feature

Figure 1. (A) Potential infection sources of a percutaneous intravascular device. Medical devices introduce a vulnerable biointerface into normally
well protected organs and vasculature. Contamination can come from (1) infusate (2) from nonsterile catheter materials, (3) the skin, or (4) from
distant hematogenous infections. (B) Dynamic biofilm life cycle on a medical device: (1) transport and initial attachment of microbes, (2)

■ ■
irreversible adhesion or attachment, (3) microcolony formation, (4) maturation of the biofilm, and (5) detachment and dispersion of the cells.

BIOFILM BASICS BIOCHEMICAL COMPOSITION OF BIOFILMS

Microorganisms associated with biofilm infections range from Small populations of bacterial cells in isolation are a challenging
Gram negative bacteria, such as Pseudomonas aeruginosa, target for chemical analysis because they contain miniscule
Escherichia coli, and Proteus mirabilis to Gram positive, such volumes of lipids, proteins, nucleic acids, and other
as Staphylococcus aureus. Many of these bacteria are found on biomolecules. In contrast, the extracellular matrix (ECM)
the skin (S. epidermidis and S. aureus), in the water (E. coli produced by a biofilm community can be 10 times the mass of
and Ps. aeruginosa), or in improperly cleaned and sterilized individual cells and is thus an attractive target for chemical
equipment, such as infrequently cleaned water lines for ven- detection and quantification methods that are faster and less
labor intensive than plating and culturing. Extracellular
tilators and dental offices. Yeast, such as Candida albicans
polymeric substances (EPS) that make up the ECM are
and C. parapsilosis, is another common cause of nosocomial insoluble and protect microbial members of the community
infections that can form biofilm on devices and lead to clinical against desiccation, UV damage, metals, and other harmful
infections.3 molecules. EPS serve as a base for adhesion, a “glue” to keep
There are five major stages in biofilm colony formation cells in a community close together, and a scaffold for further
(Figure 1B): (1) transport and initial attachment of microbes, growth. The physicochemical properties of the matrix vary
(2) irreversible adhesion or attachment, (3) microcolony depending on the species and environmental conditions such
formation, (4) maturation of the biofilm, and (5) detachment as temperature, shear, and nutrient availability.8 Biofilm EPS
and dispersion of the cells.4−6 In the first stage, planktonic are complex polymers, making them difficult to isolate and charac-
bacteria, single cells that float or swim in a fluid environment, terize. Techniques such as centrifugation, filtration, complexation,
are transported to the biointerface. The first cells adhere to the and precipitation are adapted uniquely for diverse biofilm com-
surface initially through weak, reversible adhesion by van der positions. These methods require large or thick biofilm samples
Waals and electrostatic forces. In stage two, the first cells attract and likely introduce sample bias for water-soluble components.9
other microorganisms to attach by providing adhesion sites and Three main EPS components have been found in biofilms:
building the extracellular matrix to hold the biofilm together. polysaccharides, DNA, and proteins. While polysaccharides
Communication between cells occurs via cell signaling mole- have an important influence on EPS properties, a number of
cules and quorum sensing. In the third stage of colony forma- proteins are also identified as EPS and can far exceed
tion, the biofilm grows by a combination of attracting other polysaccharides on a mass basis. Proteins serve important
microorganisms and division of existing cells. The biofilm is roles as nonspecific adhesins10 and compose flagella, pili, and
considered “mature” (stage four) when it develops both intra- fimbriae. Enzymes and toxic amyloid proteins with cross-β
cellular and intercellular signaling. Finally, cells spread and structures are also found ubiquitously.9 In recent years, DNA in
biofilms has also been investigated. A key discovery was the fact
colonize new surfaces by swarming and seeding-, clumping-,
that a common enzyme, DNase, is able to degrade many clinical
and surface-dispersal.
biofilm isolates in vitro. The origins of DNA in biofilms is not
The viability and growth of adherent microorganisms have a entirely clear, although it may be from a lysed subpopulation of
strong dependence on chemical properties at the biointerface cells.8 As cells at the outer layer of a biofilm are killed by
such as the type of metal, plastic, etc.7 The surface texture and environmental factors or drugs, the DNA that remains may
the shape of the device, components of the surrounding media become part of the matrix. Other biomolecules found in
(e.g., pH and ionic strength), and prevailing local hemodynamic biofilms include humic substances, lipids, and surfactants.9
conditions can also affect bacterial adhesion. In addition to
material factors, the rate and extent of biofilm formation on
indwelling devices is impacted by biofouling, the number of
■ BIOFILM “COMMUNITY BENEFITS”
Microbes living in a biofilm derive benefits that are not available
microorganisms initially contaminating the device, the genus in planktonic life. These include physicochemical advantages,
and species of the microorganisms, the biological environment multispecies synergisms, and rapid gene transfer. Biofilms
(host serum and platelets, temperature, and circulating drugs), increase bacterial resistance to antibiotics. The structure of the
and the host’s immune system. The complex interplay of these biofilm itself conveys numerous advantages over an unpro-
factors is poorly understood, and statistically significant clinical tected planktonic cell. At the simplest level, the high density of
data is lacking. EPS hinders access of immune system defenses such as
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Analytical Chemistry Feature

Table 1. Analytical Methods to Explore Colonization and antibodies and macrophages. It also slows the diffusion of
Biofilms antibiotics, possibly giving microbes more time to make genetic
and metabolic changes that favor survival.11 The EPS can bind
drugs through sorption or matrix components can chemically
react with drugs, resulting in covalent bonds that immobilize
the drug and prevent it from reaching its target in cells.
Secreted catalase protects microbes against hydrogen peroxide
and betalactamase protects microbes against lactam anti-
biotics.12 Because of low oxygen and nutrient concentrations
deep in the matrix, microbes living there may go into a reduced
metabolic state. In the stationary phase, long-lived persister
cells appear to be unaffected at concentrations of antibiotics
that are far above normal minimum bactericidal concentrations
(MBCs).13
Clinical investigation of medical device biofilms often shows
more than one active species. Gene transfer in biofilms is
enhanced and can lead to increased survival, virulence, and even
antibiotic resistant strains. In the human body, resistance might
be transferred from apathogenic strains found in the oral and
intestinal flora to highly virulent strains with which a host is
infected.12 Genetic information can be transferred through
conjugative transmission of plasmids (cell−cell contact through
specialized pili). A second process, transformation by chro-
mosomal DNA, involves integration via homologous recombi-
nation. For this to occur, bacteria must have “natural
competence”, the ability to enter a genetically programmed
state in order to uptake macromolecular DNA from closely
related organisms.14

■ MEASURING BIOFILMS AT THE INTERFACE

Analytical chemistry is a crucial tool for understanding biofilms
on medical devices. Analytical methods are needed to
understand how biofilms form, their biochemical composition,
and how much is being formed. For in vitro and in vivo
situations, we need to be able to detect biofilm forming bacteria
and their ECM and know how quickly they are forming, how
well prophylaxis and antibiofilm technologies work to prevent
their proliferation, and the effectiveness of antimicrobial drugs
and device coatings. Both conventional and emerging analytical
techniques (Table 1) are being employed in these efforts.
Challenges of In Vitro Biofilm Analysis. Compared to
the planktonic state of bacteria, biofilms appear at first glance to
be an easier target for quantification because their localization
on a single colonized surface is similar to a preconcentration
step. This perceived advantage, however, evaporates when one
considers the complexity and heterogeneity of biofilm structure.
The morphology of biofilms presents a challenge for analysis
because it falls somewhere between surface-only techniques and
bulk material (volume) techniques. Biofilms can vary widely in
thickness but are often described on the order of 10−100 μm.
Most surface analytical techniques cannot report on the overall
composition of these structures when they have thicknesses of
up to several hundred micrometers. Conversely, the perform-
ance of volume analytical techniques is limited by the low
amount of material available in the film.
The complex and rapidly evolving nature of a colony over
time is also a challenge for quantification and comparison be-
tween samples. In biofilms coexisting populations of micro-
organisms are held together by an ECM that is a heterogeneous
mixture. As the film matures, the composition changes over
time and exhibits differences between the developmental stages.
Multiple species may be present and the interaction may

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Analytical Chemistry Feature

depend on available nutrients and environmental factors. A Together with SEM, it can provide additional topological
common problem is that clinical biofilm forming strains information about the biofilm ECM.
become planktonic when cultured repeatedly using techniques There are a number of other imaging methods, including
that select for planktonic cells. When used in in vitro experiments, magnetic resonance imaging and scanning transmission X-ray
these strains may no longer produce biofilms with the same microscopy, that are being used for successful assessment of
properties as their clinical counterpart. biofilms.19
Systematic in vitro studies require the reproducible formation Genetic Assays for Clinical Diagnostics and Epidemi-
of biofilms on medical device surfaces. There are currently four ology. Clinical investigators favor genetic assays because they
American Society for Testing and Materials (ASTM) standard are a relatively universal format, and the information obtained
methods for growing and measuring biofilms: a drip flow on rDNA allows for extensive epidemiological characterization.
reactor (E2647), a flow reactor (E2562), a CDC reactor The genes for virulence traits, toxins, adhesins, and antibiotic
(E2196), and a modified microplate method (E2799). Each resistance are most commonly targeted. This type of
method is used for a different in vitro device model. The CDC information is an advantage in device-related infections because
reactor can hold eight large sample coupons and has been used it can help distinguish epidemic strains from less threaten-
extensively for testing medical device materials.15 The micro- ing ones. Pulsed field gel electrophoresis (PFGE) is the gold
plate method is the first method to incorporate miniaturization standard for genetic assays but suffers from lack of
for multiplexed studies and relies on an orbital incubator to reproducibility between laboratories.20 Polymerase chain
provide shear flow.16 The last step of this method requires reaction (PCR) is one of the most commonly used methods
plating and culturing to obtain quantitative bacterial counts, in clinical settings and has about 63−100% sensitivity (a value
which is an offline process that will limit the true achievable used in clinical diagnostics that is related to a test’s ability to
throughput. While these methods are good for reproducibly identify positive results) for detecting prosthetic joint
growing biofilms, in vitro protocols for these formats need to be infections. Drawbacks of PCR are low specificity, a high false
developed that can better predict biofilm formation on medical positive rate, and challenges with contamination.21 Fluores-
devices in vivo. cence in situ hybridization (FISH) is another commonly used
Challenges of Clinical In Situ or In Vivo Biofilm method. While FISH allows for detection of multiple species
Analysis. The level of difficulty in clinical in situ or in vivo biofilms, the preparation procedure is quite extensive and the
biofilm analysis depends on the analytical objective. For some dehydration may result in poor quantitation. Other genetic
cases, such as colonization of an easily exchangeable medical methods used include ribotyping, high-resolution melting
device, it is sufficient to remove the device and directly sample analysis, DNA sequencing, and DNA arrays.20 One potential
for the presence of a biofilm in situ. Under these circumstances, drawback of using genetic information to measure biofilms
analytical methods can be developed to detect abundant ECM universally is that it is unlikely that a single genetic factor can be
components, such as polysaccharides, proteins derived from correlated with biofilm growth. For this reason it is less
bacterial cell appendages, DNA, or even signaling molecules. appropriate for quantifying differences between biofilm growth
For implanted devices, obtaining access to the biointerface on different materials or measuring the effect of antibiofilm
in vivo is more difficult and clinicians currently rely on diagnostic technologies.22 In cases where rapid results are not necessary,
imaging or body fluid/tissue samples for signs of inflammation conventional plating methods are also less expensive and more
and infection to determine if the device needs to be explanted. reliable than genetic assays.
More challenging than just detection of biofilm is an in-depth Mass Spectrometry for Sample Analysis and Imaging.
biochemical analysis of clinical samples, which requires Various forms of mass spectrometry (MS) (Figures 2 and 3)
surveying, identification, and quantitation of the major have the right combination of sensitivity and selectivity to
microorganism subpopulations in the biofilm. Such an endeavor contribute to laboratory and explanted device analysis and
needs to be undertaken for all major device biofilms and will potentially clinical diagnostics. Detection modalities can be
likely require combined use of multiple analytical methods. broken down into bulk sample analysis and imaging techniques.
Microscopy in Research and Explanted Device For bulk sample analysis, diagnostics (detection of the
Investigations. Microscopy is a commonly used tool for presence of species specific biofilms) might be achieved by
analyzing structural details of biofilms in vitro (in flow cells and identifying a set of biomarkers, whereas in-depth analysis
on coupons).17 Confocal laser scanning microscopy (CLSM) requires the use of the systematic methods of proteomics and
with fluorescent stains, antibodies, and lectins is ideal to metabolomics. The potential to use MS for detection of biofilm
characterize biofilms up to ∼60 μm thickness. Scanning elec- forming bacteria and their ECM materials in patient samples is
tron microscopy (SEM) has been used to verify that other enticing. Extensive efforts have been made to develop low mass
types of assays are correlated with a real physical change in biomarkers for planktonic microorganisms. Pyrolysis and
morphology, density, and substructures of biofilms. An protein fingerprints have even been identified using matrix-
important limit of SEM is that sample preparation for high assisted laser desorption ionization (MALDI) in the high mass
vacuum involves fixing the cells, which may damage intricate region.23 Unfortunately, because of the presence of differ-
biofilm structures. Cryo-SEM and environmental SEM (ESEM) ent phenotypes and the extensive amount of ECM in bio-
have been used to study unfixed biofilms without collapse of films, these approaches have not yet directly translated into a
the extracellular matrix. Ruthenium red, which binds strongly to universal laboratory diagnostic test. On the other hand,
negatively charged polysaccharides, can provide increased conventional MS-based proteomics and metabolomic methods
contrast in ESEM.18 Atomic force microscopy (AFM) is also are well suited to tackle the complex challenges of microbial
a useful tool for measuring physical properties of biofilms. It has biochemistry. Proteomic methods lend themselves well to the
been used for monitoring bacterial adhesion on different analysis of gene expression and post-translational modifications
surfaces, interactions between cells, and measuring the strength in biofilms. MS-based proteomics methods have been used to
of adhesion by bacterial adhesins and other macromolecules. assess changes in protein levels at different biofilm stages,24 to
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Analytical Chemistry Feature

Figure 2. (A) Bright field and fluorescence microscope images (left) and a region of the MALDI mass spectrum with m/z 840 molecular ion (right)
of Lyngbya bouillonii filaments show the production of apratoxin A.29 (B) Diffusion of rifampicin in Staphylococcus epidermidis biofilms followed by
LDPI-MS (top). The amplified section of the mass spectrum shows the rifampicin molecular ion at m/z 822. The image (bottom) indicates the
diffusion of rifampicin from the left edge.33

identify overexpressed proteins,25 and indirectly to measure molecular classes are, in part, complementary. Ion beams can be
genetic exchange.26 In metabolomics, mass spectrometry is well focused to less than ∼0.05 μm enabling subcellular resolution
suited to identify and quantitate a large percentage of the for SIMS MS,31 whereas for MALDI and LDPI with low beam
typical microbe metabolome (500−1 000 distinct chemical divergence and aspherical focusing optics a close to diffraction
species). For example, the consensus metabolic network of limited resolution below ∼1 μm can be achieved.32 In practice,
Saccharomyces cerevisiae, iMM904, consists of 8 compartments, however, because of relatively large matrix crystal sizes,
713 compartment-specific metabolites, and 1402 reactions.27 diffusion during sample preparation and to keep the acquisition
MS has also been used in combination with separation tech- time at acceptable levels, MALDI imaging is mostly performed
niques to identify metabolites and networks in microorganisms. with ∼100 μm resolution. For LDPI-MS, the 349 nm Nd:YLF
Using MS to study the colocalization of secondary metabolites desorption laser beam has a focal diameter of 20 μm for
is especially useful for identifying natural product drug can- postionization with an excimer laser operating with fluorine gas
didates. More specific studies have taken advantage of unique at 157 nm33 (Figure 2B) or 300 μm for postionization with a
MS methods such as stable isotope labeling; for example, to synchrotron in the vacuum-UV.34 Because of the significant
look at how bacteria enter dormant growth modes in biofilms divergence of mid-IR laser beams used in LAESI, focusing and
during starvation.28 A combination of fluorescence microscopy thus the spatial resolution of imaging is limited to ∼200 μm.
and MALDI-MS has revealed the presence of a secondary meta- This limitation can be circumvented by the application of a
bolite and potential cancer cell toxin, apratoxin A, in filamentous sharpened optical fiber for the delivery of the laser pulse to
cyanobacteria (see Figure 2A).29 ablate cells within a ∼30 μm spot.35 In DESI, the spatial
Currently there are over half a dozen MS modalities available resolution is determined by the size of the most active area
for biofilm imaging, including secondary ion mass spectrometry within the spray where most of the sample ions are produced.
(SIMS),30 MALDI, laser desorption with postionization Molecular mass limitations for the imaged compounds tend
(LDPI), desorption electrospray ionization (DESI), and laser to be most rigorous for the instruments with the highest spatial
ablation electrospray ionization (LAESI). They are based on resolution. For SIMS, the upper limit is 300 Da for
the use of primary photon (MALDI, LDPI, and LAESI) or conventional primary ions, whereas it is ∼1 500 for the novel
charged particle beams (SIMS and DESI) for sampling and ion cluster ion sources. Many of the single microbial cell analysis
production with the optional addition of ionization enhance- and imaging experiments rely on stable isotope labeling.
ment via intercepting the produced neutrals by high energy Postionization with vacuum-UV in LDPI is also limited to
photons (vacuum-UV from laser or synchrotron sources for molecular masses up to ∼1 000 Da. DESI extends the high
LDPI) or highly charged particles (electrospray in the case of mass limit for detection to ∼66 kDa,36 whereas MALDI and
LAESI). The application of imaging MS to biofilms is a LAESI can produce ions up to ∼100 kDa. The latter has
relatively new area of research with most studies focusing on demonstrated lateral imaging,37 depth profiling,38 as well as 3D
the performance characteristics of the different methods. imaging.35
Two performance metrics by which MS imaging methods Because of the relatively recent introduction of imaging MS
can be compared are spatial resolution and analyte molecular to biofilm analysis, the strengths and weaknesses of the various
mass limitations. Better spatial resolution allows for clear approaches are still being explored. Quantitation of the imaged
images of small biofilm structures, whereas the mass limitation molecular species remains a key challenge.
describes the uppermost molecular weight limit of biofilm Living Cell Analysis with Mass Spectrometry. The
molecules that can be detected. The capabilities of these development of new atmospheric pressure or ambient MS ion
techniques in terms of spatial resolution and the accessible sources such as desorption electrospray ionization (DESI) and
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Analytical Chemistry Feature

Figure 3. (A) In situ mass spectrum of a very small colony (n < 616 ± 76) from functioning Anabaena sp. PCC7120 cyanobacteria produced by the
LAESI ambient ionization method reveals the composition of the phycobiliprotein complex in the antennae. A cyanobacterial filament is shown in
the inset with a 20 μm scale bar.41 (B) Mating Tetrahymena thermophila pair viewed by a differential interference contrast microscope (left, scale bar
is 25 μm) and analyzed through SIMS (right). Mass spectra and the image in the inset show the differences in the distributions of phosphocholine
and 2-aminoethylphosphonolipid between the cell bodies and the cell-to-cell junction.42

direct analysis in real time (DART) offers the ability to study the need for fluorescent stains. Only a few stains can be used
biofilms in their native state. The ability to directly probe simultaneously, resulting in analysis of 1−2 biofilm
microbial systems using some of these approaches has been components at most. Raman microscopy can overcome this
demonstrated. Imaging by DESI MS was used to explore the limitation because it provides a spectrum of information related
distribution of allelochemicals and other secondary metabolites to chemical bonds in a format similar to CLSM. Numerous
in bacterial cultures,39 while the relatively scarcely studied biofilm chemical groups can potentially be analyzed by Raman.
microbial volatiles lend themselves to analysis by DART.40 Ideally, Raman might be used to identify biofilms from different
LAESI MS has been proposed for the investigation of lateral species or strains by identification of a chemical fingerprint.
and depth heterogeneity of biofilms. For example, the subunit One example of this is a group developing Raman-based
composition of phycobilisomal antenna proteins and numerous identification of S. epidermis biofilms43 that was able to
metabolites from a very small population (n < 616) of distinguish between two strains of S. epidermis using principal
Anabaena sp. PCC7120 cyanobacteria were determined by component analysis. The sensitivity of Raman is a major
LAESI MS (Figure 3A).41 limitation, and several groups have used surface enhanced
Because of the lack of chemical species amplification in mass Raman spectroscopy (SERS) to improve the analysis of biofilms
spectrometric approaches, the analysis of single cells is volume using two different approaches. In the simplest method, the
limited. Hundreds of components in plant or large animal cells observation chamber is filled with a colloidal suspension of
(>50 μm) can be directly analyzed, but the analysis of a hydroxylamine hydrochloride reduced silver colloids.44 Using
bacterial cell, typically <1 μm or 0.5 fL volume, by MS remains this strategy, the investigators were able to obtain greatly
a challenge. On the basis of molecular ions or their fragments, enhanced signal compared to unenhanced Raman and monitor
currently only SIMS and matrix enhanced SIMS are capable of the biofilm growth for weeks. They identified Raman bands for
submicrometer resolution chemical imaging enabling the carbohydrates, proteins, DNA/RNA, carotenoids, and lipids in
analysis of individual microbial cells and subcellular compo- the biofilm matrix. The limitation of this method is that the
nents. In one notable example, structural changes in the cell Raman signal enhancement is limited to the surface of the bio-
membrane of mating Tetrahymena thermophila were recognized film near the colloids. Because of the complex three-dimensional
by time-of-flight SIMS measurements (Figure 3B).42 With the nature of biofilms, the method may oversimplify biofilm mea-
emergence of enhanced sensitivity techniques, continued surements. Another strategy is to coculture biofilms with the
development in the field of single cell MS is expected. nanoparticles, allowing surface enhancement throughout the
Raman Spectroscopic Imaging. In addition to methods depth of the biofilm (Figure 4).45
already proven for biofilm analysis, there are a number of Microarrays and Microfluidics. Microarrays and micro-
other analytical technologies with great potential. These fluidics are changing the way in which biological research is
technologies can provide new information about biofilms performed, and the field of biofilms is no exception. Microarray
that was either difficult to obtain or simply not possible with tools borrowed from the “omics” are enabling higher
current techniques. For example, CLSM is limited because of throughput testing. A slide based array with 768 distinct

Figure 4. (A) SEM image of E. coli biofilm prepared with silver nanoparticles.45 (B) Time dependent surface enhanced Raman spectra obtained from
E. coli cultivated with nanoparticles.45

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Analytical Chemistry Feature

Figure 5. (A) Far left and center left: Crop from fluorescence scan of 768 spot array of fungal biofilms stained with FUN 1.46 (B) Surface chemistry
used to make collagen coated arrays on glass slides for biofilm growth.46 (C) Photo of microchip for cell population measurements. Blow-up shows
proliferation chamber with electrochemical sensors.50

biofilm patches was developed to monitor fluorescence changes strategies and instrumental methods could potentially increase
in response to antifungal compounds (Figure 5A,B).46 The the sensitivity and specificity of current clinical tests. Micro-
method takes advantage of established surface chemistry and fluidics can decrease sample requirements and make these
arraying techniques to fabricate collagen patches on which techniques more affordable and reliable by integrating on-chip
biofilms form. sample preparation and handling. Current molecular diagnostics
Microfluidics can be used to observe biofilms, measure some are limited by the fact that they require sample fluid or tissue,
physical parameter, or apply physical/chemical stimuli and which may not correlate with biofilm on the surface of implanted
capture response characteristics. Semimicrofluidic flow cells are medical devices. Molecular genomics can hint at the potential of
commonly used for cultivation of biofilms. More advanced a specific strain based on genetic expression, but they do not
culture, such as simultaneous coculture of osteoblasts and prove that the strain is alive and thriving or identify the type and
bacteria, has also been performed in a semimicrofluidic 3D tissue amount of biofilm. If analytical chemists can develop chemical
model to better mimic the in vivo environment of orthopedic analysis for aspirates from around an implant, it may be possible
implants.47 Quorum sensing molecules are easier to detect in to confirm biofilm growth more directly without an invasive
small volumes because of limited diffusive dilution, surgery or removal of the device. This Feature mentions some
so detection is a good match for microfluidic chambers.48 potential target analytes, such as components of the ECM or
Although many microfluidics are closed channel format, open- signaling molecules produced by colonizing microbes.
channel microfluidic devices may be necessary for some biofilm For in situ analysis of biofilms on explanted devices, the
analysis. Open channels allow for combination of microfluidics burgeoning development in mass spectrometric methods holds
with powerful instrumental analysis methods such as imaging MS great promise. As new atmospheric pressure techniques are
or synchrotron infrared spectromicroscopy49 to chemically image perfected and become commercially available, investigators may
biofilm dynamics in real time. More sophisticated “lab-on-a-chip” be able to perform chemical imaging of live clinical biofilms in
devices with multiple layers and integrated electronics are able to order to elucidate the variety and complexity of multispecies
measure multiple physical properties of biofilms. For example, communities on a large scale (biochemical architecture) and at
bacterial population dynamics were measured in a device with the single cell and subcellular level (chemical cytometry).
both optical detection of population numbers and electro- For in vitro analysis, there are many existing techniques
chemical measurement of respiration (Figure 5C).50 ranging from microscopy to microwell assays. Because most of
Biofilms have also been probed on microchips with physical these methods are highly refined, the limits of in vitro analysis
and chemical methods. Exposure to gradients generated in are related to realism and reproducibility rather than
microchips allows for instantaneous evaluation of a continuous instrumental performance. In particular, relating outcomes of
range of concentrations. Bacteria have been exposed to oxygen in vitro assays to in vivo colonization and infection is a major
gradients generated on-chip and screened for a response.51 The challenge. Multivariate analysis could be used to unravel the
ability to create unique cellular size-scale architecture also
numerous variables and make more reliable in vitro−in vivo
allows for testing of biofilm physical parameters such as
correlations. These efforts require standard methods to grow
adhesion forces, which were studied by varying the flow in a
reproducible biofilms and test a multitude of variables related to
microfluidic device.52

■
device, microorganism, and host environment. Microfluidics
may play a key role in helping increase the throughput of in
FUTURE DIRECTIONS vitro testing and minimizing the time and cost associated with
Despite increasing use of antimicrobials and attention to sterility, bacterial culture and plating on a large scale. Because its scale is
microbial threats are not subsiding. Long-term, drug resistance is similar to that of individual cells, the microfluidic environment
developing faster than we can invent new drugs.53 Microbial may also present unique advantages to probe cells and detect
colonization and the resulting biofilms are a major source of drug low concentrations of analytes.
resistance, and the biointerface of many medical devices is a Increased understanding of the unique scientific, clinical, and
vulnerable target.54 regulatory challenges of medical device biofilms will help spur
Detection of biofilms in vivo has benefited from modern much needed innovation in the field. There are many
molecular methods, but there is still great need for more rapid opportunities for analytical chemists to play a crucial role in
and reliable analysis. Recently developed amplification this endeavor.
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Analytical Chemistry

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Feature

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ACKNOWLEDGMENTS
D.; DiBartolo, G.; Tyson, G. W.; Allen, E. E.; Ram, R. J.; Detter, J. C.;
Artistic renderings in figures were created by Robert Gates. The Richardson, P.; Thelen, M. P.; Hettich, R. L.; Banfield, J. F. Nature
authors acknowledge Dinesh Patwardhan, Director of DCMS 2007, 446, 537−541.
for his supervisory oversight on this work and thank Samantha (27) Herrgard, M. J.; Swainston, N.; Dobson, P.; Dunn, W. B.; Arga,
Spindel, Peter Nemes, and Thelma Valdes for their review of K. Y.; Arvas, M.; Bluthgen, N.; Borger, S.; Costenoble, R.; Heinemann,
the manuscript. A.V. appreciates the financial support from the M.; Hucka, M.; Le Novere, N.; Li, P.; Liebermeister, W.; Mo, M. L.;
Chemical Sciences, Geosciences and Biosciences Division, Oliveira, A. P.; Petranovic, D.; Pettifer, S.; Simeonidis, E.; Smallbone,
Office of Basic Energy Sciences, Office of Science, U.S. K.; Spasic, I.; Weichart, D.; Brent, R.; Broomhead, D. S.; Westerhoff,
Department of Energy (Grant DE-FG02-01ER15129) and the H. V.; Kirdar, B.; Penttila, M.; Klipp, E.; Palsson, B. O.; Sauer, U.;
George Washington University Selective Excellence Fund. Oliver, S. G.; Mendes, P.; Nielsen, J.; Kell, D. B. Nat. Biotechnol. 2008,
Support from the Department of Energy does not constitute 26, 1155−1160.
(28) Bajad, S. U.; Lu, W. Y.; Kimball, E. H.; Yuan, J.; Peterson, C.;
an endorsement of the views expressed in the article.

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