Theranostics 2017, Vol.

7, Issue 7 1795

Ivyspring
International Publisher
Theranostics
2017; 7(7): 1795-1805. doi: 10.7150/thno.19217
Review

Current and emerging techniques for antibiotic

susceptibility tests
Karan Syal1, Manni Mo1, 3, Hui Yu1, Rafael Iriya1, 3, Wenwen Jing1, Sui Guodong4, Shaopeng Wang1, 2,
Thomas E. Grys5, Shelley E. Haydel6, 7, and Nongjian Tao1,2,3
1. Center for Biosensors and Bioelectronics, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA;
2. State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China;
3. School of Electrical, Computer and Energy Engineering, Arizona State University, Tempe, Arizona 85287, USA;
4. Institute of Biomedical Science, Fudan University, Shanghai, China;
5. Department of Laboratory Medicine and Pathology, Mayo Clinic, Phoenix, Arizona 85054, USA;
6. Center for Immunotherapy, Vaccines, and Virotherapy, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA;
7. School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA.

 Corresponding author: [email protected]

© Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.

Received: 2017.01.16; Accepted: 2017.03.03; Published: 2017.04.10

Abstract
Infectious diseases caused by bacterial pathogens are a worldwide burden. Serious bacterial
infection-related complications, such as sepsis, affect over a million people every year with
mortality rates ranging from 30% to 50%. Crucial clinical microbiology laboratory responsibilities
associated with patient management and treatment include isolating and identifying the causative
bacterium and performing antibiotic susceptibility tests (ASTs), which are labor-intensive,
complex, imprecise, and slow (taking days, depending on the growth rate of the pathogen).
Considering the life-threatening condition of a septic patient and the increasing prevalence of
antibiotic-resistant bacteria in hospitals, rapid and automated diagnostic tools are needed. This
review summarizes the existing commercial AST methods and discusses some of the promising
emerging AST tools that will empower humans to win the evolutionary war between microbial
genes and human wits.
Key words: AST methods, antibiotic susceptibility tests

Introduction
Antibiotic-resistant bacterial pathogens are a clinical task is prescribing the patient with effective
global health epidemic, spreading at a rapid rate. In antibiotics, which requires rapid diagnosis of the
the US alone, these pathogens cost billions of dollars resistant infections and antibiotic susceptibility
in healthcare, with 2 million hospitalizations and testing (AST) [6].
23,000 deaths annually [1]. This epidemic is AST is widely used clinically to determine
accelerated by widespread misuse of antibiotics in antibiotic resistance profiles of bacterial isolates, to
clinics and agriculture over the last few decades, guide antibiotic treatment decisions, and predict
allowing bacteria to evolve and develop means of therapeutic outcome [5, 7]. Currently, AST is usually
resistance [2,3]. Resistant bacteria are widely found in performed in a clinical microbiology lab, which
the community and can also be acquired via necessitates transportation of the patient samples
nosocomial infections, post-surgery complications, from the healthcare provider to the lab. Susceptibility
and contaminated food [3, 4]. Resistant bacterial testing requires a pure culture of the offending
infections can also cause sepsis, which has mortality pathogen, a process which may take several days.
rates ranging from 30% to 50% [5]. Considering the This delay prolongs the time to diagnosis of resistant
life-threatening condition of a septic patient, a key bacteria and decisions for appropriate and effective

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Theranostics 2017, Vol. 7, Issue 7 1796

antibiotic therapy. Delays in timely administration of becoming a useful analytic modality is the
appropriate therapeutics lead to increased patient Epidemiological Cutoff Value for resistance (ECOFFs)
mortality, poor clinical outcomes [5], and use of [11]. This relates to the MIC values of a population of
broad-spectrum antibiotics, the latter of which isolates of a particular organism against a particular
promotes antibiotic resistance. To survive this drug. It can be helpful to determine intrinsic
evolutionary war against bacteria, we must pursue resistance that is present in some strains of a bacterial
technologies that can rapidly perform AST to enable species.
personalized therapies (narrow-spectrum antibiotic The current culture-based AST tools rely on
administration) at the earliest possible treatment time-consuming culturing techniques, followed by
stage. disk diffusion [7] and broth dilution susceptibility
After receipt of the patient sample (collected on testing [8], resulting in several days before MIC values
day 0), the clinical microbiologist must isolate the are determined and reported. Paradigm-shifting AST
potential pathogen by streaking the sample on technologies must overcome the current bottleneck
selective culture media and incubating the inoculated associated with the slow culturing steps. Ideally, they
media overnight (or longer) to enable growth. From a would be directly applicable on clinical samples
primary growth plate (day 1), isolated colonies must without the need for selection and/or enrichment on
be obtained by subculture. Once isolated colonies day 1, and, preferably, be able to deliver results at the
from the pathogenic organism are available (day 2), point of care (i.e., at the patient’s bedside). In addition
the bacterial inoculum is prepared and standardized to low cost and ease of operation requirements,
(day 2) prior to performing AST via disk diffusion [7] additional features, such as identification of bacterial
or broth dilution [8] methods (detailed later). strains before AST and the ability to perform AST of
The minimum inhibitory concentration (MIC) is polymicrobial infections, will also help improve
defined as the lowest concentration of antibiotic patient outcomes and reduce the selection of
required preventing bacteria growth and is used to additional resistant organisms.
determine if the infected pathogen is susceptible or In the present mini-review, we summarize the
resistant to an antibiotic [7–9]. It is important to note current technologies, discuss the emerging
that the MIC does not necessarily imply bacterial technologies, and provide scientific opinions on
death, but rather lack of growth. Thus, the MIC differs future AST technologies. Given the vast number of
from the Minimum Bactericidal Concentration (MBC), publications in this area, we mainly focus on
a useful value which is seldom determined in a phenotypic AST methods. Even with this focus, we
clinical laboratory because of the additional effort will unintentionally and inevitably exclude many
required. A breakpoint is defined as the concentration exciting emerging technologies in the scientific
of an antibiotic that enables interpretation of AST to literature due to limited page and scope. Fortunately,
define isolates as susceptible, intermediate, or several reviews [7, 9, 12, 13] on related topics have
resistant [8, 10]. If the determined MIC is less than or been published, thus enabling readers to identify
equal to the breakpoint, then the bacterial isolate is topics that are inadvertently not included here.
considered susceptible to the antibiotic. Clinical
breakpoints for different antibiotics and bacteria are Current Technologies
reviewed and updated annually by national In 1928, Alexander Fleming discovered a mold
organizations, such as the Clinical Laboratory that prevented the growth of staphylococci on an agar
Standards Institute (CLSI) in the USA and the plate (Fig. 1a). The mold produced an active
European Committee on Antimicrobial Susceptibility substance, penicillin, which become the first antibiotic
Testing (EUCAST) [8]. In addition to characterizing and usher in the antibiotic era, a critically important
bacterial isolates collected from individual patients, milestone in modern medicine [14]. Antibiotics are
MIC is used in epidemiological monitoring of the commonly used to treat bacterial infections, to reduce
evolution of antibiotic-resistant bacteria. Increasing the possibility of infections (e.g., during invasive
MIC values for an antibiotic over a period of time may surgeries) in hospitals, and to promote growth in food
indicate acquired antibiotic resistance for a given animals. The widespread use of antibiotics has
bacterial species [8]. MIC values serve as an important accelerated the pace at which bacteria become
parameter to determine phenotypic resistance in resistant to antibiotics. While antibiotic-resistant
bacterial cells, to monitor the global resistance bacteria are rapidly evolving, diagnostic technologies
surveillance, and to determine the effectiveness of that can characterize the infection, guide treatment,
new antibiotics. MIC values obtained by the current minimize unnecessary use of antibiotics, and
AST techniques also serve as a gold standard to customize therapeutic strategies for specific patients
evaluate new AST methods. Another concept that is have been slow. The mainstream technologies still

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rely on measuring bacterial growth in presence of are commonly used in clinical microbiology labs.
antibiotics over a few days using methods such as
agar dilution assays (E-test and disk diffusion), broth Broth Dilution Assay
dilution assays, and automated systems from various An MIC test can also be performed using broth
manufacturers. These technologies rely on detecting macrodilution, whereby broth volumes for testing
bacterial growth, which is not conceptually different each antibiotic concentration are at least 1 mL.
from how Fleming first discovered penicillin. Following incubation for 20-24 h, the MIC is the
lowest concentration of antibiotic that completely
Agar Dilution, Disk Diffusion and inhibits bacterial growth and therefore lacks visible
Antimicrobial Gradient Assays turbidity [8]. Due to the laborious nature of the broth
In the agar dilution assay, bacteria are inoculated macrodilution approach, the assay has been
into an agar medium containing different antibiotic miniaturized and standardized by use of small,
concentrations. While agar dilution testing offers plastic, disposable microdilution trays which contain
reproducible results, agar dilution plates are laborious 96 wells to allow minimal volume (e.g.: 0.1 mL) and
to prepare and have short shelf lives. In many clinical pre-determined antibiotic concentrations [7]. Many
microbiology laboratories, agar disk diffusion is commercially-available systems use automatic
routinely used for testing common, rapidly growing inoculating devices, but microwells may also be
bacterial pathogens [7]. The disk diffusion assay inoculated with multichannel pipettors. Broth
involves inoculating the bacteria, enriched from microdilution results may be determined visually or
clinical samples by overnight growth on selective through automated instruments.
media, onto a Mueller-Hinton agar plate, followed by Automation of the broth microdilution assay
placing commercially-prepared filter paper disks instruments provides more precise, reliable, and
impregnated with predetermined concentrations of quantitative AST. There are four
an antibiotic onto the surface of the agar commercially-available automated or semi-automated
medium.[12]The agar plate containing the bacteria instruments MicroScan WalkAway, Vitek-2, BD
inoculum and antibiotics disks are further incubated Phoenix automated system, and Sensititre [7,10]. Each
at 35-37°C in ambient air or 5% CO2 for 16-24 hours, of these instruments consists of the following: 1) A
depending on the suspected bacterium. During this single-use AST cassette, which can be a microdilution
incubation, the antibiotics diffuse into the agar with tray/test panel/card containing different antibiotics
antibiotic concentration decreasing with increasing at different concentrations; 2) an AST instrument,
distance from the disk. Antibiotic susceptibility is which reads multiple cassettes over a period of time
determined by measuring the diameter of the zones of (usually overnight) to give AST results. These
bacterial inhibition around the antibiotic disks and automated AST instruments require bacterial isolates
comparing the diameter with disk diffusion obtained through routine culture from the patient
interpretive criteria updated annually by CLSI [12,15]. samples.
While the disk diffusion test (Fig. 1b) is technically Microscan Walkaway AST cassette, based on
easy, inexpensive, and flexible, it provides only standard 96-well microdilution trays, is capable of
categorical results (e.g., susceptible, intermediate, handling 40-96 trays with automated
resistant). Since quantitative MIC results relaying the sample-handling robotics, where the antibiotic
degree of susceptibility may be necessary in some susceptibility test uses a photometer to detect
cases, the gradient diffusion method offers similar bacterial turbidity in the trays over 4.5-18 hours [7, 8,
flexibility and simplicity to disk diffusion and 16]. The Vitek-1/Vitek-2 AST instruments developed
determines quantitative MICs. In the Etest, a common by bioMérieux, use a smaller AST cassette, called an
commercially-available gradient test, the assays are AST card, in the 45-64 well plate format. Each Vitek-2
performed similarly to the disk diffusion approach AST instrument is capable of handling 30-240 AST
except that a thin plastic strip with a continuous cards and detects turbidity with bacterial growth over
exponential gradient of antibiotic is used to generate 4-10 hours to reveal AST results. The BD Phoenix is an
diffusion of the antimicrobial agent into the automated microbiology system that consists of a
agar-based medium. After overnight incubation large AST instrument capable of reading turbidity
allows bacterial growth and antibiotic diffusion, an and colorimetric changes of up to 99 AST cassettes
inhibition ellipse is visible (Fig. 1c). The quantitative (called panels). The BD system requires an average of
MIC corresponds to the point on the strip whereby the 6-16 hours, starting from incubating pure bacterial
antimicrobial concentration is no longer inhibiting cultures, to obtain MIC for the bacteria. The Sensititre
bacterial growth, thus revealing the inhibitory system by Thermo Scientific uses the standard 96-well
concentration. The disk diffusion and Etest methods microdilution panels (AST cassettes), which are

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 Theranostics 2017, Vol. 7, Issue 7 1798

inoculated by the Sensititre Autoinoculator, and is require the use of isolated bacteria grown in pure
capable of handling 64 panels. Bacterial growth in culture, and the susceptibility tests are based on
each panel is detected from the fluorescent intensity measuring bacterial growth and turbidity changes. As
monitored over 18-24 hours post incubation. a result, these automated technologies remain
Automated AST instruments, representing inherently slow and are severely limited by the low
current state of the art technologies, are extensively sensitivity of the current detection methods.
used in clinical microbiology labs in the US. Furthermore, they are limited in the number of
Compared to manual methods, these instruments antibiotics and concentrations tested and lack the
provide a streamlined workflow and quantitative capability of analyzing polymicrobial samples or
results, thus simplifying MIC determinations for heterogeneous response of bacterial populations to
pathogenic bacteria isolated from clinical samples the antibiotics.
[17]. However, these automated instruments still

Figure 1. Evolution of agar dilution methods for determining antibiotic susceptibility from the discovery of antibiotics (a) to currently used disk
diffusion (b) and Etest (c) assays. a) Photograph showing lack of staphylococcal colonies in the vicinity of the Penicillium mold adapted from Alexander Fleming’s original
research paper on the discovery of penicillin. b) E-Test uses gradient antibiotic concentrations to determine MIC of antibiotics. c) Disk diffusion assays involve placing
multiple antibiotic-impregnated disks onto an agar surface inoculated with bacteria and measuring the diameter of zones of inhibition to qualitatively determine
antibiotic susceptibility. Figure 1a Adapted from – Alexander Fleming. On the Antibacterial Action of Cultures of a Penicillum, with Special Reference to their use in
the isolation of B. Influenze. Br J ExpPathol. 1929 Jun; 10(3): 226–236Disk diffusion assay image produced by John Popovich, Haydel Lab, ASU.E-test image produced
by Rachael Liesman.

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Figure 2. Rapid AST using an emerging imaging based tool. a) Schematic comparison of traditional AST using broth microdilution and imaging-based AST
demonstrates how tracking single cell divisions can produce rapid results compared to traditional optical density (OD) tools which are limited by their sensitivity to
measure only higher bacterial concentrations. b) Setup of a 96-well plate modified into a microfluidic agarose chip for concurrent addition of bacteria and antibiotics
followed by microscopic imaging. c) Schematic of steps involved in adding bacteria and antibiotics and imaging a localized area to observe changes. From Choi J, Yoo
J, Lee M, Kim E-G, Lee JS, Lee S, et al. A rapid antimicrobial susceptibility test based on single-cell morphological analysis. Sci Transl Med 2014; 6:267ra174 Reprinted
with permission from AAAS.

biosensors [24, 25] have been pursued. For example,

Emerging Technologies optical detection of bacterial growth via the cell
Newer AST techniques, which are currently and lengths and numbers [18, 20, 26], forward light
actively being pursued by commercial entities for scattering [25], and measuring vibrational amplitude
clinical translation, are considered as emerging changes of magnetic beads [24, 27], have been
technologies for the purpose of this review. With the proposed. Micro-channel resonators have also been
increasing clinical demand for rapid AST, various used to detect nanoscale fluctuations associated with
new AST techniques based on optical imaging bacteria growth [21]. Quantitating molecular or
[18–20], micro-channel resonators [21–23] and other biochemical markers, such as 16SrRNA [28], ATP [29],

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Theranostics 2017, Vol. 7, Issue 7 1800

and luciferase [30], in bacterial cells are also being algorithms to quantify changes in the area occupied
used for rapid AST. These approaches can by a growing population of cells. However, unlike
significantly improve the current commercial AST other high resolution imaging methods, oCelloScope
technologies, but they still rely on culturing, which is does not capture the growth of individual cells, but a
not universally applicable for anaerobes, population of cells in liquid fluids and thus eliminates
slow-growing bacteria, and non-cultivable the need to attach bacterial cells to an inert surface.
microorganisms. Additionally, most of these Coupling of imaging-based tools with
emerging technologies still require substantial sample microfluidics has been reported for rapid AST.
preparation and pre-treatment steps, such as bacterial Bacterial cells are first captured in microfluidic
enrichment from patient samples, and cell lysis to chambers [35], micro channels [36], or droplets [37, 38]
extract biochemical markers. and then imaged to detect changes in the cell number
[20, 39], size [18], morphology [40] and viability [37,
Imaging-based AST 41] in the presence of antibiotics in order to perform
Multiplexed automated digital microscopy AST. Novel imaging approaches, such as measuring
(MADM) [19, 31] is an automated microscope being changes in rotational frequency of magnetic beads
developed to provide rapid identification and AST of (which is proportional to cell mass) [24, 27, 42] and
clinical samples. MADM separates bacterial cells from electro-kinetic loading of single cells, [36] have been
other substances in the clinical samples (e.g., blood or applied to AST using smartphone cameras and other
urine) using gel filters and attaches purified bacterial imaging devices [41].
cells to the surface sensing surface using Although the imaging-based AST tools shorten
electro-kinetic loading [31]. After surface attachment, the detection time from days to a few hours, these
fluorescent in-situ hybridized (FISH) probes are used technologies still use replication-dependent
to identify bacterial cells within an hour, followed by methodologies that have a primary culture step (e.g.
AST [31]. To perform AST, MADM measures bacterial growth from a blood culture bottle or growth on a
growth every 10 minutes as clonal aggregates primary culture plate). These dependencies limit the
multiply in Mueller-Hinton media. Since resistant application of imaging-based methods to
cells will grow in Mueller-Hinton media with slowly-growing pathogens, such as Mycobacterium
antibiotics and sensitive cells will be inhibited or tuberculosis. To perform AST on pathogens directly
killed, expansion and measurement of clonal masses (i.e.: without a culture step) from clinical samples, it is
over time (compared to growth controls) are used to necessary to separate bacteria from the patient sample
generate growth curves and determine susceptibility. matrix, and then measure a cellular attribute that is
MADM also uses cell morphokinetic image analysis independent of replication.
for differentiating bacterial species in polymicrobial
infections, thus expanding clinical capability and Non-imaging AST
reducing of the cost of multiple assays. While the Non-imaging methods that measure the physical
MADM imaging approach for measuring the bacteria or biochemical signature of bacterial cells have been
growth rate is faster than traditional approaches and proposed for AST. BacterioScan detects forward laser
represents a significant step forward from the current light scattering (FLLS) [25, 43] analyzes the angular
commercial tools, its universality to all antibiotics variation in the intensity of the scattered light, and
remains to be addressed [18]. determines the number and size of bacterial cells
Another imaging tool capable of rapid AST is suspended in a solution. FLLS can measure bacterial
single-cell morphological analysis (SCMA) [18, 32]. concentrations as low as 103cfu/ml, which is more
SCMA (Fig. 2) uses bright-field microscopy to sensitive than other optical methods and traditional
determine antibiotic-induced morphological changes automated instruments, and may enable rapid AST
in single bacterial cells and enable rapid AST. The (within a few hours). The FLLS technology can
captured images are processed using an automated perform AST directly on urine samples with minimal
image-processing algorithm to quantify the area and sample preparation, thus enabling point-of-care
number of growing bacterial cells. The classification applications. Disadvantages of FLLS include the use
algorithm processes several morphological of a replication-dependent approach to measure AST,
characteristics to produce antimicrobial susceptibility inability to differentiate bacteria from cell
data. Another optical imaging technique is sedimentation, lack of single cell resolution, and
oCelloScope [33, 34], which is based on imaging inability to differentiate bacterial species for
growth of a population of bacterial cells in a fluid polymicrobial analysis.
sample with antibiotics over a period of time. The LifeScale develops microchannel resonators for
recorded images are then processed using imaging rapid AST, where the microchannel resonators are

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individual microcantilevers. The technology measures rapid automated testing. Further the extracted DNA
the mass of the bacterial cells upon passage through contains DNA from both alive/dead cells leading to a
the microfluidics channels inside of the higher false positive rate for these techniques [49].
micro-cantilevers [23]. Microchannel resonators Other disadvantages include the need of previously
permit quantitation of bacterial cells and measure known sequences, micro-heterogeneity in the 16s
mass changes of the individual cells to assess RNA within a species [50], lack of correlations
antibiotic activity [21, 23, 44]. The advantages of between genotypic and phenotypic resistance [51],
microchannel resonator AST are the ability to perform and inability of performing tests on clinical samples.
sensitive mass and morphology measurements on Other biochemical signatures, such as ATP and
single bacterial cells and the promise of AST within~3 NADH, have been studied as AST biomarkers with
hours. However, the applicability of the approach to electrochemical amplification [29, 52–54]. These
clinical samples remains to be fully established. biochemical signatures are indicators of the metabolic
activities of bacteria, thus providing critical
Biochemical AST information on bacterial viability. While some of these
While the AST technologies described above techniques are capable of providing rapid AST within
detect physical and morphological features of a few hours, these techniques currently lack
bacteria, tools that measure molecular and sensitivity to perform AST at lower antibiotic
biochemical signatures, such as changes in 16s RNA concentrations and dilution ranges. Further, the
[28, 45], DNA [46, 47] and ATP [29], of growing universal application of the probe molecules to
bacterial cells have also been studied. A multiple strains and antibiotics is also questionable.
biosensor-based AST (b-AST) assay being developed While promising, these emerging approaches require
by Genefluidics measures bacterial growth via further studies and evaluations.
quantitating 16s rRNA molecules, which are specific
for each bacterial species [28, 45]. After DNA probes Future Technologies
hybridize specifically to 16S rRNA molecules, an The emerging technologies, being actively
electrochemical signal permits amplification and pursued by commercial entities discussed above,
quantitative detection. This approach allowed for AST promise rapid AST within a few hours. Furthermore,
as short as ~4 hours using clinical urine samples from some of the technologies can be directly applied to
patients experiencing a urinary tract infection. patient samples without any sample pretreatment.
Smarticles technology in development by Roche However, further shortening the test time and
Diagnostics [30] introduces recombinant applying them to slowly growing organisms will
bacteriophages with DNA probes, such that a specific require innovative approaches. We discuss future
binding of DNA probes inside the bacterial cells leads technologies that can meet these requirements below.
to luciferase expression. Luciferase expression Microcantilevers have been recently used to
produces light, which is used to quantify the number perform rapid AST [22, 55, 56], whereby bacterial cells
of bacterial cells and perform rapid AST. Real-time are attached to a microcantilever and deflection of the
PCR is another molecular approach, which quantifies microcantilever associated with the micromotions of
copies of bacterial DNA and correlates this value with the bacterial cells, is detected as the signature of
bacterial growth in a sample. This technique targets bacterial metabolism. This approach has led to AST
highly conserved regions of bacterial chromosomal within two hours for Escherichia coli and Staphylococcus
DNA to ensure species specificity and has been aureus strains for different antibiotics [22, 55]. The
applied to various combinations of antibiotics and correlation between the micromotions and viability
bacterial species. Another approach detects bacterial (metabolism) has been studied for both prokaryotic
genetic fingerprints that are detected upon exposure and eukaryotic cells [57]. While sensitive, the
to antibiotics rather than relying on a single specific micromotion of bacterial cells producing
gene or DNA sequence in the other approaches microcantilever deflections is affected by flowing
described above. liquids, and recent reports have also indicated
While the nucleic acid-based biochemical assays, inefficient transfer of antibiotics to immobilized
such as real-time PCR, can give faster results than the bacterial cells under laminar flow conditions [23].
current techniques, it has several disadvantages such Furthermore, the sensitive cantilever deflections are
as relying on high bacterial concentrations to extract caused by bacterial cells attached to the tip, so the
sufficient DNA, manual sample handling steps such small area of the tip limits the number of bacterial
as lysing bacterial cells to extract nucleic acids.[48] cells which are adsorbed to the surface, potentially
These manual steps make clinical adaptation of these preventing the application of the technique to lower
technologies difficult, where the need of the hour is bacterial concentrations present in clinical samples

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[23]. It is unclear how this approach can be applied many bacterial cells simultaneously, providing high
directly to complex matrix of clinical samples and throughput quantification of AST with single cell
polymicrobial systems. Given that eukaryotic cells can detection capability. In addition, PIT could potentially
also cause cantilever fluctuations [57], sample be used to spatially resolve and identify bacterial cells
preparation for this technique might need extraction even in a complex matrix of urine, serum, and other
of bacterial cells from complex matrix along with a body fluid samples, which is critical for developing
longer incubation of bacterial cells to attach the sensor PIT into a practical solution for testing real patient
surface, especially for low bacterial load patient samples.
samples. Flow cytometry (FC) measures changes in
A plasmonic imaging and tracking (PIT) morphology, cellular numbers, and viability via
technique has been used to track 3D motions of single labeling to perform AST [63–65]. After a dye is used to
bacterial cells associated with metabolic viability, thus stain viable cells, individual cells flow through a
leading to rapid AST [58, 59]. The PIT setup is built on channel into a reader zone, where light scattering is
an inverted optical microscope, where light from a used to measure morphology and
luminescence diode is directed onto the sensor chip excitation/emission spectra of cells is used to assess
made of gold-coated glass film with immobilized cell counts and viability. Multiple research studies
bacterial cells. Fig. 3a shows a few snapshots of the have shown the application of this technology using
plasmonic image, which reveal large fluctuations in various dyes [64, 65] applied to multiple bacterial
the image contrast of a bacterial cell. The image species and antibiotic combinations. Although flow
contrast fluctuations are due to the bacterial cell cytometry can produce rapid AST with 2-3 hours, it is
movement normal to the sensor surface (Z-direction) not a widely used technique yet. Possible
[60, 61], due to bacterial metabolism. Additionally, disadvantages are lack of use in complex patient
PIT has been shown to track the 3D movement of samples, staining inefficiency of dyes, presence of
single bacterial cells and sub-cellular organelles in 3D autofluorescence, inability to differentiate cellular
with < 5 nm spatial resolution and <1 millisecond damage caused by bactericidal or bacteriostatic
(ms) temporal resolution [61, 62]. This unprecedented antibiotics, and lack of clinical databases for
capability enables fast tracking of the 3D movement of validation [66].

Figure 3. Future technologies measuring bacterial nano-motion as a measure of bacterial metabolism to perform antibiotic susceptibility. a)
Snapshots of bacteria z-micro-motion. Panels a1-a4 show time differential images captured at various time points which show contrast of the bacteria versus the
background. The observation of the small contrast is due to micro-motions of the live bacterial cells. b) Z-displacement vs. time - The positions of minimum contrast
(a1 and a3) correspond to bacterial z-position farther away from the surface. The position of maximum contrast (a2) corresponds to z-position closest to the surface.
c) z-displacement plot of a dead bacterial cell (no motion) showing a standard deviation of 0.15 nm. Reprinted with permission from Syal K, Iriya R, Yang Y, Yu H,
Wang S, Haydel SE, et al. Antimicrobial Susceptibility Test with Plasmonic Imaging and Tracking of Single Bacterial Motions on Nanometer Scale. ACS Nano 2015;
10:845–852 Copyright 2017 American Chemical Society.

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Table 1. Summary of AST Technologies

AST Technologies Summary of Method Time of Direct on Real MIC FDA Reference
AST patient sample Approved
Current Technologies
Solid Media Cultures
1. Agar Dilution Assay Bacteria inoculated on agar plates with antibiotic discs of 16-24 Hours No Yes/No Yes [7]
different concentrations
2. Disk Diffusion Bacteria inoculated on agar plates with a single antibiotic disk 16-24 Hours No Yes/No Yes [7, 12]
3. E-test Bacteria inoculated on agar plates with a graded antibiotic 16-24 Hours No Yes Yes [7, 12]
concentration strips
Liquid Media Cultures
1. Broth Dilution Assay Bacteria inoculated in liquid media with different antibiotics to 12-24 Hours No Yes Yes [7, 10]
monitor growth
2. Automated Instruments
a) MicroScanWalkAway Measure bacterial growth in the presence of antibiotics by 4.5-18 No Yes Yes [8, 16]
recording bacterial turbidity using a photometer Hours
b) Vitek-1/Vitek-2 Measure bacterial growth in the presence of antibiotics by 6-11 Hours No Yes Yes [17 ,70]
recording bacterial turbidity using a photometer
c) BD Phoenix Record bacterial growth in the presence of antibiotics by 9-15 Hours No Yes Yes [71]
recording bacterial turbidity and colorimetric changes
d) Sensititre Record bacterial growth with antibiotics by measuring 18-24 Hours No Yes Yes [7]
fluorescence
Emerging Technologies
Imaging Based Tools
1. Multiplexed automated Image single bacteria growing into colonies with antibiotics and 3-5 Hours Yes (Urine, Yes Yes [31, 72, 73]
digital microscopy (MADM) quantify growth rates Blood)
2. Single-cell morphological Image single bacterial cell's morphology changes on antibiotic 3-4 Hours Yes (Urine) Yes No [32, 74]
analysis (SCMA) action
3. oCelloscope Measure growth of bacterial cells using low resolution optical 1-4 Hours Yes (Urine) Yes No [33]
system
Non-Imaging Based Tools
1. BacterioScan FLLS Measures bacterial numbers and sizes on antibiotic action 3-10 Hours Yes (Urine) Yes No [25, 43]
2. LifeScaleMicochannel Count bacterial cells and morphology changes on single cells > 3 Hours No Yes No [44]
Resonator post antibiotic action
3. Genefluidics Count 16s RNA increase as a proxy to bacterial growth 4 Hours Yes (Urine) Yes/No No [45, 75]
4. Smarticles Bacteriophages which express luciferase on growing cells - - - No [30]
Future Technologies
1. AFM Cantilever Measure cantilever fluctuations originating from bacterial < 2 Hours No Yes No [22]
motion as a proxy for metabolism
2. PIT Image and Quantify sub-nanometer motion of bacterial cells < 2 Hours Yes Unknown No [59]
3. Flow Cytometry Count viable bacterial cells using dyes 2-3 hours No Yes No [63]
4. IMC Heat signature of growing cells 3-14 Hours Yes Yes No [68]

Figure 4. Comparison of speed and direct application on clinical samples of current and emerging AST technologies

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Isothermal micro calorimetry (IMC) is a novel Acknowledgements

technique that measures cumulative heat and
We thank Gordon and Betty Moore Foundation,
generates heat curves of growing bacterial cells [67].
and CNSF (#2137008 and #2137902) for financial
Heat curves of growing bacteria are similar to the
support. We thank Rachael Liesman for providing
growth curve measured by standard turbidity
picture of E-test and John Popovich for providing the
detection instruments. Since the lower limit of
picture of disk-diffusion assay.
detection for IMC is ~ 104cfu/ml, the approach
enables a faster AST [67]. IMC produces AST results Competing Interests
within 3 hours using patient urine samples [68] and
The authors have declared that no competing
has been effective with various bacterial species,
interest exists.
including Mycobacterium tuberculosis [69], E. coli, and
S. aureus [67]. Although this new analytical tool uses a References
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