Accepted Article

DR. JAN MAREK (Orcid ID : 0000-0003-3708-0967)

Article type : Review Article

Antimicrobial susceptibility testing: currently used methods and

devices and the near future in clinical practice

Marketa Benkovaa,c, Ondrej Soukupb,c, Jan Mareka,c*

aDepartment of Epidemiology, bDepartment of Toxicology and Military Pharmacy, Faculty of

Military Health Sciences, University of Defence, Trebesska 1575, 500 05 Hradec Kralove, Czech
Republic
cBiomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec
Kralove, Czech Republic

*Corresponding author: Jan Marek; Department of Epidemiology, Faculty of Military Health

Sciences, University of Defence, Trebesska 1575, 500 05 Hradec Kralove, Czech Republic;
marekjjja@gmail.com

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/JAM.14704
This article is protected by copyright. All rights reserved
 Rapid identification of pathogen and its resistance to antimicrobial drugs, and subsequent
Accepted Article
appropriate antimicrobial treatment are essential for correct patient outcomes. Conventional
detection methods of bacterial resistance, such as disk-diffusion, broth microdilution and
automated instruments, are constantly widely used and primarily standardized. Nevertheless, the
results cannot be obtained earlier than 48 hours after receiving a sample, which may lead to
prolonged use or overuse of broad-spectrum antibiotics. Hence, there is a drive to develop and
introduce novel, faster, standardized, sensitive and specific methods with reliable results into
routine microbiological laboratory practice. Recently developed matrix-assisted laser
desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has been introduced in
recent years into laboratory practice, and methods based on microfluidics and microdroplets might
be introduced in the near future. This review is focused on the methods and instruments in use
both currently and in the foreseeable future, applicable to determine antimicrobial efficacy in
clinical microbiology laboratories.

Keywords: antibiotic susceptibility test, antimicrobial susceptibility testing, dilution method,

microcalorimetry, MALDI-TOF MS, microfluidic, microdroplet

Introduction

The performance of antimicrobial susceptibility testing (AST) of bacterial pathogens is an

important task to determine susceptibility to the antimicrobial agents and to detect possible drug
resistance in clinical microbiology laboratories. Empirical antibiotic therapy continues to be
effective for some bacteria: for example, there has not yet been any reported resistance of
Streptococcus pyogenes to penicillin. However, AST of the individual bacterial isolates is crucial
for species with acquired resistance determinants, such as extended spectrum beta-lactamase
producing Enterobacteriaceae, Streptococcus pneumoniae, Pseudomonas spp., Staphylococcus
spp., and Enterococcus spp. (Jorgensen and Ferraro 2009). The resistance has led to increasingly
frequent empiric treatment, such as glycopeptides and broad-spectrum beta-lactams, e.g.
carbapenems and piperacillin-tazobactam. However, the overuse of these reserved antimicrobial
drugs has led to the appearance and spread of multi-resistant microorganisms (Fair and Tor 2014;
Petersen et al. 2019).

This article is protected by copyright. All rights reserved

Accepted Article In the case of routine methods for AST, it usually takes at least 24 hours to obtain growth
of bacterial colonies and an additional 24 hours to obtain isolate characterization, namely
biochemical identification and phenotypic AST (Altaie and Dryja 1994; Faro et al. 2016). The
methods generally used afford qualitative results, using the susceptible, intermediate or resistant
categories, but some of them provide quantitative results, expressed as minimum inhibitory
concentration (MIC) (Jenkins and Schuetz 2012). The MIC is defined as the lowest drug
concentration that inhibits visible growth of a microorganism after a certain incubation. Typical is
overnight incubation, but this period can be prolonged for species such as anaerobes, which
require extended incubation time for growth under defined conditions. The range of antimicrobial
compound concentrations is usually performed in two-fold dilution and reported in mg l-1 or µg
ml-1. The specific MICs (breakpoints) distinguish susceptible and treatable from resistant and
untreatable microorganisms. However, breakpoints can vary according to the examined species
and the antibiotics (Andrews 2001; Wiegand et al. 2008). The standards of interpretation are
published by authorities such as the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) (EUCAST 2019a) and the Clinical and Laboratory Standards Institute (CLSI) located
in the USA (CLSI 2019).
Some of the phenotypic AST methods, which detect phenotypic resistance by measuring
bacterial growth in the presence of the antimicrobials being tested, have survived the development
of modern diagnostic tools in the clinical microbiology laboratories, such as disk diffusion and
broth microdilution methods (Idelevich and Becker 2019). The phenotypic AST has two
advantages compared to genotypic methods. The first advantage is the opportunity to predict drug
resistance as well as susceptibility. The second is the ability to enumerate the level of
susceptibility of a pathogen to the antimicrobial agent (quantitative AST) (Reller et al. 2009).
However, the classical manual methods, such as the disk diffusion assay, the Etest, agar and broth
dilution methods, are very time-consuming (Hudzicki 2009). The conventional methods, which
require a defined inoculum of an isolated bacterial culture, evaluate the growth of bacteria visually
or automatically. However, the culture is incubated for 16 to 20 hours, and some species require
even longer incubation (72 hours or more), according to breakpoints calibrated by EUCAST and
CLSI (Maurer et al. 2017). On the other hand, novel strategies for rapid classical AST concentrate
on more sensitive detection of growth, and rapid availability of AST results means reliable
prediction of success in antimicrobial treatment and improve outcomes for patients. Delays in
appropriate antibiotic therapy lead to prolonged hospitalization and higher patient mortality and

This article is protected by copyright. All rights reserved

 are associated with increased costs (Llor and Bjerrum 2014). Therefore, it has been important to
Accepted Article
accelerate the classical phenotypic methods or establish novel and faster techniques for phenotypic
AST.
Commercial semi-automated or automated instruments, such as the Vitek 2 (bioMérieux,
Marcy–L’Étoile, France), the Phoenix (Becton Dickinson Diagnostic Systems, Sparks, Maryland),
MicroScan (Beckman Coulter, Brea, California) etc., reduce incubation and hands-on times. These
automated devices, using optical systems for measuring subtle changes, determine bacterial
growth and antimicrobial susceptibility (Fredborg et al. 2013) and can produce results in a shorter
time period (6 to 12 hours) than conventional manual assessment (Reller et al. 2009). The
disadvantages and limitations include a complicated pre-analytical process, the requirement for a
relatively enormous number of viable organisms, the limited microorganism spectrum in the
database, and the costs (van Belkum and Dunne 2013).
To improve the sensitivity and speed of detection of bacterial resistance, novel techniques
such as several biosensors based on either chip calorimetry (Lerchner et al. 2011), electrical
conductivity (Spiller et al. 2006), millifluidic droplet analysis (Baraban et al. 2011) and surface
plasmon resonance (Chiang et al. 2009) have been developed. Regrettably, these methods allow
only a single-sample analysis. Real-time polymerase chain reactions (Espy et al. 2006),
microarrays (Miller and Tang 2009), mass spectrometry (Yiyan Li et al. 2017) and flow cytometry
(Broeren et al. 2013) have been evolved due to their high sensitivity, but they require special
probes, expensive facilities and technically qualified staff. For example, Fredborg et al. introduced
a real-time optical AST method, the oCelloScope. It is a small portable platform, based on time-
lapse imaging of 96 combinations of bacteria-antibiotics simultaneously, which provides real-time
detection of bacterial growth and antimicrobial effectiveness. This platform uses low bacterial
concentrations, provides reliable results and high sensitivity, and includes internal verification of
results. Results from the instrument can be obtained within 6 minutes in monocultures and in 30
minutes in complex samples (Fredborg et al. 2013).
Besides contemporary methods currently in use for routine AST, van Belkum and Dunne
(van Belkum and Dunne 2013) demonstrated and summarized also near-future alternative and
long-term alternative methodologies. To the near-future alternative, the following methods were
categorized: cantilever technology (weighing bacterial cells by changes in cantilever vibrations);
fluorescence-activated cell sorting (FACS; sizing and measuring differential fluorescence between
living and dead cells); magnetic bead spin (changes in spin of beads in a magnetic field as a

This article is protected by copyright. All rights reserved

 function of the number of attached bacteria); matrix-assisted laser desorption ionization - time of
Accepted Article
flight mass spectrometry (MALDI-TOF MS; detection of antibiotic degradation products);
microdroplets (monitoring of growth or substrate conversion in nanoliter droplets); and next-
generation sequencing of all cellular DNA/RNA. The second group of promising long-term
alternative methods to be tentatively used in the future involved: apoptosis markers (detection of
compounds produced upon programmed cell death); bacteriophage amplification (detection of
phage reproduction in living cells only); colorimetric detection of cell respiration; electronic noses
(direct detection of volatile organic compounds); impedance measurements (changes in electrical
characteristics of cell suspension); infrared (IR) spectroscopy (absorption characteristics of
bacteria exposed to IR light); liquid chromatography-electron spray ionization mass spectrometry
(LC-ESI MS; proteomics of living or dead cells and resistance proteins); metabolomics including
reactive oxygen species assessment (detection of changes in intracellular composition focused on
small molecules); microsound measurements (measuring of vibrational differences between living
and dead cells); nuclear magnetic resonance (NMR; assessment of the molecular composition of
complex mixtures); Raman spectroscopy (absorption characteristics of bacteria exposed to laser
light); and RNA sequencing (definition of gene expression differences by sequencing).
In this review, various AST methods, which are currently clinically used (such as
conventional methods, automated devices, microcalorimetry and methods based on polymerase
chain reactions) or potentially applicable in the foreseeable future (methods based on microfluidics
and microdroplets, MALDI-TOF MS), are described and their advantages and disadvantages are
summarized. Recent still expensive and complex optical and mechanical techniques are excluded
as they are not immediately clinically applicable.

1. Classical AST methods

1.1. Diffusion methods

1.1.1. Agar disk-diffusion method

Agar disk-diffusion assay, developed in 1940 (Heatley 1944), is one of the oldest methods
for routine AST and remains one of the most popular manual techniques for AST in clinical

This article is protected by copyright. All rights reserved

 microbiology laboratories (Matuschek et al. 2014). The main advantages are simplicity,
Accepted Article
reproducibility, ease in modifying antimicrobial disks, the possibility for use as a screening test
against numerous isolates, and last but not least low cost (Le Page et al. 2015).
Mueller-Hinton agar plates (90 mm diameter) are inoculated with a standardized inoculum
of the test microorganism (corresponding to 0.5 McFarland turbidity standard). Up to 12
commercially-prepared paper disks (approximately 6 mm in diameter) with desired concentrations
of the tested agent are placed on the inoculated agar surface. Agar plates are incubated under
suitable conditions, typically for 16-24 hours at 35-37 °C (CLSI 2018a; EUCAST 2019b).
The diameter of the growth-inhibition zones around each antibiotic disk is then measured
in millimetres and the diameter of the disk is also included in the result. This is performed
manually using a sliding caliper or a ruler which is held on the back of the inverted agar plate
(Lalitha 2004). Also the automatic system, ADAGIO Automated System (Bio-Rad Laboratories,
Inc., Hercules, CA, USA) (Idelevich et al. 2016; Strauss et al. 2020) or SIRscan automatic zone
reader (i2a, Montpellier Cedex, France) (Hombach et al. 2013), has already been marketed. The
disk-diffusion test provides qualitative results by categorizing the bacterial susceptibility as
susceptible, intermediate or resistant, and is not appropriate for determination of MIC (Nijs et al.
2003).
However, the diameter of the inhibition growth zone is related to MIC (Fig. 1) for a
particular combination of bacteria/antimicrobial drug, and an approximate MIC can be calculated
by comparing the inhibition zones with algorithms (Nijs et al. 2003; Laboratory Methods and
Strategies for Antimicrobial Susceptibility Testing 2015). The inhibition zone correlates inversely
with the MIC values - the larger the zone of growth inhibition, the lower is the antimicrobial drug
concentration required to inhibit the growth. However, the diffusibility of a given compound must
be taken into account (Bruin et al. 2013).

1.1.2. Antimicrobial gradient method

The antimicrobial gradient strip method combines the principles of the dilution and
diffusion methods. The Etest (bioMérieux, Marcy–L’Étoile, France), MIC Test Strip (Liofilchem,
Inc., Waltham, Massachusetts), M.I.C.Evaluator (Oxoid, Basingstoke, England) and Ezy MIC
Strip (HiMedia Laboratories Pvt. Ltd., Mumbai, India) are a commercially-available versions of
this method (Jönsson et al. 2018).

This article is protected by copyright. All rights reserved

Accepted Article The agar medium is previously inoculated with a bacterial lawn of the tested
microorganism (Wiegand et al. 2008). Thin test strips which are impregnated with an increasing
concentration gradient of the dried antimicrobial drug from one end to the other, are then laid on
the agar surface (Lalitha 2004). After overnight incubation, the MIC values are read by viewing
the strips from the top of the petri dish and determined at the point of intersection of the strip with
an elliptical growth inhibition zone.
As many as 5 or 6 strips (Fig. 2) may be placed in a radial pattern on the surface of the agar
plate (Citron et al. 1991). This test is rapid, easy to use and routinely used. However, it is a
relatively expensive method (Wiegand et al. 2008). Strips cost approximately $2-3 each, and
hence this approach becomes costly if more than a few drugs are tested. The method is best
applicable for only 1 or 2 drugs and for fastidious organisms requiring enriched medium or a
special incubation atmosphere. This method can be used for the determination of antibacterials,
antifungals and antimycobacterials (Citron et al. 1991; Huang et al. 1992; Jorgensen et al. 1994).

1.2. Dilution methods

Dilution methods are reference methods for the AST and are used to determine the MIC
values of the antimicrobial agents being tested on agar plates (agar dilution) or in broth medium
(broth microdilution or macrodilution) (EUCAST 2019a). Both the agar and broth dilution
methods may be used to quantitatively measure the antimicrobial activity against fastidious or
non-fastidious bacteria, yeasts and filamentous fungi (Balouiri et al. 2016).
Dilution methods have been used for the comparative testing of new antimicrobial agents,
after equivocal results of disk tests, for resistance surveillance, for susceptibility testing on strains
where disk tests may be unreliable, and when the clinical management requires quantitative results
(EUCAST 2019a; ISO 20776-1 2019).
Many guidelines have been approved for dilution AST of bacteria and fungi. CLSI and
EUCAST provide the most-recognized standards. Nevertheless, the standardized broth
microdilution methodology recommended by EUCAST and CLSI is fully aligned with the
international standard ISO 20776-1 (ISO 20776-1 2019). Standardization and careful control are
demanded for intra-laboratory and inter-laboratory reproducibility, inasmuch as the results may be
influenced by the method used (CLSI 2018b).

This article is protected by copyright. All rights reserved

Accepted Article 1.2.1. Broth macrodilution

The broth macrodilution (or tube-dilution) method was one of the earliest AST methods,
but should be superseded by the broth microdilution technique (Moreno et al. 2013). Its principal
disadvantages include the relatively large amount of space and reagents required, its labour-
intensive nature, and the possibility of errors in preparation of the antibiotic solutions for each test.
However, the advantage of this method is in providing quantitative results - the MIC (Jorgensen
and Ferraro 2009).
The procedure is performed by preparing two-fold dilutions of the antimicrobial agent
(expressed in µg ml-1) in a liquid growth medium dispensed in test tubes containing a minimum
volume (2 ml) of the standardized microbial suspensions adjusted to 0.5 McFarland turbidity scale
(Balouiri et al. 2016). Following overnight incubation at 37°C for 24 hours (bacterial strains) or at
25 °C for 4-10 days (fungal strains), the tubes are examined for the presence of visible microbial
growth by turbidity. The lowest concentration of the antimicrobial agent where the growth was
completely inhibited (no turbidity) represents the MIC (Salem and Ali 2016). The precision of
broth macrodilution was considered to be plus/minus one two-fold dilution concentration, due to
the manual preparation of the dilutions (CLSI 2018b).

1.2.2. Broth microdilution

Due to the miniaturization of the test, the broth microdilution method became more
practical and popular. This technique is performed by using small sterile disposable polystyrene
microtitration plates, usually containing 96 wells. Each well contains a volume between 0.1 - 0.2
ml and hence it allows testing of approximately 12 antibiotics over a range of 8 two-fold dilutions
on one plate (Wiegand et al. 2008; Moreno et al. 2013; ISO 20776-1 2019). The tray is filled with
serial two-fold dilutions of the antimicrobial agents, and then the plate is inoculated with a diluted
microbial suspension standardized to an optical density of 0.5 McFarland scale to achieve a final
microbial concentration of 1-5 x 105 CFU ml-1 (colony forming unit ml-1) (Brown-Elliott et al.
2012; Brook et al. 2013). The wells are mixed and the plate is incubated under suitable conditions
depending on the microorganism being tested (Balouiri et al. 2016). The MIC is determined
visually or by automated viewing device, a photometric analysis device at 620 nm (Moreno et al.
2013). The minimum bactericidal (MBC) and fungicidal (MFC) concentrations are determined by

This article is protected by copyright. All rights reserved

 subculturing from the microtitration plates on Müeller-Hinton agar (applies to bacteria, incubated
Accepted Article
for 24 h at 37 °C) and Sabouraud agar plates (applies to fungi, incubated for 48 h at 35 °C). MBC
and MFC values demonstrate the lowest concentration of an antimicrobial compound that reduces
the viability of the initial bacterial or fungal inoculum by ≥ 99.9 %. The test is conducted in
triplicate (de Oliveira et al. 2011).
The advantages of broth microdilution include reproducibility, the small amount of sample
required, and the low cost allowing large numbers of replicates. The method is more efficient and
easier than the macrodilution method (Moreno et al. 2013). The disadvantages of the method
include difficulty in the detection of contamination, inoculum viability and the inhibitory effect of
co-solvents used (e.g. dimethyl sulfoxide) (de Oliveira et al. 2011).

1.2.3. Agar dilution

The agar dilution method is a manual method not frequently used in the researched
literature (Moreno et al. 2013). This technique is standardised by the National Committee for
Clinical Laboratory Standards (NCCLS) (CLSI 2018b). The agar dilution method is performed by
incorporation of different concentrations of the antimicrobial agent into a molten agar medium,
usually using serial two-fold dilutions, followed by the inoculation of a standardized microbial
inoculum to the surface of the agar plate (Fig. 3) (Brook et al. 2013; Ge et al. 2013). The agar
plates are evaluated by visually comparing varying strains in the series, and thereafter, the MIC
endpoint is determined as the lowest antibiotic concentration that inhibits bacterial growth (Brook
et al. 2013).
The method is suitable for antibacterial as well as antifungal susceptibility testing and is
recommended as a standardized AST method for fastidious organisms, such as anaerobes and
Helicobacter spp. (CLSI 2016a). It has been also used for AST against Candida spp., Aspergillus
spp., Fusarium spp. and dermatophytes (Speeleveld et al. 1996; Mock et al. 1998; Menon et al.
2001; Imhof et al. 2003). The agar dilution technique is often preferred to broth dilution if a single
antimicrobial agent is tested against an array of isolates or if the compound may influence the
detection of microbial growth in a broth medium because of its coloring (CLSI 2016a).
Advantages of the agar dilution method include the capability to test several bacteria on the
same set of agar plates at the same time, and the ability to semi-automate the method using
inoculum replicators, which can transfer between 32 and 60 different bacterial inocula to a single

This article is protected by copyright. All rights reserved

 agar plate. A disadvantage of this method is that unless the method is automated it is labour-
Accepted Article
intensive and thus requires economic and technical resources (CLSI 2016b).

2. Automated and semiautomated devices

Automated bacterial identification and AST systems are widely used in clinical
microbiology laboratories which are under increasing pressure from clinicians to provide fast and
reliable AST and bacterial identification (ID) of bacterial isolates. Besides such primary goals of
these approaches, secondary goals include reduction of costs, reporting of results to a personal
computer via suitable interface, decrease in turnaround times, convenient selection of antibiotics,
resistant patterns-monitoring, and aiding in the diagnosis and treatment of infectious diseases
(Rhoads et al. 1995; Sader et al. 2006; Snyder et al. 2008).
Nevertheless, despite the advanced technology, such test systems might have limitations
and errors as well (Biedenbach et al. 1999; Steward et al. 2003; Saegeman et al. 2005), which can
have significant consequences for clinical outcomes (Micek et al. 2005). The most common
reported errors in a study on the accuracy of three automated instruments (VITEK, VITEK 2 and
MicroScan WalkAway) involved Pseudomonas aeruginosa and some species of the family
Enterobacteriaceae when these microorganisms were tested against β-lactam agents: negative
results led to updating of systems by product software changes and to recommendations to test
microorganism-antimicrobial agent combinations by alternative methods (Sader et al. 2006).
In 1973, the first generation of VITEK systems, known as AutoMicrobic System
(McDonnell Douglas Corp., St. Louis, Missouri), was developed as a by-product of US space
exploration. This instrument was designed for the detection, enumeration and identification of
bacteria and yeasts in clinical samples. It used dehydrated reagents and antimicrobials and was
composed of a disposable miniaturized plastic specimen-handling system, solid-state optics for
detection, and a minicomputer for control and processing. The enumeration and identification
results were automatically obtained in 13 hours when levels of the microorganism were more than
7 x 104 CFU (Aldridge et al. 1977). Within 10 years, the VITEK system had competitors, such as
the Autobac IDX (Pfizer Inc., Groton, Connecticut and General Diagnostics, Morris Plains, New
Jersey), the MS-2 (Abbott Diagnostics, Division of Abbott Laboratories, Dallas, Texas) and the
AutoScan-3 (MicroScan Corp., Hillsdale, New Jersey) (Almeida and Jorgensen 1983; DeGirolami

This article is protected by copyright. All rights reserved

 et al. 1983). The Autobac (Pfizer) and the MS-2 (Abbott) were first used for urine screening, but
Accepted Article
eventually for bacterial identification or AST as well (Costigan and Hollick 1984). However,
several currently available automated or semiautomated instruments have been presented recently,
for instance MicroScan WalkAway System (Beckman Coulter, Franklin Lakes, New Jersey), the
novel and more modern VITEK version - VITEK 2 (bioMérieux, Marcy–L’Étoile, France),
Sherlock Microbial Identification System (MIDI, Inc., Newark, Delaware), Sensititre AutoReader
and Sensititre ARIS2X (Trek Diagnostic Systems, Cleveland, Ohio) and the newest system BD
Phoenix 100 (BD Diagnostic Systems, Sparks, Maryland) (Eigner et al. 2005; O’Hara 2005).
Instrumentation for AST was reviewed by Felmingham and Brown in 2001 (Felmingham
and Brown 2001). They categorized the instruments by the type of AST method, e.g. disk
diffusion, agar dilution or broth dilution. Ten years later, Winstanley and Courvalin (Winstanley
and Courvalin 2011) reviewed Expert Systems in Clinical Microbiology – non-commercial and
commercial systems. Some systems include “expert software” for improving the interpretation
quality by rules-based filtering of results. The “expert” systems include Accuzone (AccuMed
International Inc., West Lake, Ohio), Wider (Francisco Soria Melguizo, Madrid, Spain), Aura
Image (Oxoid, Basingstoke, United Kingdom), Biomic Vision/V3 (Giles Scientific, Santa Barbara,
California), BioVideobact (Launch Diagnostics, Longfield, United Kingdom), Mastascan Elite
(Mast Group Ltd., Bootle, United Kingdom), Osiris (Bio-Rad, Hemel Hempstead, United
Kingdom), ProtoZONE (Don Whitley Scientific, Shipley, United Kingdom), Mini API
(bioMérieux, La-Balme-les-Grottes, France), ATB Plus Expert (bioMérieux, La-Balme-les-
Grottes, France), Vitek Legacy (bioMérieux, La-Balme-les-Grottes, France), SIRSCAN (Becton
Dickinson, Oxford, United Kingdom) and the above-mentioned Sensititre ARIS (Trek Diagnostic
Systems, Cleveland, Ohio), Phoenix (Becton Dickinson Diagnostic Systems, Sparks, Maryland),
MicroScan (Beckman Coulter, Brea, California) and VITEK 2 (bioMérieux, Marcy–L’Étoile,
France). The detailed description of all instrumental devices is out of scope of the review and only
the most significant instrumental devices are more described below.

2.1. VITEK and VITEK 2 Systems

The VITEK device (bioMérieux, Marcy–L’Étoile, France) is an automated photometric

system used for ID and AST of both gram-negative and gram-positive bacteria. The VITEK 2

This article is protected by copyright. All rights reserved

 System is the next generation of the first instrument. The original VITEK can process 32 or 120
Accepted Article
test cards at a time, and the VITEK 2 can process 60 or 120 cards. In the VITEK 2 System, it is
necessary to prepare primary suspensions by emulsification of bacterial isolates in 0.45 % saline
adjusted to the equivalent of 0.5 McFarland turbidity standard. All the steps required for ID and
AST are then performed automatically (Rhoads et al. 1995; Funke et al. 1998; Ligozzi et al. 2002;
Spanu et al. 2003). This instrument reads the kinetics of the growth every 15 minutes using an
optical system which combines photometer and multichannel fluorimeter readings to assess
fluorescence, turbidity and colorimetric signals (Ligozzi et al. 2002).

2.2. MicroScan WalkAway System

The first generation of the MicroScan WalkAway System, the AutoSCAN-3, was
introduced in 1981 by American MicroScan in Hillsdale (New Jersey). This semiautomated
instrument used microdilution trays containing frozen substrates. Two years later, in 1983, the
autoSCAN-4 had been developed. This next-generation model utilized dry panels that were not
refrigerated and an updated database. Thereafter, the autoSCAN-WalkAway was introduced in
1986. This version is automated and incorporates an incubator-reader that monitors the growth in
identification panels. In 1989, panels using non-frozen fluorogenic substrates for shorter
turnaround times penetrated the market. The present MicroScan WalkAway System (nowadays
Beckman Coulter, Brea, California), an automated and commercially available instrument, can
process 96 panels at the same time and uses LabPro data management system on a contiguous
computer. For detecting bacterial enzymatic activity, the system uses fluorogenic substrates and a
pH indicator (Ellner and Myers 1981; Rodriguez et al. 1999).

2.3. Trek Diagnostic Systems

Trek Diagnostic Systems (Cleveland, Ohio), founded in 1999, has two instrument
configurations. The first is the Sensititre AutoReader which is a fully-automatic fluorometer
reader controlled by microprocessor. Since the AutoReader has off-line incubation, it can measure
an unlimited number of test panels at one time. The second configuration, the Sensititre ARIS 2X,
is a fully-automated overnight benchtop incubating and reading system. This instrument uses

This article is protected by copyright. All rights reserved

 internal bar codes for panel identification and setting of incubation times. 96-well microdilution
Accepted Article
plates are inoculated with a Sensititre AutoInoculator, incubated for 18-24 hours, and
automatically transferred to the fluorometric AutoReader for monitoring of growth following
hydrolysis of the fluorogenic substrates. The instrument has a 64-plate capacity, was developed for
overnight susceptibility testing, and its test panels are available for gram-positive and gram-
negative bacteria (Felmingham and Brown 2001; O’Hara 2005).

2.4. BD Phoenix System

The BD Phoenix System, designed and marketed by Becton Dickinson Diagnostic Systems
(Sparks, Maryland), was introduced in 2003 for bacterial identification and AST. The BD Phoenix
AP instrument was introduced to automate the setup of the isolates, namely adjusts the turbidity of
the bacterial suspension to a 0.5 McFarland standard, prepares a dilution and adds the AST
indicator to the inoculum for susceptibility testing. By these steps, the instrument reduced the
hands-on processing time required to prepare test panels by 50% (Junkins et al. 2010). The
Phoenix has a huge incubator reader and can process 99 test panels at one time (one panel holder
is assigned to the internal thermometer), and test panels contain 84 wells for the antibiotic two-
fold dilutions (Carroll et al. 2006). Each panel is monitored every 20 minutes by colorimetric and
fluorescence readings for ID, and AST is based on redox and turbidimetric detection of growth.
The results are printed when each panel is measured. MIC results are available in 6-16 hours. Test
panels are designated for both gram-positive and gram-negative microorganisms (Richter and
Ferraro 2007; Jorgensen and Ferraro 2009).

3. Isothermal microcalorimetry

Using real-time isothermal microcalorimetry (IMC), the heat generated or consumed by

chemical or physical processes in microorganisms can be measured (von Ah et al. 2009).
Cumulative heat production increases or decreases in parallel with growth curves, and
correspondingly with lag, log and stationary phases. The total number of cells, i.e. maximum
bacterial growth rate, is represented by maximum heat production. The antibiotic dose, at first,
increases the rate of heat production due to the activity of resistant mechanisms that are energy-

This article is protected by copyright. All rights reserved

 dependent, and afterwards heat production decays due to the loss of activity and death of the
Accepted Article
bacteria (Fig. 4) (van Belkum and Dunne 2013).
Passive measuring is carried out within small sealed thermostated ampoules of 3-20 ml,
containing the microrganism, antimicrobial agent in double-diluted concentrations, and growth
medium. The minimum heat inhibitory concentration (MHIT) denotes the lowest concentration of
a drug for 50% reduction in heat flow rate, depending on the drug being tested, or 50% inhibition
of total heat production (Lewis and (Dan) Daniels 2003; von Ah et al. 2009).
IMC has been used to determine the drug susceptibility of various species, e.g. S. aureus,
E. coli (Schumacher et al. 2018), methicillin-resistant S. aureus (MRSA) (Baldoni et al. 2009),
Mycobacterium spp. (Howell et al. 2012; Boillat-Blanco et al. 2015) and Aspergillus spp. (Tafin et
al. 2012). The major advantages of the method include testing in sealed vials that moderates safety
concerns during the evaluation of high-risk organisms such as M. tuberculosis or fungal species,
and the possibility to differentiate quickly bacterial species susceptible or resistant to antibiotics
(Bonkat et al. 2012). However, no official standardization documents are available, even though
IMC results were found to be comparable to standardized methods (Braissant et al. 2014).

4. Polymerase chain reaction-based methods

Polymerase chain reaction (PCR)-based genotypic techniques, both conventional and real-
time, are commercially available as assays and automated machines. PCR relies on the
amplification of DNA sequences that are specific for a particular pathogen and its drug
susceptibility or resistance. PCR was initially used for the rapid identification and quantification of
the pathogen (Itahashi et al. 2010), but thereafter, with increased knowledge of the genetic basis of
resistance to antibiotics, PCR-based approaches have been developed to detect the presence of
genetic determinants of resistance to different antimicrobial agents (Fluit et al. 2001). A salient
example of PCR application includes the mecA gene, commonly found in MRSA, encoding a
modified penicillin-binding protein PBP2a with reduced affinity to beta-lactam antibiotics
(Wielders et al. 2002; Fishovitz et al. 2014). This method can also detect the presence of
resistance genes in gram-negative bacteria (such as carbapenemase- and cephalosporinase-
encoding genes) (Codjoe and Donkor 2017; Walker et al. 2019), and vancomycin resistance
associated with the vanA and vanB genes primarily for resistance in Enterococcus species (Cekin

This article is protected by copyright. All rights reserved

 et al. 2013). Loop-mediated isothermal amplification (LAMP) is genotypic method for the
Accepted Article
detection of antibiotic resistance and evaluation of AST based on PCR (amplification of DNA).
The gene is amplified using Bst DNA polymerase instead of Taq polymerase at a constant
temperature of 60-65 °C (Yanmei Li et al. 2017).
Real-time PCR, using hydrolysis probes, hybridization probes, or double-stranded DNA-
binding fluorescent dyes (Matsuda 2017), uses the ability to quantify the number of specific
nucleic acid copies in a clinical sample for measuring the growth of bacteria in the presence of the
antibiotic being tested, and indirectly measures phenotypic resistance. Moreover, the sample does
not always need to be purified, can be non-sterile, and may contain bacterial mixtures. In general,
PCR-based methods are efficient, reliable and fast screening tools with high specificity and
sensitivity, and with the test results available within 2 hours (Pulido et al. 2013; Schumacher et al.
2018).

5. MALDI-TOF MS

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (known as

MALDI-TOF MS) was discovered in the 1980s and the device analyzed relatively large
biomolecules (Hillenkamp and Karas 1990). However, the first attempts to use mass spectrometry
for microorganism identification date back to 1975 (Anhalt and Fenselau 1975). In recent years,
MALDI-TOF MS has been introduced into clinical microbiology laboratories as a time- and cost-
saving method based on the determination of mass/charge (m/z) ratio and strain differentiation
using their ribosomal proteins (Maxson et al. 2017). MALDI-TOF MS does not depend on
metabolic activity and can directly identify within minutes colonies of gram-positive and gram-
negative bacteria and yeasts grown on agar plates (Wieser et al. 2012).
Generally, the first step in identification using MALDI-TOF MS is co-crystallization,
where the crystal is created by mixing the microbial sample (for bacteria 104 to 106 CFU) and a
matrix solution (a benzoic acid or cinnamic acid derivative), and is formed and placed on the steel
surface of the target plate to dry at room temperature. The target plate can hold up to 384 samples.
The spotted target is then inserted into the machine and transported to the measuring chamber in
the vacuum environment. The laser beams desorb the ribosomal proteins of the bacteria or yeasts,
contributing to ionization of the proteins. Using an electromagnetic field, the ions are accelerated

This article is protected by copyright. All rights reserved

 in a pulsed fashion into a linear flight tube. The molecular mass (m) and the charge (z) determine
Accepted Article
the time of flight (TOF) of the desorbed particles. The TOF is measured using a detector at the end
of the vacuum flight tube. The generated mass spectrum is compared to reference spectra in the
database by the use of algorithms, and the resulting fingerprint is unique for an individual species
(Steensels et al. 2011; Wieser et al. 2012).
Besides, the technique has been successfully used to detect specific resistance mechanisms,
e.g. the activity of antibiotic-inactivating enzymes by quantifying enzyme activities (e.g. detection
of carbapenemases in less than 30 minutes and quantification of β-lactamases within a turnaround
time of 150 minutes) in bacteria towards antibiotics (Burrer et al. 2015; Lasserre et al. 2015) and
for direct detection of genes indicative of resistance to antibiotics (e.g. vanA and mecA) (Griffin et
al. 2012; Rhoads et al. 2016). Since the latter detection depends on the identification of specific
resistance mechanisms, the assessment is only applicable with certain antibiotics. Semi-
quantitative MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) for AST
appears to be a possible approach by measuring the relative growth rate of bacteria exposed to an
antimicrobial drug in comparison to untreated controls. For the determination of resistance, MBT-
ASTRA utilizes information on growth rate calculated from the total area under the curve (AUC)
of all peaks, and therefore, the assay does not depend on the resistance mechanism and is utilizable
with any antibiotic (Maxson et al. 2017).
MALDI-TOF MS has also been used with success in research to identify proteins and
peptides (Marvin et al. 2003). Despite the high acquisition price of the instrument, the cost of
consumables is low, and therefore the device is of benefit to laboratories handling numerous
identifications of pathogens (Steensels et al. 2011). However, application of the device into the
routine for rapid AST requires further validation, such as standardized protocols, test kits and
software (Clark et al. 2013; Maurer et al. 2017).

6. Microfluidic devices and microdroplets

In the last decade, microfluidic methods have been developed within advances in
nanotechnology and bioengineering as a miniaturization of molecular assays. These so-called “lab-
on-a-chip” platforms can be used for antibiotic susceptibility and toxicity testing (Kim et al.
2019). Devices can also be designed for several further functionalities, such as nucleic acid

This article is protected by copyright. All rights reserved

 hybridization and amplification, and cell lysis, and have been widely used in the field of single cell
Accepted Article
studies (Gao et al. 2017; Gorgannezhad et al. 2019). Functionalities can even be combined.
Microfluidic platforms are cheap and fast, provide accurate screening, benefit from efficient heat
and mass transfer and short diffusion distances, show the potential for high throughput due to a
high surface area to volume ratio, and they can be highly automated (Dai et al. 2016; Schumacher
et al. 2018; Zhu et al. 2018).
These techniques are based on micro-channels, i.e. 10-100 µm, and the extremely small
volumes of reagent and analyte in nanolitres (Zhu et al. 2018) or picolitres (Hayat and El Abed
2018) can flow through the channels on one chip. The detection methods depend on the device
used and vary widely, and are generally electrochemical, magnetic or optical/microcalorimetric
(Schumacher et al. 2018). Microfluidic AST methods have a big potential for application in
clinical laboratories due to a reduction in materials and workload, but have so far been used only
for antimicrobial research (Chen et al. 2010; Kalashnikov et al. 2012).
Owing to the small size of the chip, the method can be applied to portable devices (Dai et
al. 2016; Zhu et al. 2018). Cira et al. (Cira et al. 2012) developed a self-loading microfluidic
instrument which is a portable point-of-care device that determines the MIC with a single pipetting
step. Chambers moulded in a polydimethylsiloxane (PDMS) layer are filled with dried antibiotics
and reversibly sealed with a second layer of PDMS that contains channels connecting the
chambers. For determining the MIC, glucose and phenol red are added as a pH indicator into the
cell suspension, and the device is then incubated 18 hours. Bacteria degrade glucose (during their
growth), which produces organic acids that decrease the pH of the solution. The process can be
observed visually by a colorimetric change using ambient light (Cira et al. 2012).
Tang et al. (Tang et al. 2013) developed a microfluidic pH sensor where the pH-responsive
chitosan hydrogel is immobilized in a microfluidic channel (Fig. 5). The chitosan hydrogel can
swell and shrink in response to pH changes. The swelling and shrinking can be evaluated in real
time by Fourier transform reflective interferometric spectroscopy. The effective optical thickness
(EOT) is inversely related to the pH and quantified. Higher EOT:pH ratio means higher activity of
bacteria, whereas a lower EOT:pH ratio indicates lower bacterial activity. Bacterial growth curves
can be generated within 2 hours (Tang et al. 2013).
Choi et al. (Choi et al. 2013) demonstrated a microfluidic agarose channel (MAC), where
bacteria were integrated into agarose in a microfluidic culture chamber in the presence of
antibiotics. The growth of single cells can be tracked by microscopy. Time-lapse images were

This article is protected by copyright. All rights reserved

 analyzed by CCD (charge-coupled device) camera and the MIC determination was performed in
Accepted Article
only 3-4 hours (Choi et al. 2013).
The microdroplet method is a subcategory of microfluidics based on micro- or
nanodroplets that can be used as a small individual transport reaction vessel where
microorganisms are embedded. These inverse emulsion droplets, that can be individually
manipulated, are carried in dispersed liquid phase which is injected into the stream of a continuous
phase. Both phases are immiscible liquids (Basova and Foret 2015). When droplets contain
bacteria in sufficient numbers, the metabolic activity and viability of cells can be determined by
fluorescence measurement inside each droplet due to changes in cell number (Lu et al. 2017).
In general, microfluidic systems are characterized by low-Reynolds number flow which
indicates laminar fluid flow. A system was developed for AST consisting of 100nL aqueous
emulsion droplets containing encapsulated bacteria (103 per droplet) and different concentrations
of antimicrobial drug in a drop-maker module, with a fluorescence detector equipped with electric
valves. The fluorescence signal which is emitted from each droplet when the droplet passes the
detection system is recorded and the growth curves can be created at each drug concentration by
measuring over time. However, for evaluation as a routine AST method, it is necessary to develop
reference databases of MIC and assess the reproducibility (Baraban et al. 2011).

7. Conclusion

In this review, currently-used or potential methods for AST in routine laboratories were
described and summarized. In the pursuit of decreased time-to-result and empirical treatment of
patients, constant development of novel methods for AST and microorganism identification is
necessary. However, the use of traditional methods, such as broth microdilution and disk
diffusion, is consistently required in clinical practice to obtain the correct results according to the
standardized protocols of EUCAST and CLSI, or for comparison against the results of novel
techniques. However, there is a requirement to have a sufficiently grown bacterial inoculum for
the subsequent antimicrobial testing, and therefore the time to patient outcome is prolonged.
Automated systems have benefits in hands-on time reduction and fast, reliable microorganism
identification and AST results. The major advantage of IMC involves the determination of
susceptible versus resistant species in a short time, but the disadvantage is that official validated
protocols are unavailable even though IMC results are comparable to standardized methods. The

This article is protected by copyright. All rights reserved

 PCR (e.g. LAMP technique) enables evaluation of AST and detects the occurrence of genetic
Accepted Article
determinants of resistance. However, it should be point out that the occurrence of resistance genes
does not always result in phenotypic resistance since a specific resistance genotype can be partly
or fully disconnected from the phenotype. In general, PCR-based methods are efficient, reliable
and fast with high specificity and sensitivity, and with the test results available within 2 hours.
Recently, MALDI-TOF MS is being introduced into routine practice. It is able to identify the
proteins of pathogens and appears to be a promising highly specific technique with reduced time
and low cost of consumables, but regrettably this method is not yet endowed with standardized
protocols, software and equipment for AST, and the acquisition costs and its maintenance are
high. The miniaturization of microfluidic and microdroplet methods is a significant benefit
because of lower material costs and possible portability. While microfluidics methods have been
only used in AST research and require further improvement, the techniques have a huge potential
to make a breakthrough into microbiological laboratories. However, the latest optical and
mechanical devices are still expensive and complex (excluded from the review), and thus
nonapplicable into clinical laboratories in the foreseeable future.

8. Acknowledgements
The work was supported by the Ministry of Health of the Czech Republic (NV18-09-00181)
and by the Ministry of Education, Youth and Sports of the Czech Republic (student grant
SV/FVZ201607). The authors are grateful to Ian McColl MD, PhD for assistance with the
manuscript.

9. Conflict of interest
No conflict of interest declared.

This article is protected by copyright. All rights reserved

 10. References
Accepted Article
von Ah, U., Wirz, D. and Daniels, A. (2009). Isothermal micro calorimetry - a new method for
MIC determinations: results for 12 antibiotics and reference strains of E. coli and S. aureus. BMC
Microbiol 9:106. doi: https://doi.org/10.1186/1471-2180-9-106.

Aldridge, C., Jones, P., Gibson, S., Lanham, J., Meyer, M., Vannest, R. and Charles, R. (1977).
Automated Microbiological Detection-Identification System. J Clin Microbiol 6(4):406–413.

Almeida, R.J. and Jorgensen, J.H. (1983). Rapid determination of novobiocin resistance of
coagulase-negative staphylococci with the MS-2 system. J Clin Microbiol 17(3):558–560.

Altaie, S.S. and Dryja, D. (1994). Detection of group B Streptococcus comparison of solid and
liquid culture media with and without selective antibiotics. Diagn Microbiol Infect Dis 18(3):141–
144. doi: https://doi.org/10.1016/0732-8893(94)90082-5.

Andrews, J.M. (2001). Determination of minimum inhibitory concentrations. J Antimicrob

Chemother 48 Suppl 1:5–16.

Anhalt, J.P. and Fenselau, Catherine. (1975). Identification of bacteria using mass spectrometry.
Anal Chem 47(2):219–225. doi: https://doi.org/10.1021/ac60352a007.

Baldoni, D., Hermann, H., Frei, R., Trampuz, A. and Steinhuber, A. (2009). Performance of
Microcalorimetry for Early Detection of Methicillin Resistance in Clinical Isolates of
Staphylococcus aureus. J Clin Microbiol 47(3):774–776. doi: https://doi.org/10.1128/JCM.02374-
08.

Balouiri, M., Sadiki, M. and Koraichi Ibnsouda, S. (2016). Methods for in vitro evaluating
antimicrobial activity: A review. J Pharm Anal 6(2):71–79. doi:
https://doi.org/10.1016/j.jpha.2015.11.005.

Baraban, L., Bertholle, F., Salverda, M.L.M., Bremond, N., Panizza, P., Baudry, J., … Bibette, J.
(2011). Millifluidic droplet analyser for microbiology. Lab Chip 11(23):4057–4062. doi:
https://doi.org/10.1039/c1lc20545e.

Basova, E.Y. and Foret, F. (2015). Droplet microfluidics in (bio)chemical analysis. Analyst
140(1):22–38. doi: https://doi.org/10.1039/c4an01209g.

This article is protected by copyright. All rights reserved

 van Belkum, A. and Dunne, W.M. (2013). Next-Generation Antimicrobial Susceptibility Testing.
Accepted Article
J Clin Microbiol 51(7):2018–2024. doi: https://doi.org/10.1128/JCM.00313-13.

Biedenbach, D.J., Marshall, S.A. and Jones, R.N. (1999). Accuracy of cefepime antimicrobial
susceptibility testing results for Pseudomonas aeruginosa tested on the MicroScan WalkAway
System. Diagn Microbiol Infect Dis 33(4):305–307. doi: https://doi.org/10.1016/S0732-
8893(98)00159-X.

Boillat-Blanco, N., Tafin, U.F., Jaton, K. and Trampuz, A. (2015). Susceptibility testing of
Mycobacterium abscessus by isothermal microcalorimetry. Diagn Microbiol Infect Dis 83(2):139–
143. doi: https://doi.org/10.1016/j.diagmicrobio.2015.06.006.

Bonkat, G., Bachmann, A., Solokhina, A., Widmer, A.F., Frei, R., Gasser, T.C. and Braissant, O.
(2012). Growth of Mycobacteria in Urine Determined by Isothermal Microcalorimetry:
Implications for Urogenital Tuberculosis and Other Mycobacterial Infections. Urology
80(5):1163.e9-1163.e12. doi: https://doi.org/10.1016/j.urology.2012.04.050.

Braissant, O., Müller, G., Egli, A., Widmer, A., Frei, R., Halla, A., Wirz, D., Gasser, T.C.,
Bachmann, A., Wagenlehner, F. and Bonkat, G. (2014). Seven hours to adequate antimicrobial
therapy in urosepsis using isothermal microcalorimetry. J Clin Microbiol 52(2):624–626. doi:
https://doi.org/10.1128/JCM.02374-13.

Broeren, M.A.C., Maas, Y., Retera, E. and Arents, N.L.A. (2013). Antimicrobial susceptibility
testing in 90 min by bacterial cell count monitoring. Clin Microbiol Infect 19(3):286–291. doi:
https://doi.org/10.1111/j.1469-0691.2012.03800.x.

Brook, I., Wexler, H.M. and Goldstein, E.J.C. (2013). Antianaerobic Antimicrobials: Spectrum
and Susceptibility Testing. Clin Microbiol Rev 26(3):526–546. doi:
https://doi.org/10.1128/CMR.00086-12.

Brown-Elliott, B.A., Nash, K.A. and Wallace, R.J. (2012). Antimicrobial Susceptibility Testing,
Drug Resistance Mechanisms, and Therapy of Infections with Nontuberculous Mycobacteria. Clin
Microbiol Rev 25(3):545–582. doi: https://doi.org/10.1128/CMR.05030-11.

This article is protected by copyright. All rights reserved

 Bruin, J.P., Diederen, B.M.W., IJzerman, E.P.F., Den Boer, J.W. and Mouton, J.W. (2013).
Accepted Article
Correlation of MIC value and disk inhibition zone diameters in clinical Legionella pneumophila
serogroup 1 isolates. Diagn Microbiol Infect Dis 76(3):339–342. doi:
https://doi.org/10.1016/j.diagmicrobio.2013.03.001.

Burrer, A., Findeisen, P., Jaeger, E., Ghebremedhin, B., Grundt, A., Ahmad-Nejad, P., …
Neumaier, M. (2015). Rapid detection of cefotaxime-resistant Escherichia coli by LC-MS. Int J
Med Microbiol 305(8):860–864. doi: https://doi.org/10.1016/j.ijmm.2015.08.004.

Carroll, K.C., Borek, A.P., Burger, C., Glanz, B., Bhally, H., Henciak, S. and Flayhart, D.C.
(2006). Evaluation of the BD Phoenix automated microbiology system for identification and
antimicrobial susceptibility testing of staphylococci and enterococci. J Clin Microbiol 44(6):2072–
2077. doi: https://doi.org/10.1128/JCM.02636-05.

Cekin, Y., Erman Daloğlu, A., Oğünç, D., Ozhak Baysan, B., Dağlar, D., Inan, D., … Colak, D.
(2013). Evaluation of vancomycin resistance 3 multiplexed PCR assay for detection of
vancomycin-resistant enterococci from rectal swabs. Ann Lab Med 33(5):326–330. doi:
https://doi.org/10.3343/alm.2013.33.5.326.

Chen, C.H., Lu, Y., Sin, M.L.Y., Mach, K.E., Zhang, D.D., Gau, V., … Wong, P.K. (2010).
Antimicrobial Susceptibility Testing Using High Surface-to-Volume Ratio Microchannels. Anal
Chem 82(3):1012–1019. doi: https://doi.org/10.1021/ac9022764.

Chiang, Y.-L., Lin, C.-H., Yen, M.-Y., Su, Y.-D., Chen, S.-J. and Chen, H. (2009). Innovative
antimicrobial susceptibility testing method using surface plasmon resonance. Biosens Bioelectron
24(7):1905–1910. doi: https://doi.org/10.1016/j.bios.2008.09.020.

Choi, J., Jung, Y.-G., Kim, J., Kim, S., Jung, Y., Na, H. and Kwon, S. (2013). Rapid antibiotic
susceptibility testing by tracking single cell growth in a microfluidic agarose channel system. Lab
Chip 13(2):280–287. doi: https://doi.org/10.1039/c2lc41055a.

Cira, N.J., Ho, J.Y., Dueck, M.E. and Weibel, D.B. (2012). A self-loading microfluidic device for
determining the minimum inhibitory concentration of antibiotics. Lab Chip 12(6):1052–1059. doi:
https://doi.org/10.1039/c2lc20887c.

This article is protected by copyright. All rights reserved

 Citron, D.M., Ostovari, M.I., Karlsson, A. and Goldstein, E.J. (1991). Evaluation of the E test for
Accepted Article
susceptibility testing of anaerobic bacteria. J Clin Microbiol 29(10):2197–2203.

Clark, A.E., Kaleta, E.J., Arora, A. and Wolk, D.M. (2013). Matrix-assisted laser desorption
ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical
microbiology. Clin Microbiol Rev 26(3):547–603. doi: https://doi.org/10.1128/CMR.00072-12.

CLSI (2016a). CLSI document M45 – Methods for Antimicrobial Dilution and Disk Susceptibility
Testing of Infrequently Isolated or Fastidious Bacteria, 3rd Edition.

CLSI (2016b). Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently
Isolated or Fastidious Bacteria, CLSI guideline M45. 3rd Edition.

CLSI (2018a). CLSI Guideline M02 – Performance Standards for Antimicrobial Disk
Susceptibility Tests. 13th Edition.

CLSI (2018b). CLSI Guideline M07 – Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria That Grow Aerobically. 11th Edition.

CLSI (2019). CLSI document M100 – Performance Standards for Antimicrobial Susceptibility
testing. 29th Edition.

Codjoe, F.S. and Donkor, E.S. (2017). Carbapenem Resistance: A Review. Med Sci (Basel) 6(1):1.
doi: https://doi.org/10.3390/medsci6010001.

Costigan, W.J. and Hollick, G.E. (1984). Use of the Autobac IDX system for rapid identification
of Enterobacteriaceae and nonfermentative gram-negative bacilli. J Clin Microbiol 19(2):301–302.

Dai, J., Hamon, M. and Jambovane, S. (2016). Microfluidics for Antibiotic Susceptibility and
Toxicity Testing. Bioengineering (Basel) 3(4). doi:
https://doi.org/10.3390/bioengineering3040025.

DeGirolami, P.C., Eichelberger, K.A., Salfity, L.C. and Rizzo, M.F. (1983). Evaluation of the
AutoSCAN-3, a device for reading microdilution trays. J Clin Microbiol 18(6):1292–1295.

This article is protected by copyright. All rights reserved

 Eigner, U., Schmid, A., Wild, U., Bertsch, D. and Fahr, A.M. (2005). Analysis of the comparative
Accepted Article
workflow and performance characteristics of the VITEK 2 and Phoenix systems. J Clin Microbiol
43(8):3829–3834. doi: https://doi.org/10.1128/JCM.43.8.3829-3834.2005.

Ellner, P.D. and Myers, D.A. (1981). Preliminary evaluation of the autoSCAN-3, an instrument
for automated reading an interpretation of microdilution trays: identification of aerobic gram-
negative bacilli. J Clin Microbiol 14(3):326–328.

Espy, M.J., Uhl, J.R., Sloan, L.M., Buckwalter, S.P., Jones, M.F., Vetter, E.A., Yao, J.D.C.,
Wengenack, N.L., Rosenblatt, J.E., Cockerill, F.R. and Smith, T.F. (2006). Real-time PCR in
clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 19(1):165–
256. doi: https://doi.org/10.1128/CMR.19.1.165-256.2006.

EUCAST (2019a). The European Committee on Antimicrobial Susceptibility Testing. Breakpoint

tables for interpretation of MICs and zone diameters. Version 9.0.

EUCAST (2019b). The European Committee on Antimicrobial Susceptibility Testing. Disk

Diffusion Method for Antimicrobial Susceptibility Testing - Version 7.0.

Fair, R.J. and Tor, Y. (2014). Antibiotics and bacterial resistance in the 21st century. Perspect
Medicin Chem 6:25–64. doi: https://doi.org/10.4137/PMC.S14459.

Faro, J., Mitchell, M., Chen, Y.-J., Kamal, S., Riddle, G. and Faro, S. (2016). Development of a
Novel Test for Simultaneous Bacterial Identification and Antibiotic Susceptibility. Infect Dis
Obstet Gynecol 2016:5293034–5293034. doi: https://doi.org/10.1155/2016/5293034.

Felmingham, D. and Brown, D.F.J. (2001). Instrumentation in antimicrobial susceptibility testing.

J Antimicrob Chemother 48:81–85. doi: https://doi.org/10.1093/jac/48.suppl_1.81.

Fishovitz, J., Hermoso, J.A., Chang, M. and Mobashery, S. (2014). Penicillin-binding protein 2a
of methicillin-resistant Staphylococcus aureus. IUBMB life 66(8):572–577. doi:
https://doi.org/10.1002/iub.1289.

Fluit, A.C., Visser, M.R. and Schmitz, F.J. (2001). Molecular detection of antimicrobial resistance.
Clin Microbiol Rev 14(4):836–871. doi: https://doi.org/10.1128/CMR.14.4.836-871.2001.

This article is protected by copyright. All rights reserved

 Fredborg, M., Andersen, K.R., Jorgensen, E., Droce, A., Olesen, T., Jensen, B.B., Rosenvinge,
Accepted Article
F.S. and Sondergaard, T.E. (2013). Real-Time Optical Antimicrobial Susceptibility Testing. J Clin
Microbiol 51(7):2047–2053. doi: https://doi.org/10.1128/JCM.00440-13.

Funke, G., Monnet, D., deBernardis, C., von Graevenitz, A. and Freney, J. (1998). Evaluation of
the VITEK 2 system for rapid identification of medically relevant gram-negative rods. J Clin
Microbiol 36(7):1948–1952.

Gao, Y., Gesenberg, C. and Zheng, W. (2017). Chapter 17 - Oral Formulations for Preclinical
Studies: Principle, Design, and Development Considerations. In: Qiu, Y., Chen, Y., Zhang, G. G.
Z., Yu, L. and Mantri, R. V. (eds.). Developing Solid Oral Dosage Forms (Second Edition).
Boston: Academic Press, pp. 455–495.

Ge, B., Wang, F., Sjoelund-Karlsson, M. and McDermott, P.F. (2013). Antimicrobial resistance in
Campylobacter: Susceptibility testing methods and resistance trends. J Microbiol Methods
95(1):57–67. doi: https://doi.org/10.1016/j.mimet.2013.06.021.

Gorgannezhad, L., Stratton, H. and Nguyen, N.-T. (2019). Microfluidic-Based Nucleic Acid
Amplification Systems in Microbiology. Micromachines (Basel) 10(6):408. doi:
https://doi.org/10.3390/mi10060408.

Griffin, P.M., Price, G.R., Schooneveldt, J.M., Schlebusch, S., Tilse, M.H., Urbanski, T., …
Venter, D. (2012). Use of matrix-assisted laser desorption ionization-time of flight mass
spectrometry to identify vancomycin-resistant enterococci and investigate the epidemiology of an
outbreak. J Clin Microbiol 50(9):2918–2931. doi: https://doi.org/10.1128/JCM.01000-12.

Hayat, Z. and El Abed, A.I. (2018). High-Throughput Optofluidic Acquisition of Microdroplets in

Microfluidic Systems. Micromachines (Basel) 9(4):183. doi: https://doi.org/10.3390/mi9040183.

Heatley, N.G. (1944). A method for the assay of penicillin. Biochem J 38(1):61–65. doi:
https://doi.org/10.1042/bj0380061.

Hillenkamp, F. and Karas, M. (1990). Mass-Spectrometry of Peptides and Proteins by Matrix-

Assisted Ultraviolet-Laser Desorption Ionization. Methods Enzymol 193:280–295. doi:
https://doi.org/10.1016/0076-6879(90)93420-P.

This article is protected by copyright. All rights reserved

 Hombach, M., Zbinden, R. and Böttger, E.C. (2013). Standardisation of disk diffusion results for
Accepted Article
antibiotic susceptibility testing using the sirscan automated zone reader. BMC Microbiol
13(1):225. doi: https://doi.org/10.1186/1471-2180-13-225.

Howell, M., Wirz, D., Daniels, A.U. and Braissant, O. (2012). Application of a Microcalorimetric
Method for Determining Drug Susceptibility in Mycobacterium Species. J Clin Microbiol
50(1):16–20. doi: https://doi.org/10.1128/JCM.05556-11.

Huang, M.B., Baker, C.N., Banerjee, S. and Tenover, F.C. (1992). Accuracy of the E test for
determining antimicrobial susceptibilities of staphylococci, enterococci, Campylobacter jejuni,
and gram-negative bacteria resistant to antimicrobial agents. J Clin Microbiol 30(12):3243–3248.

Hudzicki, J. (2009). Kirby-Bauer Disk Diffusion Susceptibility Test Protocol. American Society
for Microbiology; Washington, DC: Dec 8, 2009. [Online] https://www.asm.org/Protocols/Kirby-
Bauer-Disk-Diffusion-Susceptibility-Test-Pro.

Idelevich, E.A. and Becker, K. (2019). How to accelerate antimicrobial susceptibility testing. Clin
Microbiol Infect 25(11):1347–1355. doi: https://doi.org/10.1016/j.cmi.2019.04.025.

Idelevich, E.A., Becker, K., Schmitz, J., Knaack, D., Peters, G. and Köck, R. (2016). Evaluation of
an Automated System for Reading and Interpreting Disk Diffusion Antimicrobial Susceptibility
Testing of Fastidious Bacteria. PloS One 11(7):e0159183–e0159183. doi:
https://doi.org/10.1371/journal.pone.0159183.

Imhof, A., Balajee, S.A. and Marr, K.A. (2003). New methods to assess susceptibilities of
Aspergillus isolates to caspofungin. J Clin Microbiol 41(12):5683–5688. doi:
https://doi.org/10.1128/jcm.41.12.5683-5688.2003.

ISO 20776-1 (2019). Susceptibility testing of infectious agents and evaluation of performance of
antimicrobial susceptibility test devices — Part 1: Broth micro-dilution reference method for
testing the in vitro activity of antimicrobial agents against rapidly growing aerobic bacteria
involved in infectious diseases.

Itahashi, M., Higaki, S., Fukuda, M. and Shimomura, Y. (2010). Detection and Quantification of
Pathogenic Bacteria and Fungi Using Real-Time Polymerase Chain Reaction by Cycling Probe in

This article is protected by copyright. All rights reserved

 Patients
Accepted Article With Corneal Ulcer. Arch Ophthalmol 128(5):535–540. doi:
https://doi.org/10.1001/archophthalmol.2010.66.

Jenkins, S.G. and Schuetz, A.N. (2012). Current concepts in laboratory testing to guide
antimicrobial therapy. Mayo Clin Proc 87(3):290–308. doi:
https://doi.org/10.1016/j.mayocp.2012.01.007.

Jönsson, A., Jacobsson, S., Foerster, S., Cole, M.J. and Unemo, M. (2018). Performance
characteristics of newer MIC gradient strip tests compared with the Etest for antimicrobial
susceptibility testing of Neisseria gonorrhoeae. APMIS 126(10):822–827. doi:
https://doi.org/10.1111/apm.12887.

Jorgensen, J.H. and Ferraro, M.J. (2009). Antimicrobial Susceptibility Testing: A Review of
General Principles and Contemporary Practices. Clin Infect Dis 49(11):1749–1755. doi:
https://doi.org/10.1086/647952.

Jorgensen, J.H., Ferraro, M.J., McElmeel, M.L., Spargo, J., Swenson, J.M. and Tenover, F.C.
(1994). Detection of penicillin and extended-spectrum cephalosporin resistance among
Streptococcus pneumoniae clinical isolates by use of the E test. J Clin Microbiol 32(1):159–163.

Joynson, J.A., Forbes, B. and Lambert, R.J.W. (2002). Adaptive resistance to benzalkonium
chloride, amikacin and tobramycin: the effect on susceptibility to other antimicrobials. J Appl
Microbiol 93(1):96–107. doi: https://doi.org/10.1046/j.1365-2672.2002.01667.x.

Junkins, A.D., Arbefeville, S.S., Howard, W.J. and Richter, S.S. (2010). Comparison of BD
Phoenix AP Workflow with Vitek 2. J Clin Microbiol 48(5):1929. doi:
https://doi.org/10.1128/JCM.00111-10.

Kalashnikov, M., Lee, J.C., Campbell, J., Sharon, A. and Sauer-Budge, A.F. (2012). A
microfluidic platform for rapid, stress-induced antibiotic susceptibility testing of Staphylococcus
aureus. Lab Chip 12(21):4523–4532. doi: https://doi.org/10.1039/c2lc40531h.

Kim, S., Masum, F. and Jeon, J.S. (2019). Recent Developments of Chip-based Phenotypic
Antibiotic Susceptibility Testing. Biochip J 13(1):43–52. doi: https://doi.org/10.1007/s13206-019-
3109-7.

This article is protected by copyright. All rights reserved

 Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing (2015). Available at:
Accepted Article
https://clinicalgate.com/laboratory-methods-and-strategies-for-antimicrobial-susceptibility-testing-
2/ [Accessed: 22 November 2019].

Lalitha, M.K. (2004). Manual on Antimicrobial Susceptibility Testing. (Under the Auspices of
Indian Association of Medical Microbiologists). American Society for Microbiology; Washington,
DC.

Lasserre, C., De Saint Martin, L., Cuzon, G., Bogaerts, P., Lamar, E., Glupczynski, Y., … Tande,
D. (2015). Efficient Detection of Carbapenemase Activity in Enterobacteriaceae by Matrix-
Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Less Than 30 Minutes.
J Clin Microbiol 53(7):2163–2171. doi: https://doi.org/10.1128/JCM.03467-14.

Le Page, S., van Belkum, A., Fulchiron, C., Huguet, R., Raoult, D. and Rolain, J.-M. (2015).
Evaluation of the PREVI® Isola automated seeder system compared to reference manual
inoculation for antibiotic susceptibility testing by the disk diffusion method. Eur J Clin Microbiol
Infect Dis 34(9):1859–1869. doi: https://doi.org/10.1007/s10096-015-2424-8.

Lerchner, J., Mueller-Hagen, D., Roehr, H., Wolf, A., Mertens, F., Mueller, R., … Klare, I.
(2011). Chip-calorimetric evaluation of the efficacy of antibiotics and bacteriophages against
bacteria on a minute-timescale. J Therm Anal Calorim 104(1):31–36. doi:
https://doi.org/10.1007/s10973-010-1241-7.

Lewis, G. and (Dan) Daniels, A.U. (2003). Use of isothermal heat-conduction microcalorimetry
(IHCMC) for the evaluation of synthetic biomaterials. J Biomed Mater Res B Appl Biomater
66B(2):487–501. doi: https://doi.org/10.1002/jbm.b.10044.

Li, Yanmei, Fan, P., Zhou, S. and Zhang, L. (2017). Loop-mediated isothermal amplification
(LAMP): A novel rapid detection platform for pathogens. Microb Pathog 107:54–61. doi:
https://doi.org/10.1016/j.micpath.2017.03.016.

Li, Yiyan, Yang, X. and Zhao, W. (2017). Emerging Microtechnologies and Automated Systems
for Rapid Bacterial Identification and Antibiotic Susceptibility Testing. SLAS Technol 22(6):585–
608. doi: https://doi.org/10.1177/2472630317727519.

This article is protected by copyright. All rights reserved

 Ligozzi, M., Bernini, C., Bonora, M.G., de Fatima, M., Zuliani, J. and Fontana, R. (2002).
Accepted Article
Evaluation of the VITEK 2 system for identification and antimicrobial susceptibility testing of
medically relevant gram-positive cocci. J Clin Microbiol 40(5):1681–1686. doi:
https://doi.org/10.1128/JCM.40.5.1681-1686.2002.

Llor, C. and Bjerrum, L. (2014). Antimicrobial resistance: risk associated with antibiotic overuse
and initiatives to reduce the problem. Ther Adv Drug Saf 5(6):229–241. doi:
https://doi.org/10.1177/2042098614554919.

Lu, H., Caen, O., Vrignon, J., Zonta, E., El Harrak, Z., Nizard, P., Baret, J-C. and Taly, V. (2017).
High throughput single cell counting in droplet-based microfluidics. Sci Rep 7(1):1366–1366. doi:
https://doi.org/10.1038/s41598-017-01454-4.

Marvin, L.F., Roberts, M.A. and Fay, L.B. (2003). Matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry in clinical chemistry. Clin Chim Acta 337(1–2):11–21. doi:
https://doi.org/10.1016/j.cccn.2003.08.008.

Matsuda, K. (2017). Chapter Two - PCR-Based Detection Methods for Single-Nucleotide

Polymorphism or Mutation: Real-Time PCR and Its Substantial Contribution Toward
Technological Refinement. In: Makowski, G. S. (ed.). Adv Clin Chem. Elsevier, pp. 45–72.

Matuschek, E., Brown, D.F.J. and Kahlmeter, G. (2014). Development of the EUCAST disk
diffusion antimicrobial susceptibility testing method and its implementation in routine
microbiology laboratories. Clin Microbiol Infect 20(4):O255-266. doi:
https://doi.org/10.1111/1469-0691.12373.

Maurer, F.P., Christner, M., Hentschke, M. and Rohde, H. (2017). Advances in Rapid
Identification and Susceptibility Testing of Bacteria in the Clinical Microbiology Laboratory:
Implications for Patient Care and Antimicrobial Stewardship Programs. Infect Dis Rep 9(1):6839.
doi: https://doi.org/10.4081/idr.2017.6839.

Maxson, T., Taylor-Howell, C.L. and Minogue, T.D. (2017). Semi-quantitative MALDI-TOF for
antimicrobial susceptibility testing in Staphylococcus aureus. PLoS One 12(8):e0183899. doi:
https://doi.org/10.1371/journal.pone.0183899.

This article is protected by copyright. All rights reserved

 Menon, T., Umamaheswari, K., Kumarasamy, N., Solomon, S. and Thyagarajan, S.P. (2001).
Accepted Article
Efficacy of fluconazole and itraconazole in the treatment of oral candidiasis in HIV patients. Acta
Trop80(2):151–154. doi: https://doi.org/10.1016/S0001-706X(01)00170-X.

Micek, S.T., Lloyd, A.E., Ritchie, D.J., Reichley, R.M., Fraser, V.J. and Kollef, M.H. (2005).
Pseudomonas aeruginosa bloodstream infection: importance of appropriate initial antimicrobial
treatment. Antimicrob Agents Chemother 49(4):1306–1311. doi:
https://doi.org/10.1128/AAC.49.4.1306-1311.2005.

Miller, M.B. and Tang, Y.-W. (2009). Basic concepts of microarrays and potential applications in
clinical microbiology. Clin Microbiol Rev 22(4):611–633. doi:
https://doi.org/10.1128/CMR.00019-09.

Mock, M., Monod, M., Baudraz-Rosselet, F. and Panizzon, R.G. (1998). Tinea capitis
Dermatophytes: Susceptibility to Antifungal Drugs Tested in vitro and in vivo. Dermatology
197(4):361–367. doi: https://doi.org/10.1159/000018032.

Moreno, P.R.H., da Costa-Issa, F.I., Rajca-Ferreira, A.K., Pereira, M.A.A. and Kaneko, T.M.
(2013). Native Brazilian Plants Against Nosocomial Infections: A Critical Review on their
Potential and the Antimicrobial Methodology. Curr Top Med Chem 13(24):3040–3078.

Nijs, A., Cartuyvels, R., Mewis, A., Peeters, V., Rummens, J.L. and Magerman, K. (2003).
Comparison and evaluation of Osiris and Sirscan 2000 antimicrobial susceptibility systems in the
clinical microbiology laboratory. J Clin Microbiol 41(8):3627–3630. doi:
https://doi.org/10.1128/JCM.41.8.3627-3630.2003.

O’Hara, C.M. (2005). Manual and automated instrumentation for identification of

Enterobacteriaceae and other aerobic gram-negative bacilli. Clin Microbiol Rev 18(1):147–162.
doi: https://doi.org/10.1128/CMR.18.1.147-162.2005.

de Oliveira, L.A., de Souza-Moreira, T.M., Cefali, L.C., Chiari, B.G., Correa, M.A., Borges Isaac,
V.L., Nunes Salgado, H.R. and Linhari Rodrigues Pietro, R.C. (2011). Design of antiseptic
formulations containing extract of Plinia cauliflora. Braz J Pharm Sci 47(3):525–533. doi:
https://doi.org/10.1590/S1984-82502011000300010.

This article is protected by copyright. All rights reserved

 Pendland, S.L., Martin, S.J., Chen, C., Schreckenberger, P.C. and Danziger, L.H. (1997).
Accepted Article
Comparison of charcoal- and starch-based media for testing susceptibilities of Legionella species
to macrolides, azalides, and fluoroquinolones. J Clin Microbiol 35(11):3004–3006.

Petersen, M.W., Perner, A., Sjövall, F., Jonsson, A.B., Steensen, M., Andersen, J.S., … Møller,
M.H. (2019). Piperacillin/tazobactam vs carbapenems for patients with bacterial infection:
Protocol for a systematic review. Acta Anaesthesiol Scand 63(7):973–978. doi:
https://doi.org/10.1111/aas.13382.

Pulido, M.R., Garcia-Quintanilla, M., Martin-Pena, R., Miguel Cisneros, J. and McConnell, M.J.
(2013). Progress on the development of rapid methods for antimicrobial susceptibility testing. J
Antimicrob Chemother 68(12):2710–2717. doi: https://doi.org/10.1093/jac/dkt253.

Reller, L.B., Weinstein, M., Jorgensen, J.H. and Ferraro, M.J. (2009). Antimicrobial Susceptibility
Testing: A Review of General Principles and Contemporary Practices. Clin Infect Dis
49(11):1749–1755. doi: https://doi.org/10.1086/647952.

Rhoads, D.D., Wang, H., Karichu, J. and Richter, S.S. (2016). The presence of a single MALDI-
TOF mass spectral peak predicts methicillin resistance in staphylococci. Diagn Microbiol Infect
Dis 86(3):257–261. doi: https://doi.org/10.1016/j.diagmicrobio.2016.08.001.

Rhoads, S., Marinelli, L., Imperatrice, C. and Nachamkin, I. (1995). Comparison of Microscan
Walkaway System and Vitek System for Identification of Gram-Negative Bacteria. J Clin
Microbiol 33(11):3044–3046.

Richter, S.S. and Ferraro, M.J. (2007). Susceptibility testing instrumentation and computerized
expert systems for data analysis and interpretation. In: Murray P.R., Baron E.J., Jorgensen J.H.,
Landry M.L., Pfaller M.A. Manual of Clinical Microbiology. 9th ed. Washington, D.C.: ASM
Press, pp. 245–256.

Rodriguez, L.A., Vivas, J., Gallardo, C.S., Acosta, F., Barbeyto, L. and Real, F. (1999).
Identification of Hafnia alvei with the MicroScan WalkAway system. J Clin Microbiol
37(12):4186–4188.

This article is protected by copyright. All rights reserved

 Sader, H.S., Fritsche, T.R. and Jones, R.N. (2006). Accuracy of three automated systems
Accepted Article
(MicroScan WalkAway, VITEK, and VITEK 2) for susceptibility testing of Pseudomonas
aeruginosa against five broad-spectrum beta-lactam agents. J Clin Microbiol44(3):1101–1104.
doi: https://doi.org/10.1128/JCM.44.3.1101-1104.2006.

Saegeman, V., Huynen, P., Colaert, J., Melin, P. and Verhaegen, J. (2005). Susceptibility testing
of Pseudomonas aeruginosa by the Vitek 2 system: A comparison with Etest results. Acta Clin
Belg60(1):3–9. doi: https://doi.org/10.1179/acb.2005.002.

Salem, M.S. and Ali, M.A.M. (2016). Novel Pyrazolo[3,4-b]pyridine Derivatives: Synthesis,
Characterization, Antimicrobial and Antiproliferative Profile. Biol Pharm Bull 39(4):473–483.
doi: https://doi.org/10.1248/bpb.b15-00586.

Schumacher, A., Vranken, T., Malhotra, A., Arts, J.J.C. and Habibovic, P. (2018). In vitro
antimicrobial susceptibility testing methods: agar dilution to 3D tissue-engineered models. Eur J
Clin Microbiol Infect Dis 37(2):187–208. doi: https://doi.org/10.1007/s10096-017-3089-2.

Snyder, J.W., Munier, G.K. and Johnson, C.L. (2008). Direct comparison of the BD Phoenix
system with the MicroScan WalkAway system for identification and antimicrobial susceptibility
testing of Enterobacteriaceae and nonfermentative gram-negative organisms. J Clin Microbiol
46(7):2327–2333. doi: https://doi.org/10.1128/JCM.00075-08.

Spanu, T., Sanguinetti, M., Ciccaglione, D., D’Inzeo, T., Romano, L., Leone, F. and Fadda, G.
(2003). Use of the VITEK 2 system for rapid identification of clinical isolates of Staphylococci
from bloodstream infections. J Clin Microbiol 41(9):4259–4263. doi:
https://doi.org/10.1128/jcm.41.9.4259-4263.2003.

Speeleveld, E., Gordts, B., Landuyt, H.W.V., Vroey, C.D. and Raes-Wuytack, C. (1996).
Susceptibility of clinical isolates of Fusarium to antifungal drugs. Mycoses 39(1‐2):37–40. doi:
https://doi.org/10.1111/j.1439-0507.1996.tb00081.x.

Spiller, E., Schöll, A., Alexy, R., Kümmerer, K. and Urban, G.A. (2006). A sensitive microsystem
as biosensor for cell growth monitoring and antibiotic testing. Selected Papers from
TRANSDUCERS ’05. Sens Actuators A Phys 130–131:312–321. doi:
https://doi.org/10.1016/j.sna.2006.02.051.

This article is protected by copyright. All rights reserved

 Steensels, D., Verhaegen, J. and Lagrou, K. (2011). Matrix-Assisted Laser Desorption Ionization-
Accepted Article
Time of Flight Mass Spectrometry for the Identification of Bacteria and Yeasts in a Clinical
Microbiological Laboratory: A Review. Acta Clin Belg66(4):267–273. doi:
https://doi.org/10.2143/ACB.66.4.2062567.

Steward, C.D., Mohammed, J.M., Swenson, J.M., Stocker, S.A., Williams, P.P., Gaynes, R.P.,
McGowan Jr, J.E. and Tenover, F.C. (2003). Antimicrobial susceptibility testing of carbapenems:
multicenter validity testing and accuracy levels of five antimicrobial test methods for detecting
resistance in Enterobacteriaceae and Pseudomonas aeruginosa isolates. J Clin Microbiol
41(1):351–358. doi: https://doi.org/10.1128/jcm.41.1.351-358.2003.

Strauss, M., Zoabi, K., Sagas, D., Reznik-Gitlitz, B. and Colodner, R. (2020). Evaluation of Bio-
Rad® discs for antimicrobial susceptibility testing by disc diffusion and the ADAGIOTM system
for the automatic reading and interpretation of results. Eur J Clin Microbiol Infect Dis 39(2):375–
384. doi: https://doi.org/10.1007/s10096-019-03735-4.

Tafin, U.F., Clauss, M., Hauser, P.M., Bille, J., Meis, J.F. and Trampuz, A. (2012). Isothermal
microcalorimetry: a novel method for real-time determination of antifungal susceptibility of
Aspergillus species. Clin Microbiol Infect 18(7):E241–E245. doi: https://doi.org/10.1111/j.1469-
0691.2012.03854.x.

Tang, Y., Zhen, L., Liu, J. and Wu, J. (2013). Rapid Antibiotic Susceptibility Testing in a
Microfluidic pH Sensor. Anal Chem 85(5):2787–2794. doi: https://doi.org/10.1021/ac303282j.

Walker, G.T., Quan, J., Higgins, S.G., Toraskar, N., Chang, W., Saeed, A., Sapiro, V., Pitzer, K.,
Whitfield, N., Lopansri, B.K., Motyl, M. and Sahm, D. (2019). Predicting Antibiotic Resistance in
Gram-Negative Bacilli from Resistance Genes. Antimicrob Agents Chemother 63(4):e02462-18.
doi: https://doi.org/10.1128/AAC.02462-18.

Wiegand, I., Hilpert, K. and Hancock, R.E.W. (2008). Agar and broth dilution methods to
determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc
3(2):163–175. doi: https://doi.org/10.1038/nprot.2007.521.

This article is protected by copyright. All rights reserved

 Wielders, C.L.C., Fluit, A.C., Brisse, S., Verhoef, J. and Schmitz, F.J. (2002). mecA gene is
Accepted Article
widely disseminated in Staphylococcus aureus population. J Clin Microbiol 40(11):3970–3975.
doi: https://doi.org/10.1128/jcm.40.11.3970-3975.2002.

Wieser, A., Schneider, L., Jung, J. and Schubert, S. (2012). MALDI-TOF MS in microbiological
diagnostics-identification of microorganisms and beyond (mini review). Appl Microbiol
Biotechnol 93(3):965–974. doi: https://doi.org/10.1007/s00253-011-3783-4.

Winstanley, T. and Courvalin, P. (2011). Expert Systems in Clinical Microbiology. Clin Microbiol
Rev 24(3):515–556. doi: https://doi.org/10.1128/CMR.00061-10.

Zhu, X.-D., Chu, J. and Wang, Y.-H. (2018). Advances in Microfluidics Applied to Single Cell
Operation. Biotechnol J 13(2):1700416. doi: https://doi.org/10.1002/biot.201700416.

This article is protected by copyright. All rights reserved