3.0 4.

Review

Current and Emerging Methods of

Antibiotic Susceptibility Testing

Zeeshan A. Khan, Mohd F. Siddiqui and Seungkyung Park

Special Issue
Diagnosis of Bacterial Pathogens
Edited by
Dr. Sabine Dittrich

https://doi.org/10.3390/diagnostics9020049
 diagnostics
Review
Current and Emerging Methods of Antibiotic
Susceptibility Testing
Zeeshan A. Khan † , Mohd F. Siddiqui † and Seungkyung Park *
School of Mechanical Engineering, Korea University of Technology and Education, Cheonan,
Chungnam 31253, Korea; [email protected] (Z.A.K.); [email protected] (M.F.S.)
* Correspondence: [email protected]; Tel.: +82-41-560-1149; Fax: +82-41-560-1253
† These authors contributed equally to this work.




Received: 18 March 2019; Accepted: 28 April 2019; Published: 3 May 2019


Abstract: Antibiotic susceptibility testing (AST) specifies effective antibiotic dosage and formulates
a profile of empirical therapy for the proper management of an individual patient’s health against
deadly infections. Therefore, rapid diagnostic plays a pivotal role in the treatment of bacterial infection.
In this article, the authors review the socio-economic burden and emergence of antibiotic resistance.
An overview of the phenotypic, genotypic, and emerging techniques for AST has been provided
and discussed, highlighting the advantages and limitations of each. The historical perspective on
conventional methods that have paved the way for modern AST like disk diffusion, Epsilometer
test (Etest), and microdilution, is presented. Several emerging methods, such as microfluidic-based
optical and electrochemical AST have been critically evaluated. Finally, the challenges related with
AST and its outlook in the future are presented.

Keywords: Antibiotic susceptibility tests; resistance; phenotypic; genotypic; bacteria

1. Introduction
Antibiotic resistance is defined as the genetic ability of bacteria to encode the resistance genes
that counterfeit the inhibitory effect of potential antibiotics for survival [1]. It can be developed either
intrinsically by natural recombination and integration into the bacterial genome, or it can be acquired
through horizontal gene mutation events such as conjugation, transformation, and transduction [2].
The prominent events in the generation of bacterial resistance include inactivation of the porin channel,
modification of antibiotic targets, and neutralizing antibiotic efficacy through enzymatic action [3].
Thus, the understanding of the genetic makeover and the morpho-anatomical changes in bacteria are
of prime importance to counteracting the resistance mechanism.
The discovery of antibiotics was paradigm-altering, as it was not only an effective tool against
chronic infections but also opened new avenues for drug industries. Global antibiotic statistics
suggested an increase of 35% in the antibiotic consumption between 2000 and 2010, and the current
antibiotic industry stands at USD 39.8 billion (up to 2015). Russia, India, China, Brazil, and South Africa
are major contributing countries, where 76% of the rise in antibiotic consumption has been estimated [4].
The changes experienced in the enhanced consumption of antibiotics over the past decade remain
unprecedented, and this is chiefly a result of the emergence of new diseases. Alternately, the increase
in antibiotic consumption and industrialization might be due to overuse or misuse of antibiotics
recommended by physicians/self-medication at the time of infection [5,6]. A recent report on the
casualties related to antibiotic resistance by the world health organization (WHO) depicted an alarming
700,000 lives per year currently, and predicts a disturbing 10 million/year by 2050, ensuring that
antibiotic resistance will be the most prevalent cause of death [7]. Adding to this, WHO also forewarns
the severity of antibiotic resistance, stating that “it threatens the achievements of modern medicine,

Diagnostics 2019, 9, 49; doi:10.3390/diagnostics9020049 www.mdpi.com/journal/diagnostics

Diagnostics 2019, 9, 49 2 of 17

a post-antibiotic era—in which common infections and minor injuries can kill—is a very real possibility
for the 21st century” [8].
Minimum inhibitory concentrations (MICs) of various antimicrobial susceptibility testing (AST) are
categorized by various international agencies. These MIC guidelines determine whether an antibiotic is
susceptible or not. The Clinical and Laboratory Standards Institute (CLSI) provides the most popular
guidelines, which are based on pharmacokinetic–pharmacodynamic (PK-PD) properties and mechanisms
of resistance [9]. Most European countries follow the MIC cut-offs based on PK-PD properties, and the
epidemiological MIC cut-offs (ECOFFS) as determined by the European Committee on Antimicrobial
Susceptibility Testing (EUCAST). The MIC breakpoints recommended by EUCAST are generally higher
than the CLSI. Because of the modifications in the guidelines, the results are substantially changed,
such as higher ceftazidime resistance in Klebsiella pneumonia and ESBL-producing Escherichia coli (E. coli).
Moreover, the CLSI guidelines re accessible for non-members as a package of three documents for USD
500 annually, while EUCAST guidelines are freely available on the EUCAST website [9].
A sharp surge in bacteria-encoding resistance is occurring worldwide, jeopardizing the efficacy
of antibiotics that have saved millions of lives [10]. The antibiotics which have threatened bacteria
for decades are under grave threat themselves. Owing to large societal repercussions of multidrug
resistance and the significantly reduced development of drugs, it is mandatory to determine the
microbes which need more attention than others for drug development. Consequently, WHO has
developed a priority list of the pathogens, and stratified the list into critical, high, and medium priorities.
Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumanii, carbapenem-resistant and
third-generation cephalosporin-resistant Enterobacteriaceae were placed in critical-priority bacteria.
The high priority bacteria included vancomycin-resistant Enterococcus faecium, methicillin-resistant
Staphylococcus aureus (MRSA), clarithromycin-resistant Helicobacter pylori, fluoroquinolone-resistant
Campylobacter spp., penicillin-resistant Streptococcus pneumoniae, ampicillin-resistant Haemophilus
influenzae, and fluoroquinolone-resistant Shigella spp. [11] (Figure 1). Resistance against beta-lactam
antibiotics like penicillin is widespread, while resistance against other drugs, such as vancomycin and
fluoroquinolone, are less frequently observed (Supplementary Table S1). The infections arising from
resistant bacteria, because of mutations, might present themselves with harsher symptoms than their
predecessors (Supplementary Table S1). Although novel drugs have shown much promise against
these resistant bacteria, their rapid diagnostic is still a huge concern (Supplementary Table S1).
In this review, we concisely discuss the advantages and limitations of various tools for
AST (Supplementary Table S2). We first review the phenotypic methods for AST like diffusion,
dilution, and automated AST tools. Central aspects of genotype-based AST methods, including
susceptibility diagnostics based by polymerase chain reaction (PCR) and DNA microarray, are addressed.
Then, an overview of several emerging approaches such as fluorescent, colorimetric, and electrochemical
microfluidic sensors, with their related caveats, are discussed (Supplementary Table S2).
Diagnostics 2019, 9, 49 3 of 17

Figure 1. Representation of the final ranking of antibiotic-resistant bacteria, adapted with permission
from Tacconelli et al. [11]. CR = carbapenem resistant. 3GCR = third-generation cephalosporin
resistant. VR = vancomycin resistant. MR = meticillin resistant. ClaR = clarithromycin resistant.
FQR = fluoroquinolone resistant. PNS = penicillin non-susceptible. AmpR = ampicillin resistant.

2. Phenotypic AST Methods

2.1. Diffusion
The disk diffusion method is the gold standard for confirming the susceptibility of bacteria.
Standardized disk diffusion was introduced by Bauer and Kirby’s experiments in 1956, after finalizing
all aspects of optimization by changing physical conditions [12]. In this method, the isolated
bacterial colony is selected, suspended into growth media, and standardized through a turbidity
test. The standardized suspension is then inoculated onto the solidified agar plate, and the
antibiotic-treated paper is tapped on the inoculated plate. The disc containing the antibiotic is
allowed to diffuse through the solidified agar, resulting in the formation of an inhibition zone after
the overnight incubation at 35 ◦ C. Thereafter, the size of the inhibition zone formed around the
paper disc is measured; the size of the inhibition zone corresponds to the concentration of antibiotic
(Figure 2) [12,13]. Assessing and determining the susceptibility of bacteria generally takes 16–24 h.
Several diffusion-based experiments have been performed prior to the standardized disk diffusion
method. In the 1920s, Fleming was the pioneering contributor to AST. Fleming’s gutter method was
the first method of antibiotic analysis where antibiotic was dispensed into a gutter made on solid
agar that allowed the antibiotics to diffuse through it [14]. A modification to this design, called the
“Oxford cup method,” was subsequently developed by Abraham et al. in 1941, where the gutter
was replaced with a glass cup for diffusion [15]. Simultaneously, in the 1940s, Pope (1940), Foster
and Woodruff (1943), and Vincet and Vincet (1944) used an antibiotic-impregnated paper disc for
the diffusion of antibiotics [16,17]. These methods were hindered by inaccurate analysis due to
evaporation, difficulty in handling, sterilization, and cumbersome operation [18]. Moreover, a single
antibiotic was focused on susceptibility testing (i.e., penicillin). In later years, the introduction
of effective drugs and convenient means of susceptibility testing have prevailed with the increase
Diagnostics 2019, 9, 49 4 of 17

of the deadly infections. Therefore, variations of the method have been adopted to expand its
versatility and utility. In 1947, Hoyt, Levine, and Bondi introduced penicillin tablets and the standard
6.5-mm disk method separately to emphasize multiple targets [19,20]. In the 1950s, experiments
by Gould and Bowie (1952) and Stokes (1955) enabled the differentiation between susceptible and
resistant bacteria through the multiple disk diffusion technique [21,22]. All the proposed methods
were inaccurate, unsuitable, and unreliable for routine testing because of discrepancies in results
obtained from different labs [23]. Hence, in 1961, several organizations (especially WHO) made
several efforts to address the need for a standardized method for antibiotic susceptibility testing.
Later, in the year 1966, Bauer and Kirby’s method was confirmed as a standard method for susceptibility
testing. This method has potential for the routine testing of susceptibility in clinical laboratories.
Furthermore, the method is widely accepted because it offers a simple, cost-effective protocol for
the detection of multiple targets [24]. However, along with these advantages, it also has some
significant drawbacks: only semi-automation is available (Sirscan), insufficient data availability for
many bacteria (strains of Pseudomonas, Bacillus, and Corynebacterium), and it has a poor performance
when analyzing slow-growing and fastidious bacteria [25,26]. Influenced by many physiochemical
factors like evaporation, solubility, pH, temperature, and nutrient media, additional limitations restrict
its suitability for accurate diagnostics [27].
Recently, the emergence of various instruments for analyzing the zone of inhibition has added
to the reliability of the disk diffusion results by reducing variability due to operator handling and
interpretation. The camera or scanner takes the picture, and the inbuilt image analysis software
displays the zone of inhibition and compares the obtained results with the various guidelines present
in the database. Accuzone (AccuMed International, Hillsboro, Oregon, USA), Biomic (Giles Scientific,
Santa Barbara, California, USA), Mastascan Elite (Mast, Bootle, UK), and Sirscan (Becton Dickinson,
Oxford, UK) are a few of the instruments capable of analyzing the zone of inhibition, but all differ in
data input, analysis, ease of use, and presentation of results [28].
Diagnostics 2019, 9, 49 5 of 17

Figure 2. Representation of various conventional antibiotic susceptibility testing methods. (a) Disk
diffusion, demonstrating of inhibition zones, adapted from Sageerabanoo [29]. (b) Etest gradient
disk diffusion, adapted from Sader [30], under terms of the Creative Commons attribution license.
(c,d) Broth macro and micro dilution, showing bacterial susceptibility based on optical density and
(e) Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MADI-TOF MS),
adapted from the MALDI Biotyper system (Bruker, Billerica, Massachusetts, United States), Laboratory
Information System (LIS) and Laboratory Information Management System (LIMS).

2.2. Dilution
Dilution was one of the earliest tools in microbiological practice, starting in the early 1870s, and it
allows the growth and identification of bacterial populations in suspension [31]. Pasteur, Lister, Koch,
and Ehrlich were listed as the pioneers in the field of bacteriology, and they worked on the concept
of macrodilution [32]. William Roberts and John Tyndall further contributed to the macrodilution
method and observed bacterial growth in a diluted medium [33]. The two basic types of dilution are
microdilution and macrodilution, wherein broth and agar are the most commonly used mediums.
In broth dilution, consecutive two-fold dilutions (1, 2, 4, 8, and 12 µL) of antibiotics are made and
dispensed into micro-centrifuge tubes containing bacterial growth medium, followed by making up the
final volume by adding the medium and incubating overnight at 35 ◦ C. Finally, the growth examination
is carried out for setting the breakpoint through the turbidity of culture media (Figure 2) [34,35]. In agar
Diagnostics 2019, 9, 49 6 of 17

dilution, antibiotics are diluted into the agar medium, followed by plate formation and application of
bacterial cells to the surface of the agar plate.
In the early 20th century, various scientists made efforts to introduce serial dilution. They set the
dilution factor in terms of geometric progression, and derived the generalized mathematical equation for
interpreting the dilution results [36–40]. In 1929, Alexander Fleming performed the serial dilution technique
to understand the activity of antibiotics. In this technique, two-fold dilution of antibiotics is mixed with a
pre-inoculated liquid medium to determine antibiotic actions by checking the turbidity. In 1942, Fleming
modified the previous protocol by using pH instead of turbidity to identify antibacterial activity. In the
same year, Rammelkamp and Maxon introduced broth macro dilution, or the “tube dilution method”,
which is regarded as the standardized dilution method for both minimum inhibitory concentration (MIC)
and AST. CLSI recommends guidelines to set the breakpoints. The first attempt regarding AST was made
by Schmith and Reymann using agar medium during the 1940s [41].
Microdilution is a miniaturized prototype of the macrodilution method where susceptibility
testing is performed on disposable 96-well microtiter plates, where each well has a sample capacity of
~0.1 mL (Figure 2) [42]. To dispense the samples into microwells, mechanized dispensers are used to
avoid the handling error. After overnight incubation, growth and MIC are assessed through specialized
optical instruments. This method has been well standardized for most fastidious bacteria [13].
The central drawback of dilution methods is the requirement of a large volume of reagents.
Apart from that, other potential limitations include: experimental space, tedious dilution steps
(macrodilution), the possibility of false positive results due to long incubation times [43], chances of
cross-contamination, bacterial incompatibility for growth, and the inability of discriminating viable and
nonviable bacteria. Maintaining the recommended optimum testing parameters like pH, temperature,
media, and length of incubation are additional hurdles, and a control viability plate is mandatory in
tests to achieve practical clinical relevance [44].

2.3. Etest
Epsilometer testing (Etest) is another significant development for the routine analysis of widespread
antibiotic resistance in bacteria. In the late 1980s, Bolmström and Eriksson developed this test [45].
AB BIODISK manufactured the first Etest plastic strip to inspect multiple antibiotics on a single platform
in 1991 (Figure 2) [45]. Etest plastic strips are coated with pre-defined antibiotic concentrations, and the
corresponding interpretive MIC ranges are marked on the surface and back of the strip, respectively.
For detection, multiple strips are placed on a pre-inoculated streaked agar plate, followed by an
overnight incubation; elliptical inhibition zones appear around the strips, indicating the MIC at
the intersection point between the inhibition zone and the strip edge [46]. The simplicity, accuracy,
and reliability of the Etest makes it appropriate and convenient for Food and Drug Administration
(FDA) approved commercialization [47]. The ability of convenient interpretations of MIC under diverse
physical conditions made the Etest a preferential method over standardized disk diffusion and dilution
techniques in clinical laboratories for AST [30,48].
In the 1990s, series of comparative studies with the other standardized techniques, for instance,
agar dilution and diffusion, and broth dilution, established the significance of the Etest. Many strains
of H. pylori, Neisseria gonorrhoeae, Enterococcus spp., and many other clinical isolates were tested
by Etest and compared with standard methods, resulting in a good correlation in the range of
91%–99% [49–51]. Recently, in 2016, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant
S. aureus (MRSA) isolates were examined with an Etest to determine the MIC of ceftaroline. The results
were compared with broth microdilution (BMD) and showed an excellent agreement of more than
95% [52,53]. Multiple cultures of Campylobacter spp. against seven antibiotics were also evaluated by
Etest to determine their resistance [54]. All these results demonstrated the reliability and importance
of the Etest in evaluating MICs of a wide range of antibiotics over the present standardized methods,
especially for slow-growing bacteria (Campylobacter jejuni, H. pylori) and rare fastidious bacteria
(S. pneumoniae and Neisseria spp.). One of the significant advantages of the Etest is its sensitivity; it can
Diagnostics 2019, 9, 49 7 of 17

detect extended-spectrum beta-lactamase (ESBL), even in a trace amount [55]. Additionally, accurate
resistance strains can easily be easily quantified in laboratories/hospitals due to the stable concentration
gradient of antibiotics marked on the Etest strip.
Besides several advantages, there are some limitations that cannot be ignored, primarily related to
the inaccurate and inconsistent behavior of the Etest for certain antibacterial agents, such as Penicillin,
ciprofloxacin, ofloxacin, and rifampicin [56]. Some additional demerits that make the Etest complicated
for routine test analyses are: pH-sensitive coated antibiotics, expensive batch performance, strip
storage, and laboratory set-up for proper plate inoculation and incubation [57].

2.4. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)
MALDI-TOF MS, introduced in 2000, is another sensitive method for bacterial identification.
High sensitivity and accuracy are the key characteristics that make it a useful method for clinical
relevance. Assorted studies reveal its significance in discriminating MRSA, MSSA, and other bacterial
strains where susceptible and resistant bacteria have been evaluated through spectral peak analysis.
Even the subtle difference in expression profiles have been noticed in isogenic strains of S. aureus [58,59].
The efficiency of MALDI-TOF MS has been further investigated on vancomycin-resistant Enterococci,
where sensitivity higher than 90% has been recorded. Furthermore, analysis of multiple targets with
different resistant strains of Pseudomonas spp. against ciprofloxacin, tobramycin, and meropenem have
been identified efficiently [60]. The newly developed MALDI Biotyper antibiotic susceptibility test rapid
assay (MBT-ASTRA) is a more-straightforward and cost-effective modulation of MALDI-TOF MS used
for both AST and MIC determination [61]. Despite all the advantage of MALDI-TOF MS, the expensive
nature of the instrument and its maintenance are prime disadvantages for mass application.

2.5. Automated Systems

Since the dawn of automated technologies in the 1980s, antibiotic susceptibility tests have
been perpetually improvised and, hence, have superseded conventional phenotypic methods [13].
Automation, simplicity, and compactness are the major reasons for their widespread acceptance in
diagnostics. Computer integration has allowed online analysis and data sharing, which is a giant
leap for results validation, especially in remote areas [62]. Among the developed automated systems,
MicroScan WalkAway (Beckman Coulter, Inc. Atlanta, Georgia, USA) (1980), Micronaut (Merlin,
berlin, Germany) (1990), the avantage test (Abbott Laboratories, Irving, Texas, USA) (1980), Vitek 2
(bioMe’rieux, Marcy-l’Étoile, France) (2000), Phoenix (BD Diagnostics, Franklin Lakes, New jersey,
USA) (2001), and Sensititre ARIS 2X (Trek Diagnostic Systems, Oakwood Village, Ohio, USA) (2004)
are the major FDA approved systems for AST. Vitek and Pheonix detect growing bacteria on the
basis of turbidity, whereas comparable automated systems like MicroScan WalkAway (Beckman
Coulter, Inc. Atlanta, Georgia, USA) and Sensititre ARIS 2X (are based on fluorescence emission of
the growing bacteria. The resistance in gram-negative, gram-positive, and Streptococcus strains of
bacteria can easily be estimated through Phoenix, and Vitek 2, but, MicroScan WalkAway and Sensititre
ARIS 2XESBL (Trek Diagnostic Systems, Oakwood Village, Ohio, USA) are capable of detecting the
extended-spectrum beta-lactamase (ESBL)-producing strains in the species mentioned above [63].
Micronaut and Avantage are capable of accurate direct susceptibility testing for gram-positive and
gram-negative bacteria, respectively [64,65].
Detection incompatibility for many bacteria/antibiotics using Sensititre ARIS 2 and Vitek 1 have
led to the development of the updated Sensititre ARIS 2X and Vitek 2, which have better performances
and are applicable to a broader range of bacteria/antibiotics. Presently, all the automated systems
are incorporated with advanced expert system software for enhanced performance and online data
processing [28]. Every automated system has a specific panel capacity and an average time for
performing detection, which varies from 40 to 100 wells, and can vary in time such as 20, 12 and 9 h,
respectively. Certain models such as Phoenix AP, and Vitek 2Xl, are dedicated towards automated
inoculation, enhanced card capacity and compactness.
Diagnostics 2019, 9, 49 8 of 17

The aforementioned systems lack reproducibility, sensitivity, and reliability compared with the
existing traditional methods. Moreover, an inability to test a wide range of clinically relevant bacteria
(e.g., S. pneumonia), antimicrobial agents (e.g., vancomycin), and heteroresistant isolates, as well as a
limited panel capacity and the high cost of instruments and consumables, are all significant issues that
restrict these systems from frequent analysis [66].

3. Genotypic AST Methods

Molecular or genotypic AST are the effective direct methods that eliminate tedious bacterial
cultures, long incubation, chances of contamination, and the spreading of deadly infections [67]. PCR,
DNA microarray and DNA chips, and loop-mediated isothermal amplification (LAMP) are some of the
genotypic techniques for the detection of antibiotic resistance. Mutational assessment of methicillin
resistance in Staphylococcus spp., vancomycin resistance in Enterococcus spp., and multi-antibiotic
(isoniazid, rifampin, streptomycin, pyrazinamide, and the fluoroquinolones) resistance in Mycobacterium
spp. have been successfully estimated through various genotypic techniques.
PCR is one of the most efficient and rapid molecular tools for quantification and profiling of bacterially
infectious genes. The first report on PCR diagnostic application was published by Saiki et al. [68].
The general methodology of PCR includes cycles of denaturation, annealing of the primers, and elongation
of the primers by a thermostable DNA polymerase in a compatible buffer containing nucleotides, ions,
and so on. Each cycle of amplification doubles the target DNA molecule. The amplified target can
be confirmed for the presence of resistance genes through electrophoresis, southern blotting, restriction
fragment-length polymorphism, single-strand conformation polymorphism (SSCP), DNA fingerprinting,
molecular beacons, and other DNA sequencing analysis methods (Figure 3) [68,69]. Another tool developed
on the basis of PCR is LAMP, which has also been used for the evaluation of AST. In LAMP, the gene of
interest is amplified at a constant temperature of 60–65 ◦ C using a Bst DNA polymerase instead of Taq
polymerase because of strong strand displacement activity (required in isothermal techniques) [70].

-b bacterial identification from mixed

Figure 3. Digital PCR-High Resolution Melt analysis (HRM)-based
bacterial samples, reproduced with permission from [71], published by American Chemical Society,
2017. (SVM: Support-vector machine)

DNA microarrays and DNA chips are the other promising technologies utilized for screening
susceptibility [72]. DNA arrays employ cDNA fragment probes on nylon membrane, where each
DNA chip has a glass or silicon platform for probe binding. The specific hybridization of the
labeled probe with the target and its recognition help to determine the resistance. Determination of
isoniazid resistance in M. tuberculosis has been carried out successfully through DNA microarrays and
chips [73,74]. Colorimetric detection and multiplexing are the attractive features of these techniques.
Genotypic methods are generally attributed to the rapid, direct, sensitive, and specific detection
of resistance genes, but they also suffer from severe drawbacks that diminish their clinical utility.
These drawbacks include: (i) the individual antimicrobial agents to be tested need a specific assay
Diagnostics 2019, 9, 49 9 of 17

for detection; (ii) only potential/key resistance genes can be detected, which are often not relevant
due to coincidental mutations; (iii) there is a lack of sensitivity towards the patients with latent
infections, or when only a few organisms are present in a sample; (iv) the genetic mechanism/profile
for the resistance of all bacteria is not yet defined; (v) the occurrence of false-positive results due to
contamination of the test sample might be expected; (vi) they require expensive reagents and machinery
with specific maintenance conditions; and most importantly (vii) all the tools have a prerequisite of
skilled personnel [67].

4. Emerging Methods for AST

Microfluidics-based diagnostics are one of the most promising emerging tools for AST.
Microfluidics is an evolving field characterized by the manipulation of fluids in micro-volume,
thereby offering portability, cost-effectiveness, multiplexing, reproducibility, and a controllable
environment in an in vitro system [12]. The concept was first introduced in the semiconductor and
micro-electromechanical systems (MEMS) industries, then further extended to the field of biomedical
research [75]. Integrated microfluidic devices, generally referred to as micro total analysis systems
(µTAS), are proficient in performing molecular diagnostics [76].
The quantity of samples has always been the biggest challenge for biological studies in general,
and pathological analysis in particular. Since microfluidics is capable of dealing with the minimal
quantity of samples, it has therefore emerged as a promising tool for pathologists. Currently, microfluidics
platforms are capable of single-cell analysis, and can even analyze the single-cell interrogation of signaling
networks in cultured cell lines. In the past decades, numerous conventional and automated strategies
have been successfully embraced to diagnose the ever-increasing antibiotic resistance in bacteria [77].
Nevertheless, the most-practiced techniques, such as disk diffusion and microdilution, have a high
chance of cross-contamination, laborious protocols, lengthy processing times, improper power supplies,
and cumbersome set-ups that render their application in resource-limited regions, and unfortunately, these
regions have a higher rate of resistance too [2,6]. The rise of microfluidics as a diagnostic tool has shown
promise in addressing the abovementioned shortcomings [78,79].
Progression in optical imaging has developed various image sensors with high sensitivity and
resolution that are capable of biological analysis. These imaging systems are frequently used for
morphological and growth studies of bacteria. Generally, owing to real-time analysis and minimum
culture dependency, microfluidic devices coupled with an optical sensor can perform AST and detect
the MIC in few hours [80]. Recent reports based on single bacterial cell analysis have claimed that
optical sensor-based nanofluidic (30 nl) can finish AST within 30 min [81]. This direct imaging of single
bacterium requires simple sample preparation steps, but eliminates the tedious steps of continuous
sample injection, loading of cells, and counting-based cell identification [81].
Fluorescence proteins and dyes are commonly used for tagging resistant biomarkers.
These proteins/dyes can be of biological or chemical origin. One of the pioneer proteins used
for imaging is green fluorescent protein (GFP). GFP, obtained from jellyfish Aequoria victoria, is essential
for the noninvasive real-time monitoring of antimicrobial susceptibility [82]. Adding to this, the same
group was able to evaluate multiple antibiotic sensitivities in real-time in polymicrobial culture bacterial
strains (namely, E. coli, P. aeruginosa, and K. pneumoniae) simultaneously. Green and red fluorescent
protein tagging have been utilized for real-time growth quantification in the presence of multiple
antibiotics on a multiplexed microfluidic platform (Figure 4) [83]. All these methods are sensitive and
reliable, but creating these recombinant bacteria requires molecular handling, which is a challenge
for routine clinical observations. Fluorescent dyes are another means for optical fluorescence tagging.
These chemicals, with all the benefits of the fluorescence protein, can avoid stearic hindrance due to
their small size, and elimination of cloning or transformation. SYTOX green and resazurin (alamarBlue;
inactive precursor of resazurin) are two common fluorescence indicator dyes used for viability testing
in AST [84,85]. Similarly, resazurin can be utilized in colorimetric detection. When the culture media is
supplemented with antibiotic and resazurin, a uniform intense blue color is obtained in the beginning.
Diagnostics 2019, 9, 49 10 of 17

In the presence of resistance, the resazurin is reduced to resafurin, and the intense blue color changes
to pink and leuco; in the absence of resistance in bacteria, the blue color sustains. This method is also
translated to a microfluidic chip for MIC estimation for four different antibiotics against 20 clinical
strains of Escherichia and Shigella [85].

Figure 4. Demonstration of sensors for antibacterial susceptibility testing involving (a) an optical
microfluidics biosensor, showing optical detection of microbial cultures, reproduced with permission
from [83], published by RCS Advances, 2015; and (b) an electrochemical biosensor, detection was based
on hybridization of the target bacterial 16S rRNA with a detector probe, adapted with permission from
Liu [86] (under terms of the Creative Commons attribution license).

Similarly, bioluminescence or ATP bioluminescence assay (ATP-BLA) is an enzyme-based approach

mediated by luciferase enzyme that converts luciferin substrate to oxyluciferin in the presence of
ATP, leading to an emission of light [87]. By using this phenomenon, the susceptibility of 13 different
types of clinical strains present in urinary infections were evaluated against eight antibiotics on the
microfluidic plate. When bacteria grew in the presence of antibiotics, resistant bacteria resulted in
bioluminescence, whereas susceptible bacteria remained neutral. Both identification and susceptibility
were obtained within 3–6 h [88,89].
Several electrochemical devices have been developed for AST. One of the most prominent works utilized
AC electrokinetic fluid motion and Joule heating-induced temperature elevation for the electrochemical
sensing of bacterial 16S rRNA (Figure 4) [86]. Real-time and rapid detection are possible, as 16s rRNA is
highly specific for the bacterial pathogens in blood culture and does not need prior purification. Although 16S
rRNA has indeed provided critical details about sensitive bacterial analysis, genetic complexities across
kingdoms make it inappropriate for reproducible, clinically relevant, or point-of-care susceptibility testing.
The use of electroactive chemicals (redox reagents) as probe molecules can be an interesting approach to
elucidating bacterial susceptibility. In 2015, pyocyanin, a potential marker of cell viability and virulence,
was studied for the electrochemical monitoring of the susceptibility of P. aeruginosa biofilms on a microfluidic
device [90]. A good correlation between the electrical signal drop and the viability of P. aeruginosa cells in
the presence of antibiotics was successfully demonstrated. The most recent electrochemical biosensor can
perform AST within 90 min along with the label-free isolation of bacteria from whole blood samples [91].
Plastic-based microchips with printed electrodes capture the target bacteria with the help of specific
antibodies. The electrochemical response to the captured bacteria is monitored in both the presence and
absence of antibiotics.
The simplified blood culture system (SBCS) is an emerging tool in near-patient testing and
surveillance tools for blood stream infections (BSI) [92]. SBCSs uses unprocessed samples, thereby,
it requires zero sample preparation time. Furthermore, SBCSs combine detection, gram status,
and identification in blood culture (BC) instruments, thus ensuring the evaluation of susceptibility
Diagnostics 2019, 9, 49 11 of 17

within 8–12 h, whereas the conventional blood culture system requires up to 48 h because of incubation
for colony generation and susceptibility testing. Moreover, owing to its simplified and time efficient
nature, the SBCS can be used in resource-limited settings, which was not possible for conventional
BC methods due to the lack of electricity, limited culture bottles, profuse dust, improper ambient
temperature control, and lack of skilled personnel [92].

5. Challenges and Future Perspective

Primarily, the continuous flow of culturing media to feed cells is the biggest challenge for
maintaining nutrient conditions, as a slight change in growth media due to evaporation can affect the
bacterial growth and hence the accuracy of AST [93,94]. The accuracy of AST might be improved by
considering the factors altering its pharmacokinetics (diffusion, metabolism, and elimination), which
may result in unpredictable changes, and therefore, the MIC might change. Furthermore, to avoid the
indiscriminate use of antibiotics and the evolution of antibiotic resistance, the dosage scheme should
also consider the method of antibiotic administration (e.g., oral versus intravenous (IV) administration)
and the site of infection, along with the AST results [95].
The applicability of molecular biology tools such as cloning and recombinant expression has
enhanced the sensitivity of detection, and low concentrations of bacteria in clinical samples can be
evaluated, but there are some serious limitations. Creating recombinant strains with these molecular
genes is a troublesome process. Firstly, genetic analysis is cumbersome and prone to mutations due
to frequent change in the resistant behavior of bacteria [96]. Therefore, prior knowledge of specific
resistance genes before susceptibility testing is essential. Secondly, the genetic markers for all clinically
relevant bacteria is still unknown, moreover, the known targets are not universal. Thirdly, advanced
molecular biology skills and laboratory sets are important in dealing with recombinant technology.
Alternatively, fluorescence label dyes are used to avoid molecular challenges. Although the use of
dyes is simple and easy over other molecular techniques, the requirement of a high-resolution A
charge-coupled device camera (CCD) and sophisticated instruments for signal amplification and
observation are restricted to resource-limited areas. False-positive results due to changes in physical
parameters are also a major problem. Additionally, immense versatility among the culture conditions
of different bacterial species is a challenge for the development of a single platform for different bacteria
and multiplexing [97,98].
Looking beyond the imaging requirements, researchers must also focus on other relevant unanswered
questions in the context of diagnostic devices. This primarily includes what performance metrics
will be essential to lessen the exposure of contamination from hospitals or biomedical research units,
and secondarily, what advances will be necessary to reduce the incorporation of sophisticated external
circuits, pumps, and pneumatic systems in maintaining the flow continuity in microfluidic platform To
address these issues, the paper-based detection system seems to be an attractive approach in developing
a cost-effective, automated, and incinerable platform for the determination of susceptibility. In the past
decades, the emergence of paper-based microfluidics has proven its performance for biological assays [99],
and it could be a bedrock for reducing contamination through easy disposal. Additionally, the inbuilt
property of capillary action of paper can eliminate the integration of complicated pneumatic chambers
and pumps. Therefore, more research work on paper microfluidic AST would be beneficial in the future.
An absence of the simultaneous detection of multiple analytes and the need for simpler fabrication
tools are further shortcomings of microfluidics. The collaboration of academic research and commercial
firms with transparent technology distribution might offer a compelling solution for multiplexing and
for more-straightforward fabrication. Hence, efforts to expand our understanding, especially for the
development of a user-friendly device with multiplexing, would be valuable.
Smartphones and the technology that powers them are continually growing more advanced.
Coupling fluorescent/colorimetric tools with the ubiquitous and ever-evolving smartphones will
enable us with on-site monitoring, real-time database updates, and the generation of an antibiotic
susceptibility map to help us understand the geographical prevalence of resistance. The use of
Diagnostics 2019, 9, 49 12 of 17

smartphones is limited by their camera performances, which result in lower detection limits, especially
in a colorimetric assay [100]. It is exciting to speculate that ongoing advancement will bring much
higher resolution cameras coupled with better time-lapse technologies and offer morphological and
biochemical measurements.
Loop-mediated isothermal amplification polymerase chain reaction (LAMP-PCR) has shown
us the way to develop lateral-flow devices for the genetic detection of antibiotic resistance.
Carbapenem-resistance in Acinetobacter baumanii was successfully evaluated by LAMP-PCR by
amplifying the OXA-type carbapenemases and metallo-β-lactamases genes [101]. However, more
studies are warranted to establish the applicability of LAMP. In coming years, there might be a rise in
non-infecting but resistance-bearing mutants; genetic detection will be essential to screen out these
mutants, and LAMP-based lateral flow devices will serve that purpose.

6. Conclusions
While all AST methods offer qualitative assessments using susceptible, intermediate, or resistant
categories, certain methods specify qualitative and effective antibiotic dosage (e.g., minimum inhibitory
concentration) and formulate a profile of empirical therapy for the proper management of individual
patients’ health against deadly infections. A rise in antibiotic resistance is a certainty, therefore we
must develop technologies that will permit rapid AST (within an hour) and are non-invasive (saliva-
or urine-based) or minimally invasive.

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-4418/9/2/49/s1.

Author Contributions: Z.A.K., M.F.S., and S.P. wrote and edited the manuscript. All the authors read and
approved the final manuscript.
Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the
Korean government (MSIT) (NRF-2015R1C1A1A01054762).
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Blair, J.M.; Webber, M.A.; Baylay, A.J.; Ogbolu, D.O.; Piddock, L.J. Molecular mechanisms of antibiotic
resistance. Nat. Rev. Microbiol. 2015, 13, 42–51. [CrossRef] [PubMed]
2. Martínez, J.L. Antibiotics and Antibiotic Resistance Genes in Natural Environments. Science 2008, 321,
365–367. [CrossRef] [PubMed]
3. Lalitha, M. Manual on antimicrobial susceptibility testing. In Performance Standards for Antimicrobial Testing:
Twelfth Informational Supplement; CLSI: Wayne, PA, USA, 2004; Volume 56238, pp. 454–456.
4. Van Boeckel, T.P.; Gandra, S.; Ashok, A.; Caudron, Q.; Grenfell, B.T.; Levin, S.A.; Laxminarayan, R.
Global antibiotic consumption 2000 to 2010: An analysis of national pharmaceutical sales data.
Lancet Infect. Dis. 2014, 14, 742–750. [CrossRef]
5. Lee, C.-R.; Cho, I.H.; Jeong, B.C.; Lee, S.H. Strategies to Minimize Antibiotic Resistance. Int. J. Environ. Res.
Public Health 2013, 10, 4274–4305. [CrossRef]
6. Ayukekbong, J.A.; Ntemgwa, M.; Atabe, A.N. The threat of antimicrobial resistance in developing countries:
Causes and control strategies. Antimicrob. Resist. Infect. Control 2017, 6, 47. [CrossRef]
7. Brogan, D.M.; Mossialos, E. A critical analysis of the review on antimicrobial resistance report and the
infectious disease financing facility. Glob. Health 2016, 12, 8. [CrossRef]
8. Viens, A.M.; Littmann, J. Is Antimicrobial Resistance a Slowly Emerging Disaster? Public Health Ethics 2015,
8, 255–265. [CrossRef]
9. Kassim, A.; Omuse, G.; Premji, Z.; Revathi, G. Comparison of Clinical Laboratory Standards Institute and
European Committee on Antimicrobial Susceptibility Testing guidelines for the interpretation of antibiotic
susceptibility at a University teaching hospital in Nairobi, Kenya: A cross-sectional study. Ann. Clin.
Microbiol. Antimicrob. 2016, 15, 21. [CrossRef] [PubMed]
10. Ventola, C.L. The antibiotic resistance crisis: Part 1: Causes and threats. Pharm. Ther. 2015, 40, 277–283.
Diagnostics 2019, 9, 49 13 of 17

11. Tacconelli, E.; Carrara, E.; Savoldi, A.; Harbarth, S.; Mendelson, M.; Monnet, D.L.; Pulcini, C.; Kahlmeter, G.;
Kluytmans, J.; Carmeli, Y.; et al. Discovery, research, and development of new antibiotics: The WHO priority
list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 2018, 18, 318–327. [CrossRef]
12. Bauer, A.W.; Kirby, W.M.; Sherris, J.C.; Turck, M. Antibiotic susceptibility testing by a standardized single
disk method. Am. J. Clin. Pathol. 1966, 45, 493–496. [CrossRef] [PubMed]
13. Reller, L.B.; Weinstein, M.; Jorgensen, J.H.; Ferraro, M.J. Antimicrobial Susceptibility Testing: A Review of
General Principles and Contemporary Practices. Clin. Infect. Dis. 2009, 49, 1749–1755. [CrossRef]
14. Fleming, A. On the antibacterial action of cultures of a penicillium, with special reference to their use in the
isolation of B. influenzae. Br. J. Exp. Pathol. 1929, 10, 226. [CrossRef]
15. Abraham, E.P.; Chain, E.; Fletcher, C.M.; Gardner, A.D.; Heatley, N.G.; Jennings, M.A.; Florey, H.W.
Further observations on penicillin. Lancet 1941, 238, 177–189. [CrossRef]
16. Foster, J.W.; Woodruff, H.B. Microbiological aspects of penicillin: I. Methods of assay. J. Bacteriol. 1943, 46, 187.
17. Vincent, J.G.; Vincent, H.W.; Morton, J. Filter Paper Disc Modification of the Oxford Cup Penicillin
Determination. Proc. Soc. Exp. Biol. Med. 1944, 55, 162–164. [CrossRef]
18. Gavin, J.J. Analytical Microbiology: II. The Diffusion Methods. Appl. Microbiol. 1957, 5, 25.
19. Hoyt, R.E.; Levine, M.G. A Method for determining Sensitivity to Penicillin and Streptomycin.
Science (Washington) 1947, 171. [CrossRef]
20. Bondi, A., Jr.; Spaulding, E.H.; Smith, D.E.; Dietz, C.C. A routine method for the rapid determination of
susceptibility to penicillin and other antibiotics. Am. J. Med. Sci. 1947, 213, 221–225. [CrossRef]
21. Gould, J.C. The determination of bacterial sensitivity to antibiotics. Edinb. Med. J. 1952, 59, 178–199.
[PubMed]
22. Stokes, E.J. Antibiotic sensitivity testing. Br. Med. J. 1971, 2, 707. [CrossRef]
23. Mayrhofer, S.; Domig, K.J.; Mair, C.; Zitz, U.; Huys, G.; Kneifel, W. Comparison of broth microdilution, Etest,
and agar disk diffusion methods for antimicrobial susceptibility testing of Lactobacillus acidophilus group
members. Appl. Environ. Microbiol. 2008, 74, 3745–3748. [CrossRef]
24. World Health Organization. Standardization of Methods for Conducting Microbic Sensitivity Tests: Second Report of the
Expert Committee on Antibiotics [Meeting Held in Geneva from 11 to 16 July 1960]; WHO: Geneva, Switzerland, 1961.
25. Patel, J.B.; Tenover, F.C.; Turnidge, J.D.; Jorgensen, J.H. Susceptibility test methods: Dilution and disk
diffusion methods. In Manual of Clinical Microbiology, 10th ed.; American Society of Microbiology: Sterling,
VA, USA, 2011; pp. 1122–1143.
26. Hombach, M.; Zbinden, R.; Bottger, E.C. Standardisation of disk diffusion results for antibiotic susceptibility
testing using the sirscan automated zone reader. BMC Microbiol. 2013, 13, 225. [CrossRef]
27. Murray, P.R. Antibiotic Susceptibility Testing. Part I. Lab. Med. 1983, 14, 345–350. [CrossRef]
28. Felmingham, D.; Brown, D.F. Instrumentation in antimicrobial susceptibility testing. J. Antimicrob. Chemother.
2001, 48, 81–85. [CrossRef]
29. Sageerabanoo, S.; Malini, A.; Mangaiyarkarasi, T.; Hemalatha, G. Phenotypic detection of extended spectrum
β-lactamase and Amp-C β-lactamase producing clinical isolates in a Tertiary Care Hospital: A preliminary
study. J. Nat. Sci. Biol. Med. 2015, 6, 383–387. [CrossRef] [PubMed]
30. Sader, H.S.; Pignatari, A.C. E test: A novel technique for antimicrobial susceptibility testing. Sao Paulo Med. J.
Rev. Paul. Med. 1994, 112, 635–638. [CrossRef] [PubMed]
31. Guardino, R.F. Early History of Microbiology and Microbiological Methods; Parenteral Drug Association:
Wilmington, DE, USA, 2005.
32. Rittenberg, S.C. Three Centuries of Microbiology. Hubert, A. Lechevalier and Morris Solotorovsky.
McGraw-Hill, New York, 1965. viii + 536 pp. Paper, $4.95. Science 1965, 149, 530–531. [CrossRef]
33. Poupard, J.A.; Rittenhouse, S.F.; Walsh, L.R. The evolution of antimicrobial susceptibility testing methods.
In Antimicrobial Susceptibility Testing; Springer, Plenum Publishing: New York, NY, USA, 1994; pp. 3–14.
34. Ericsson, H.M.; Sherris, J.C. Antibiotic sensitivity testing. Report of an international collaborative study.
Acta Pathol. Microbiol. Scand. 1971, 90.
35. Jorgensen, J.H.; Turnidge, J.D.; Washington, J.A.; Murray, P.R.; Pfaller, M.A.; Tenover, F.C.; Baron, E.J.;
Yolken, R.H. Antibacterial susceptibility tests: Dilution and disk diffusion Methods. In Manual of Clinical
Microbiology; Geo. F. Brooks Publisher: Washington, DC, USA, 1999.
36. Phelps, E.B. A method of calculating the numbers of B. coli from the results of dilution tests. Am. J. Public Hyg.
1908, 18, 141.
Diagnostics 2019, 9, 49 14 of 17

37. McCrady, M. Tables for rapid interpretation of fermentation-tube results. Public Health J. 1918, 9, 201–220.
38. Reed, J. Report of Advisory Committee on Official Water Standards; Public Health Reports; Sage Publications,
Inc.: Teller Road, AK, USA, 1925; Volume 40.
39. Greenwood, M.; Yule, G.U. On the statistical interpretation of some bacteriological methods employed in
water analysis. J. Hyg. 1917, 16, 36. [CrossRef]
40. Fisher, R.A. Statistical methods for research workers. In Breakthroughs in Statistics; Springer, Oliver & Boyd:
New York, NY, USA, 1992; pp. 66–70.
41. Wheat, P.F. History and development of antimicrobial susceptibility testing methodology. J. Antimicrob. Chemother.
2001, 48, 1–4. [CrossRef]
42. Tang, Y.-W.; Stratton, C.W. Advanced Techniques in Diagnostic Microbiology; Springer: Nashville, TN, USA, 2012.
43. Waites, K.B.; Duffy, L.B.; Bébéar, C.M.; Matlow, A.; Talkington, D.F.; Kenny, G.E.; Totten, P.A.; Bade, D.J.;
Zheng, X.; Davidson, M.K. Standardized methods and quality control limits for agar and broth microdilution
susceptibility testing of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum.
J. Clin. Microbiol. 2012, 50, 3542–3547. [CrossRef]
44. Collins, A.M.; Craig, G.; Zaiman, E.; Roy, T.E. A comparison between disk-plate and tube-dilution methods
for antibiotic sensitivity testing of bacteria. Can. J. Public Health/Rev. Can. Sante’e Publique 1954, 45, 430–439.
45. Picard, J. Applied Veterinary Bacteriology and Mycology: Bacteriological Techniques; University of Pretoria, Afrivip:
Pretoria, South Africa, 1990.
46. Sanchez, M.L.; Jones, R.N. E test, an antimicrobial susceptibility testing method with broad clinical and
epidemiologic application. Antimicrob. Newsl. 1992, 8, 1–7. [CrossRef]
47. Joyce, L.F.; Downes, J.; Stockman, K.; Andrew, J.H. Comparison of five methods, including the PDM
Epsilometer test (E test), for antimicrobial susceptibility testing of Pseudomonas aeruginosa. J. Clin. Microbiol.
1992, 30, 2709–2713.
48. Baker, C.N.; Stocker, S.A.; Culver, D.H.; Thornsberry, C. Comparison of the E Test to agar dilution, broth
microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria.
J. Clin. Microbiol. 1991, 29, 533–538.
49. Glupczynski, Y.; Labbé, M.; Hansen, W.; Crokaert, F.; Yourassowsky, E. Evaluation of the E test for quantitative
antimicrobial susceptibility testing of Helicobacter pylori. J. Clin. Microbiol. 1991, 29, 2072–2075.
50. Van Dyck, E.; Smet, H.; Piot, P. Comparison of E test with agar dilution for antimicrobial susceptibility testing
of Neisseria gonorrhoeae. J. Clin. Microbiol. 1994, 32, 1586–1588.
51. Schulz, J.E.; Sahm, D.F. Reliability of the E test for detection of ampicillin, vancomycin, and high-level
aminoglycoside resistance in Enterococcus spp. J. Clin. Microbiol. 1993, 31, 3336–3339.
52. Cantón, R.; Livermore, D.M.; Morosini, M.I.; Díaz-Regañón, J.; Rossolini, G.M. Etest® versus broth
microdilution for ceftaroline MIC determination with Staphylococcus aureus: Results from PREMIUM,
a European multicentre study. J. Antimicrob. Chemother. 2017, 72, 431–436. [CrossRef]
53. Skov, R.; Smyth, R.; Larsen, A.R.; Bolmstrom, A.; Karlsson, A.; Mills, K.; Frimodt-Moller, N.; Kahlmeter, G.
Phenotypic detection of methicillin resistance in Staphylococcus aureus by disk diffusion testing and Etest
on Mueller-Hinton agar. J. Clin. Microbiol. 2006, 44, 4395–4399. [CrossRef] [PubMed]
54. Hariharan, H.; Sharma, S.; Chikweto, A.; Matthew, V.; DeAllie, C. Antimicrobial drug resistance as determined
by the E-test in Campylobacter jejuni, C. coli, and C. lari isolates from the ceca of broiler and layer chickens
in Grenada. Comp. Immunol. Microbiol. Infect. Dis. 2009, 32, 21–28. [CrossRef]
55. Falagas, M.E.; Karageorgopoulos, D.E. Extended-spectrum β-lactamase-producing organisms. J. Hosp. Infect.
2009, 73, 345–354. [CrossRef]
56. Frosch, M.; Maiden, M.C.J. Handbook of Meningococcal Disease: Infection Biology, Vaccination, Clinical Management;
John Wiley & Sons: Hoboken, NJ, USA, 2006.
57. Balouiri, M.; Sadiki, M.; Ibnsouda, S.K. Methods for in vitro evaluating antimicrobial activity: A review.
J. Pharm. Anal. 2016, 6, 71–79. [CrossRef]
58. Bernardo, K.; Pakulat, N.; Macht, M.; Krut, O.; Seifert, H.; Fleer, S.; Hünger, F.; Krönke, M. Identification and
discrimination of Staphylococcus aureus strains using matrix-assisted laser desorption/ionization-time of
flight mass spectrometry. Proteomics 2002, 2, 747–753. [CrossRef]
59. Hrabák, J.; Chudáčková, E.; Walková, R. Matrix-assisted laser desorption ionization–time of flight
(MALDI-TOF) mass spectrometry for detection of antibiotic resistance mechanisms: From research to
routine diagnosis. Clin. Microbiol. Rev. 2013, 26, 103–114. [CrossRef] [PubMed]
Diagnostics 2019, 9, 49 15 of 17

60. Jung, J.; Eberl, T.; Sparbier, K.; Lange, C.; Kostrzewa, M.; Schubert, S.; Wieser, A. Rapid detection of antibiotic
resistance based on mass spectrometry and stable isotopes. Eur. J. Clin. Microbiol. Infect. Dis. 2014, 33,
949–955. [CrossRef]
61. Zimmermann, S.; Burckhardt, I. Development and Application of MALDI-TOF for Detection of Resistance
Mechanisms. In MALDI-TOF and Tandem MS for Clinical Microbiology; John Wiley & Sons: Hoboken, NJ, USA,
2017; pp. 231–248.
62. Richter, S.S.; Ferraro, M.J. Susceptibility testing instrumentation and computerized expert systems for data
analysis and interpretation. In Manual of Clinical Microbiology, 10th ed.; American Society of Microbiology:
Sterling, VA, USA, 2011; pp. 1144–1154.
63. Sellenriek, P.; Holmes, J.; Ferrett, R.; Drury, R.; Storch, G.A. Comparison of MicroScan Walk-Away® , Phoenix™
and VITEK-TWO® Microbiology systems used in the identification and susceptibility testing of bacteria.
In Proceedings of the Abstr 105th General Meeting of the American Society for Microbiology, Atlanta, GA,
USA, 5–9 June 2005.
64. Wright, D.N.; Matsen, J.M.; DiPersio, J.R.; Kirk, M.; Saxon, B.; Ficorilli, S.M.; Spencer, H.J. Evaluation of four
newer antimicrobial agents in the Avantage susceptibility test system. J. Clin. Microbiol. 1989, 27, 2381–2383.
65. Wellinghausen, N.; Pietzcker, T.; Poppert, S.; Belak, S.; Fieser, N.; Bartel, M.; Essig, A. Evaluation of the Merlin
MICRONAUT System for Rapid Direct Susceptibility Testing of Gram-Positive Cocci and Gram-Negative
Bacilli from Positive Blood Cultures. J. Clin. Microbiol. 2007, 45, 789–795. [CrossRef] [PubMed]
66. Karlowsky, J.A.; Richter, S.S. Antimicrobial susceptibility testing systems. In Manual of Clinical Microbiology,
11th ed.; American Society of Microbiology: Sterling, VA, USA, 2015; pp. 1274–1285.
67. Cockerill, F.R. Genetic methods for assessing antimicrobial resistance. Antimicrob. Agents Chemother. 1999, 43,
199–212. [CrossRef]
68. Fluit, A.C.; Visser, M.R.; Schmitz, F.-J. Molecular detection of antimicrobial resistance. Clin. Microbiol. Rev.
2001, 14, 836–871. [CrossRef] [PubMed]
69. Miller, M.B.; Tang, Y.-W. Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology.
Clin. Microbiol. Rev. 2009, 22, 611–633. [CrossRef]
70. Li, Y.; Fan, P.; Zhou, S.; Zhang, L. Loop-mediated isothermal amplification (LAMP): A novel rapid detection
platform for pathogens. Microb. Pathog. 2017, 107, 54–61. [CrossRef]
71. Athamanolap, P.; Hsieh, K.; Chen, L.; Yang, S.; Wang, T.H. Integrated Bacterial Identification and Antimicrobial
Susceptibility Testing Using PCR and High-Resolution Melt. Anal. Chem. 2017, 89, 11529–11536. [CrossRef]
72. Frye, J.G.; Lindsey, R.L.; Rondeau, G.; Porwollik, S.; Long, F.; McClelland, M.; Jackson, C.R.; Englen, M.D.;
Meinersmann, R.J.; Berrang, M.E.; et al. Development of a DNA microarray to detect antimicrobial resistance
genes identified in the National Center for Biotechnology Information database. Microb. Drug Resist. 2010,
16, 9–19. [CrossRef]
73. Gryadunov, D.; Mikhailovich, V.; Lapa, S.; Roudinskii, N.; Donnikov, M.; Pan’kov, S.; Markova, O.;
Kuz’min, A.; Chernousova, L.; Skotnikova, O.; et al. Evaluation of hybridisation on oligonucleotide
microarrays for analysis of drug-resistant Mycobacterium tuberculosis. Clin. Microbiol. Infect. 2005, 11,
531–539. [CrossRef] [PubMed]
74. Huang, W.L.; Hsu, Z.J.; Chang, T.C.; Jou, R. Rapid and accurate detection of rifampin and isoniazid-resistant
Mycobacterium tuberculosis using an oligonucleotide array. Clin. Microbiol. Infect. 2014, 20, O542–O549.
[CrossRef] [PubMed]
75. Sackmann, E.K.; Fulton, A.L.; Beebe, D.J. The present and future role of microfluidics in biomedical research.
Nature 2014, 507, 181–189. [CrossRef]
76. Park, S.; Zhang, Y.; Lin, S.; Wang, T.H.; Yang, S. Advances in microfluidic PCR for point-of-care infectious
disease diagnostics. Biotechnol. Adv. 2011, 29, 830–839. [CrossRef]
77. Ibrahim, W.A.; Marouf, S.A.; Erfan, A.M.; Nasef, S.A.; Jakee, J.K.E. The occurrence of disinfectant and
antibiotic-resistant genes in Escherichia coli isolated from chickens in Egypt. Vet. World 2019, 12, 141–145.
[CrossRef] [PubMed]
78. Chin, C.D.; Laksanasopin, T.; Cheung, Y.K.; Steinmiller, D.; Linder, V.; Parsa, H.; Wang, J.; Moore, H.;
Rouse, R.; Umviligihozo, G.; et al. Microfluidics-based diagnostics of infectious diseases in the developing
world. Nat. Med. 2011, 17, 1015–1019. [CrossRef]
Diagnostics 2019, 9, 49 16 of 17

79. Martinez, A.W.; Phillips, S.T.; Whitesides, G.M.; Carrilho, E. Diagnostics for the Developing World: Microfluidic
Paper-Based Analytical Devices; ACS Publications: Sterling, VA, USA, 2009.
80. Chen, C.H.; Lu, Y.; Sin, M.L.Y.; Mach, K.E.; Zhang, D.D.; Gau, V.; Liao, J.C.; Wong, P.K.
Antimicrobial susceptibility testing using high surface-to-volume ratio microchannels. Anal. Chem. 2010, 82,
1012–1019. [CrossRef]
81. Baltekin, Ö.; Boucharin, A.; Andersson, D.I.; Elf, J. Fast Antibiotic Susceptibility Testing (FASTest) based on
single cell growth rate measurements. bioRxiv 2016. [CrossRef]
82. Webb, J.S.; Barratt, S.R.; Sabev, H.; Nixon, M.; Eastwood, I.M.; Greenhalgh, M.; Handley, P.S.; Robson, G.D.
Green fluorescent protein as a novel indicator of antimicrobial susceptibility in Aureobasidium pullulans.
Appl. Environ. Microbiol. 2001, 67, 5614–5620. [CrossRef]
83. Mohan, R.; Sanpitakseree, C.; Desai, A.V.; Sevgen, S.E.; Schroeder, C.M.; Kenis, P.J.A. A microfluidic approach
to study the effect of bacterial interactions on antimicrobial susceptibility in polymicrobial cultures. RSC Adv.
2015, 5, 35211–35223. [CrossRef]
84. Kalashnikov, M.; Lee, J.C.; Campbell, J.; Sharon, A.; Sauer-Budge, A.F. A microfluidic platform for rapid,
stress-induced antibiotic susceptibility testing of Staphylococcus aureus. Lab Chip 2012, 12, 4523–4532.
[CrossRef]
85. Elavarasan, T.; Chhina, S.K.; Ash, M.P.; Sankaran, K. Resazurin reduction based colorimetric antibiogram in
microfluidic plastic chip. Sens. Actuators B Chem. 2013, 176, 174–180. [CrossRef]
86. Liu, T.; Lu, Y.; Gau, V.; Liao, J.C.; Wong, P.K. Rapid antimicrobial susceptibility testing with electrokinetics
enhanced biosensors for diagnosis of acute bacterial infections. Ann. Biomed. Eng. 2014, 42, 2314–2321. [CrossRef]
87. Marques, S.M.; Esteves da Silva, J.C. Firefly bioluminescence: A mechanistic approach of luciferase catalyzed
reactions. IUBMB Life 2009, 61, 6–17. [CrossRef]
88. Dong, T.; Zhao, X. Rapid identification and susceptibility testing of uropathogenic microbes via
immunosorbent ATP-bioluminescence assay on a microfluidic simulator for antibiotic therapy. Anal. Chem.
2015, 87, 2410–2418. [CrossRef]
89. Ivančić, V.; Mastali, M.; Percy, N.; Gornbein, J.; Babbitt, J.T.; Li, Y.; Landaw, E.M.; Bruckner, D.A.;
Churchill, B.M.; Haake, D.A. Rapid antimicrobial susceptibility determination of uropathogens in clinical
urine specimens by use of ATP bioluminescence. J. Clin. Microbiol. 2008, 46, 1213–1219. [CrossRef]
90. Webster, T.A.; Sismaet, H.J.; Goluch, E.D. Electrochemically monitoring the antibiotic susceptibility of
Pseudomonas aeruginosa biofilms. Analyst 2015, 140, 7195–7201. [CrossRef]
91. Safavieh, M.; Pandya, H.J.; Venkataraman, M.; Thirumalaraju, P.; Kanakasabapathy, M.K.; Singh, A.;
Prabhakar, D.; Chug, M.K.; Shafiee, H. Rapid Real-Time Antimicrobial Susceptibility Testing with Electrical
Sensing on Plastic Microchips with Printed Electrodes. ACS Appl. Mater. Interfaces 2017, 9, 12832–12840.
[CrossRef]
92. Dailey, P.J.; Osborn, J.; Ashley, E.A.; Baron, E.J.; Dance, D.A.B.; Fusco, D.; Fanello, C.; Manabe, Y.C.;
Mokomane, M.; Newton, P.N.; et al. Defining System Requirements for Simplified Blood Culture to Enable
Widespread Use in Resource-Limited Settings. Diagnostics 2019, 9, 10. [CrossRef]
93. Rodriguez, F.D.; Simonsson, P.; Alling, C. A method for maintaining constant ethanol concentrations in cell
culture media. Alcohol Alcohol. 1992, 27, 309–313.
94. Dawson, A.I. Bacterial Variations Induced by Changes in the Composition of Culture Media. J. Bacteriol.
1919, 4, 133–148. [PubMed]
95. Schreckenberger, P.C.; Binnicker, M.J. Optimizing Antimicrobial Susceptibility Test Reporting.
J. Clin. Microbiol. 2011, 49, S15–S19. [CrossRef]
96. Martinez, J.L.; Baquero, F. Mutation Frequencies and Antibiotic Resistance. Antimicrob. Agents Chemother.
2000, 44, 1771–1777. [CrossRef]
97. Khan, Z.A.; Siddiqui, M.F.; Park, S. Progress in antibiotic susceptibility tests: A comparative review with
special emphasis on microfluidic methods. Biotechnol. Lett. 2019, 41, 221–230. [CrossRef]
98. van Belkum, A.; Dunne, W.M., Jr. Next-generation antimicrobial susceptibility testing. J. Clin. Microbiol.
2013, 51, 2018–2024. [CrossRef]
99. Kim, S.; Masum, F.; Jeon, J.S. Recent Developments of Chip-based Phenotypic Antibiotic Susceptibility
Testing. BioChip J. 2019, 13, 43–52. [CrossRef]
Diagnostics 2019, 9, 49 17 of 17

100. Huang, X.; Xu, D.; Chen, J.; Liu, J.; Li, Y.; Song, J.; Ma, X.; Guo, J. Smartphone-based analytical biosensors.
Analyst 2018, 143, 5339–5351. [CrossRef]
101. Vergara, A.; Zboromyrska, Y.; Mosqueda, N.; Morosini, M.I.; García-Fernández, S.; Roca, I.; Cantón, R.;
Marco, F.; Vila, J. Evaluation of a Loop-Mediated Isothermal Amplification-Based Methodology To Detect
Carbapenemase Carriage in Acinetobacter Clinical Isolates. Antimicrob. Agents Chemother. 2014, 58, 7538–7540.
[CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).