Current Technologies in Antimicrobial Susceptibility

Testing and Microbial Identification:-advantages,

limitations/challenges & Future perspective.

Summary

The emergence and spread of bacterial resistances to antibiotics is now leading to

what is known as the antibiotic crisis. This is driven mostly by the antibiotic misuse
and disabuse, rendered possible mainly due to the absence of fast and accurate
technologies for antibiotic screening and resistant bacteria identification in infectious
diseases. Technologies that provide sensitive, quick and easy read-outs, conveying
information about the optimal treatment are required. A multitude of these tests are
readily available on the market, with many more being developed. However, current
methods have their shortcomings, all lacking one or more qualities to fulfil the
missing needs.An in depth study overview and analysis of the field, with a deep focus
on the current market, will serve as a guideline for the future emerging technologies
involving susceptibility testing and antibiotic resistance evaluation.
Acknowledgment

Initially, we appreciate the department of Medical Laboratory Sciences, College of

Health Sciences, Salale University for selecting for us this term topic and facilitating
its preparation.
We would also like to thank Mr.Fedasan Alemu, our instructor, for making
arrangements and supporting us during this term paper preparation.
Table of content
List of Figure
Acronyms and Abbrevations

AMR Antimicrobial Resistence

AST Antimicrobial Susceptibility Test
AUC Area Under the Curve
CLSI
EUCAST

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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.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.Thus, the understanding of the genetic makeover and the morpho-
anatomical changes in bacteria are of prime importance to counteracting the resistance
mechanism(1).
Antimicrobial resistance (AMR) continues to be a public health threat of global
interest. Predictions show that about 10 million people will die yearly from infections
caused by AMR bacteria till 2050. But, this remains subject to how the globe reacts to
reduce the burden of AMR. Africa is one of the regions likely to contribute the
highest numbers to these global deaths(2)(12).
An important task of the clinical microbiology laboratory is the performance of
antimicrobial susceptibility testing of significant bacterial isolates(2). Antimicrobial
susceptibility testing (AST) technologies help to accelerate the initiation of targeted
antimicrobial therapy for patients with infections and could potentially extend the
lifespan of current narrow-spectrum antimicrobials[1]. Empirical therapy
continues to be effective for some bacterial pathogens because resistance mechanisms
have not been observed e.g., continued penicillin susceptibility of Streptococcus
pyogenes. Susceptibility testing of individual isolates is important with species that
may possess acquired resistance mechanisms (eg, members of the
Enterobacteriaceae, Pseudomonas species, Staphylococcus species, Enterococcus
species, and Streptococcus pneumoniae)[2].
The development of bacterial antimicrobial resistance is neither an unexpected nor a
new phenomenon. It is, however, an increasingly troublesome situation because of the
frequency with which new emerging resistance phenotypes are occurring among
many bacterial pathogens and even commensal organisms(4).The development of
bacterial resistance to these antimicrobials resulted in the need for physicians to

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request the microbiology lab to test a patient’s pathogen against various
concentrations of a given antimicrobial to determine susceptibility or resistance to that
drug(6).
Combating microbial antibiotic resistance now includes the following directions:
(1)Designing new drugs able to effectively suppress pathogens. (2) Finding ways
to slow down the spread of resistance, in particular by reducing antibiotic
consumption. (3) Developing methods to “turn off” the resistance(ref5).
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(1).
There is an unmet need for rapid and decentralized diagnostics in outpatient clinics to
reduce the misuse of antibiotics. It is important to identify the etiological pathogen
and to differentiate between viral and bacterial infections, to identify the antimicrobial
resistances in microbes, and to find out which antimicrobial agent should be used for
the cure(3).

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Laboratory methods used in Antimicrobial Susceptibility
Testing and Microbial Identification

Knowing the weaknesses and limitations of current technologies can serve as the first
stepping stone in the development of the next generation diagnostic tools(10). A
number of antimicrobial susceptibility testing (AST) methods are available to
determine bacterial susceptibility to antimicrobials. The selection of a method is based
on many factors such as practicality, flexibility, automation,cost, reproducibility,
accuracy, and individual preference(4). According to EUCAST and CLSI guidelines,
reliable antibiotic resistance diagnostics requires phenotypic testing, i.e., an
experimental test whether the microorganism grows in the presence of the
antibiotic(3).
Classical AST techniques such as broth microdilution, disk diffusion, gradient tests,
agar dilution and breakpoint tests are based on continuous exposure of a bacterial
isolate to a set of antimicrobials, followed by a visual detection of growth. The use of
advanced optoelectronic systems, fiber optics, microfluidics and indicator dyes
sensitive to redox-state or pH can further enhance the sensitivity and performance of
optical systems(3).

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Current Technologies for Rapid AST

1. Automated and Semi-Automated Devices Based on Microdilution

Susceptibility Testing

Clinical microbiology laboratories are under increasing pressure to provide fast and
reliable microbial identification (ID) and AST(ref3).Automated and semi-automated
devices for bacterial ID and AST are worthy of the task and have significantly
improved laboratory efficiency.Nowadays,automation has been successfully
implemented in most clinical microbiological laboratories to reduce turnaround times,
increase efficiency, and improve cost-effectiveness(ref3). These instruments, using
optical systems for measuring subtle changes, determine bacterial growth and
antimicrobial susceptibility and can produce results in a shorter time (6–12 h) than
conventional manual assessment.
 VITEK 2 Systems:-The first generation of VITEK system with a turnaround
time of 13 h was developed for enumeration and identification of bacteria and
yeasts in 1973. The VITEK 2 System, the next-generation of an instrument, is a
BMD-based AST system that uses 64-well plastic cards containing 17–20
antimicrobial agents. If the bacterial isolate is not previously identified, one card
is used for bacterial identification (ID card) and the other for antimicrobial
susceptibility testing (AST card). Two Vitek 2 instruments are available with test
card (ID and AST) capacities of 60 cards (Vitek 2) and 120 cards (Vitek 2 XL).
Results are reported in 4–18 h, containing MIC and category of susceptibility,
whereas the detection of AMR is facilitated by the Advanced Expert System
(AES). The currently available Vitek 2 Compact instruments can use 15, 30, and
60 cards. The main advantage of the Vitek 2 system with computer software is
the determination of susceptibility of clinically important resistant pathogens,
such as Staphylococcus aureus and Enterococcus faecalis, to an additional four to
ten antibiotics(ref3)
 Phoenix System:- The Phoenix System is widely accepted and used in clinical
microbiology laboratories for identification testing (ID) and antimicrobial
susceptibility testing (AST). The principle of determining the susceptibility is
based on the use of an oxidation-reduction indicator (resazurin dye or Alamar
blue) and the detection of bacterial growth in the presence of various

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 concentrations of the antimicrobial agent. In the Phoenix instrument, a maximum
of 100 tests can be performed by using Phoenix ID/AST combination panels (51
for ID and 85 for AST). The instrument performs automatic reading at 20 min
intervals during incubation for up to 18h and provides accurate and rapid
susceptibility results with easy workflow for the laboratory worker.In 2014, the
new panel for susceptibility of Gram-negative bacteria was introduced for the
Phoenix system to be used in combination with the BD Bruker MALDI-TO.
 MicroScan WalkAway plus System:-The MicroScan WalkAway plus System
provides accurate and rapid identification and susceptibility results for a wide
range of Grampositive and Gram-negative aerobic bacteria. The instrument
utilises three types of panel configurations: combo panels, breakpoint combo
panels, and MIC panels. There are two types of system: 40- and 96-panel capacity
models. The panels are manually inoculated, rehydrated by the RENOK
inoculator, and read automatically. The results are obtained after 4.5–18 h by
reading of rapid panels.
 MicroScan AutoScan 4:-The AutoScan 4 is a semiautomated instrument mostly
used in smaller laboratories or for the testing of supplemental antimicrobial
agents. The instrument provides simplified ID/AST testing in a highly reliable
and affordable package. The system uses the off-line incubation of the
conventional MicroScan AST panels. The panels are manually inoculated or with
the MicroScan Renok instrument and read automatically.
 MicroScan WalkAway System:-The first generation of the MicroScan
WalkAway System available on the market is the AutoSCAN-3. The new
versions of instruments Auto-ACAN-4 and AutoSCAN-WalkAway are improved
and use dry panels that do not need refrigeration. The AutoSCAN-WalkAway
system detects bacterial enzymatic activity and can process 96 panels at the same.

2. Molecular-Based Techniques for Resistance Detection

Molecular AST directly detects specific resistance genes, as well as mutations in and
expression of these genes. These molecular methods have been developed and tested
as an alternative for or complementary to conventional AST and are generally faster
than classic culture-based assays, with the test results available within one to a few
hours.Most of the molecular AST methods fall into one of the three categories:

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amplification-based, hybridization-based, or sequence-based. In amplification-based
methods, the target gene sequence is amplified to allow detection; in hybridization-
based techniques, hybridized nucleic acid probes target gene sequences allowing
detection; and in sequence-based approaches, genome sequences are analysed to
detect resistance-conferring mutations or resistance genes.

Figure 1:-The basic workflow of molecular-based techniques for antimicrobial

susceptibility testing.

2.1. Polymerase Chain Reaction

The most widely used nucleic acid amplification-based method for the detection of
specific resistance genes is polymerase chain reaction (PCR). Both real-time and
conventional PCR rely on the amplification of nucleic acid sequences that encode
resistance to an antibiotic. New PCR-based methods are being developed for the
detection of genetic determinants of resistance to a variety of antibiotics for various
bacterial species, as our knowledge about the genetic basis of antibiotic resistance
increases. Multiplex assays for simultaneous testing of multiple genetic determinants
in various bacterial species have also been developed, i.e., multiplex assays for
identifying numerous cephalosporinaseand carbapenemase-encoding genes, such as
blaKPC, blaNDM, blaIMP, blaVIM, blaAmpC, blaTEM, blaSHV, and blaOXA, or

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mecA gene-encoding methicillin resistance in MRSA. OpGen, Inc. (Rockville, MD,
USA) has recently released the multiplex-based Acuitas® AMR Gene Panel that
detects 28 genetic AMR markers, covering select drugs in nine classes of antibiotics,
from 26 different pathogens. The advantage of this test in comparison with other
commercially available molecular tests is that it also detects non-beta-lactam
resistance genes and those for what would be considered “last-resort antibiotics”, such
as colistin.
Real-time PCR (quantitative PCR, qPCR) is one of the most ubiquitous methods
found throughout clinical microbiology. Although costlier, qPCR offers several
advantages over conventional PCR, including the measurement of data in real-time,
greater sensitivity,reduced risk of carryover contamination, and greater amenity to
multiplexing. Further advantages are that many systems are partially or even
completely automated, such as GeneXpert® Instrument Systems (Cepheid Corp.,
Sunnyvale, CA, USA) and BD MAX System platform (Becton Dickinson, Franklin
Lakes, NJ, USA), which are easily operated and can be used for the detection of
carbapenemases, ESBLs, MRSA, VRE, etc.
Limitation:-The downside is that they are limited to using test assays only from
specific manufacturers,with GeneXpert® Instrument Systems requiring GeneXpert
assays (Cepheid Corp., Sunnyvale, CA, USA) and BD MAX System using Check-
Points® qPCR assays (Wageningen, The Netherlands).
Tests based on qPCR can also be used for phenotypic differentiation of resistant and
susceptible strains due to its ability to measure genome copy numbers during bacterial
growth in the presence of antibiotics. The major disadvantage is that the system
cannot provide information about the mechanism of resistance and that it requires the
previous culture, meaning that the primary clinical samples cannot be used(ref3).

2.2. DNA-Microarrays

DNA-microarrays are used to identify the presence of specific nucleic acid sequences
using complementary short oligonucleotides immobilised on a solid surface. Since
these oligonucleotides can be assembled onto solid surfaces in close proximity, this
method could detect numerous sequences in a single assay, which would allow
simultaneous, in parallel detection of different pathogens and detection of vast
numbers of different resistance genes, as well as detecting numerous distinct

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mechanisms of resistance or variants of a single mechanism present in bacterial
isolates, as opposed to PCR-based approaches. The Verigene system (Luminex
Corporation, Austin, TX, USA) has developed Blood Culture Multiplex Microarray-
Based Molecular assays for rapid diagnostics of 12 Gram-positive and 9 Gram-
negative bacteria, along with their associated resistance genes (i.e., mecA, vanA,
vanB, blaKPC, blaNDM, blaIMP, and blaVIM). In addition to qPCR-based assays,
CheckPoints® has also developed the CHECK-MDR CT103 DNA microarray for the
detection of the clinically most prevalent ESBLs and carbapenemases, as well as
mobile colistin resistance (mcr) genes in Gram-negative bacteria. Both of these
microarray tests have shown high sensitivity (94–100%) and specificity (94–98%),
and CHECK-MDR CT103 DNA microarray also showed the ability to discriminate
between carbapenemase and ESBL variants of GES-type beta-lactamase.
The advantages of currently available molecular-based methods are that they are
direct,rapid, highly sensitive, and specific, thus potentially allowing the earlier
administration of targeted therapy. Furthermore, for some methods, direct clinical
samples can be used.
Limitation:-The presence of a resistance marker does not always have to correlate
with phenotypic resistance.Additionally, the extent and intensity of gene expression
are important parameters, as some genes need different expression levels to produce
resistance. A potential solution to this issue would be the use of reverse transcription
qPCR, which relies on the measurement of gene transcripts (RNA levels) instead of
the presence of a gene.
Another drawback is that these methods can only detect resistances that are searched
for, and not novel or uncharacterised mechanisms of resistance, which could lead to
false-negative results and inappropriate classification of resistant isolates as
susceptible.
A final consideration is that these methods are not capable of defining MIC values. As
such, these methods have to be validated against phenotypic data to be useful, and
extensive resistance marker databases and innovative bioinformatics methodologies
are mandatory requirements. Nevertheless, molecular-based AST methods are a safe,
efficient, and reliable screening tool in clinical settings.

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2.3. Whole-Genome Sequencing in Antimicrobial Susceptibility Testing

Applying whole-genome sequencing (WGS) would essentially enable the detection of

all genes involved in AMR, which would help make comprehensive databases of all
species-specific resistance factors and make in silico AMR detection possible. Recent
studies showed high concordance between the resistance profiles obtained using WGS
and those obtained using phenotypic susceptibility testing, demonstrating that data
obtained from genome sequences can correlate well with phenotypic resistance in
some cases. In addition to genome-based resistome analyses, RNA-mediated
transcriptomic approaches have also been described.
Limitation:-Despite all of the advantages, WGS is not routinely performed in clinical
practice. Considering the turnaround times of WGS, the existence of unknown
resistance mechanisms, and the elevated cost compared with traditional and emerging
techniques, the use of WGS for AST is not yet part of routine practice in clinical
microbiology(ref3).

3. Mass Spectrometry

3.1 Matrix-assisted laser desorption ionization-time of flight mass spectrometry

(MALDITOF MS):- was discovered in the 1980s and introduced into the
microbiological routine as an effective tool for bacterial and yeast identification about
15 years ago. It has been applied to classify the specific bacterial protein contents and
their matching protein biomarkers because of its rapid turnaround time, low sample
volume requirements, and per-sample costs(9).
MALDI-TOF mass spectrometry is a new technology that has revolutionized
pathogen identification and has also proven to accelerate detection of antimicrobial
resistance compared to the traditional antibiotic susceptibility tests (AST) as well as
DNA amplification methodologies(5). MALDI-TOF mass spectrometry as an
analytical technique is based on the cellular proteome, which reflects gene products
and metabolic products of the organisms. Especially, this method is based on the
analysis of highly abundant, mainly ribosomal, proteins of microorganisms in the
mass range of 2,000 to 20,000 Daltons. These proteins are ionized into charged
molecules, by either addition or loss of one or more than one protons, in order to
measure the mass to charge (m/z) ratio(5).

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The technology has incorporated up to know four different methodologies:
(I) The detection of differences of mass spectra of susceptible and resistant
isolates of a given microorganism using the classical strain typing methodology;
e.g.the first evaluation of MALDI-TOF in determination of antibiotic resistance
was the case of methicillin resistant S. aureus (MRSA) and methicillin-sensitive
S. aureus (MSSA).
(II) The analysis of bacterial induced hydrolysis of β-lactam antibiotics;
e.g.MALDI Biotyper-Selective Testing of Antibiotic Resistance-Beta-Lactamase
Assay(MBT-STAR-BL Assay). Hydrolysis is detected by the observation of
specific mass shifts after a 30–180-minute incubation period of the pathogen with
the tested β-lactam antibiotic
(III) The detection of stable (non-radioactive) isotope-labeled amino acids;
e.g.MBT-RESIST assay was performed for the detection of MRSA using
oxacillin and cefoxitin as antibiotics. In the presence of these antibiotics, only
resistant strains (MRSA) are able to grow and to perform protein biosynthesis,
while susceptible strains (MSSA) stop growing under conditions of antibiotic
stress and thereby present different profile spectra compared to the setups without
antibiotics.
(IV) The analysis of bacterial growth in the presence and absence of antibiotics
using an internal standard.
Several MALDI-TOF MS-based methods have been proposed for rapid detection of
antimicrobial resistance, including monitoring antibiotic modification by bacterial
culture (e.g., beta-lactam hydrolysis, acetylation of fluoroquinolones, direct detection
of proteins involved in specific resistance mechanisms, and detection of stable isotope
labelling that requires expensive, isotopically labelled media.
The hydrolysis of the target beta-lactam antibiotic, as shown by peak disappearance,
is used to detect beta-lactamase-producing bacteria using MALDI-TOF MS. As a
result, the assay for detecting carbapenemase production automatically determines
sensitivity or resistance depending on the degree of antibiotic hydrolysis. The method
had 98% sensitivity and 100% specificity after 30 min of incubation of bacteria with
the antibiotic, with both reaching 100% after 60 min of incubation.
Limitation:-Despite all the advantage of MALDI-TOF MS, the expensive nature of
the instrument and its maintenance are prime disadvantages for mass application(1).
Most automated systems lack reproducibility,sensitivity, and reliability compared

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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 (1)
Beta-lactam resistance is only recognized when it is mediated by beta-lactamases;
alternative resistance mechanisms have not been elucidated; therefore, other tests
should confirm negative results.
Another assay for the detection of carbapenemases is a rapid and novel method using
detonation nanodiamonds (DNDs) as a platform for the concentration and extraction
of A. baumannii carbapenemase-associated proteins before MALDI-TOF MS
analysis. The sensitivity and the specificity of the proposed platform could reach 96%
and 73%, as compared with traditional imipenem susceptibility testing, and 100%
compared with PCR results. This method may detect the carbapenemases produced by
A. baumannii in 90 min and does not require the addition of a carbapenemase
substrate, as other mass spectrometric methods do. It is efficient for detecting other
carbapenemase-producing bacteria.
3.2 MALDI Biotyper-Antibiotic Susceptibility Test Rapid Assay (MBT-
ASTRA):-is an alternative MS-based method for AST which utilises semi-
quantitative MALDI-TOF MS to measure the relative growth rates of bacterial
isolates exposed to antibiotics compared with untreated controls during a short
incubation step. A software tool calculates and compares the area under the curves
(AUCs) of spectra of bacteria either exposed or not to an antibiotic. In this method, if
the microbial strain is susceptible, the AUC of the bacterial suspension with the
antibiotic will be reduced compared with that without antibiotics, whereas with a
resistant strain the AUCs with or without antibiotics will be comparable.
The main advantage of the MBT-ASTRA is that the assay does not depend on the
resistance mechanism and is utilisable with any antibiotic. Moreover, it does not
require specialised media or instrumentation, beyond the MALDI-TOF mass
spectrometer.
Limitation:-drawback of the MBT-ASTRA assay is that the concentration of
antibiotics used and the incubation time must be optimised for each species and
antibiotic combination.

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MBT-Resist assay, based on the detection of peak shift after stable isotope labelling,
is an approach that uses the following principle: bacteria are grown in parallel in two
distinct culture mediums, one containing 12C as a carbon component and the other
containing 13C. The system compares the mass spectrum of bacteria grown on an
isotope-labelled medium with antibiotics to the mass spectrum of the same strain
grown on an unlabelled medium without antibiotics. Resistant strains can thrive in the
presence of antibiotics, incorporating 13C into the polypeptide, causing a shift in the
peak to a higher m/z in the mass spectrum.
Antibiotic resistance by direct-on-target microdroplet growth assay (DOT-MGA) is a
novel approach for detecting antimicrobial susceptibility in bacteria treated with
breakpoint concentrations of antibiotic on the target plate of MALDI-TOF MS. The
best performance was obtained by recovering bacteria from positive blood cultures
and after a 4h incubation of microdroplets with or without meropenem at the
breakpoint concentration.The accuracy of the DOT-MGA achieved results
incomparable with those of the BMD assay, with a time saving of about 14 h, and
higher than combination disk tests. Due to the great speed and simple application,
MALDITOF MS would be the most suitable for endemic AMR clinical strains in
specific settings, i.e., MRSA, VRE, CRAB, CRPA, and ESBL-, AmpC-, and
carbapenemase-producing Enterobacterales.

Generally Advantages of the Current Technologies in

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing (AST) and microbial identification are crucial in

the diagnosis and treatment of infectious diseases. Current technologies in these areas
have the following advantages.
1. Rapid results: Many current technologies offer rapid results, allowing clinicians to
make informed treatment decisions quickly.
2. High accuracy: Modern technologies are highly accurate, reducing the risk of
misdiagnosis and inappropriate treatment.
3. Automation: Automation of AST and microbial identification reduces human error
and increases efficiency.

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4. Flexibility: Many technologies can be adapted for use with a range of bacterial
species, allowing for broad-spectrum testing.
5. Cost-effective: Some current technologies are cost-effective, making them
accessible to a wider range of healthcare providers.

Generally limitations of the Current Technologies in

Antimicrobial Susceptibility Testing and Microbial
Identification

1. Emergence of antibiotic-resistant bacteria: Some bacteria may be resistant to

multiple antibiotics, making it difficult to treat infections.
2. Lack of standardization:There is a lack of standardization across different AST
methods, making it difficult to compare results from different laboratories.
3. Turnaround time:Some methods may have longer turnaround times, delaying
treatment decisions.
4. Limited sensitivity:Some methods may have limited sensitivity, leading to false-
negative results.
5. Cost:Some methods may be expensive, limiting their accessibility in resource-
limited settings.

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. 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.The applicability of molecular biology tools such as cloning and recombinant
expression has enhanced the sensitivity of detection, and low concentrations of

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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. 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. 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 inmaintaining 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,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 ofmicrofluidics. 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

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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 smartphones is limited by their camera performances, which
result in lower detection limits, especially in a colorimetric assay. 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-beta-lactamases genes. 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(1).

Future prospects

1. Development of new technologies:Ongoing research is focused on developing

new technologies that offer higher accuracy, faster results, and broader applicability.
2. Integration of genomic data: The integration of genomic data into AST and
microbial identification could provide more accurate and personalized treatment
options.
3. Standardization: Efforts are underway to standardize AST and microbial
identification methods to improve comparability across different laboratories.
4. Point-of-care testing: The development of point-of-care testing could enable faster
diagnosis and treatment decisions in remote or resource-limited settings.
5. Artificial intelligence: The use of artificial intelligence could help to improve the
accuracy and speed of AST and microbial identification, particularly in complex
cases.

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