1. Introduction
Antimicrobial resistance (AMR) stands as a paramount global health crisis, demanding innovative approaches for rapid antimicrobial susceptibility testing (AST) and Amr Diagnosis. Traditional clinical microbiology methods are often slow, labor-intensive, and costly, leading to delayed diagnoses and the overuse of broad-spectrum antibiotics. This empirical approach fuels the spread of AMR, resulting in increased mortality and healthcare expenditures. To combat this growing threat, advancements in AMR diagnosis are crucial, and this review explores the latest cutting-edge methods and technologies driving progress in this vital field.
The escalating rates of AMR pose a significant economic burden and threaten the efficacy of modern medical treatments, including chemotherapy and surgical procedures. The continuous emergence of resistant strains necessitates ongoing antibiotic discovery, but equally important is the implementation of effective strategies to prolong the lifespan of existing antibiotics. Rapid and accurate AMR diagnosis is a cornerstone of these strategies, enabling targeted therapies and reducing the reliance on empirical treatments.
Studies reveal that AMR infections cause over 33,000 deaths annually in the European Union alone, with an economic impact estimated at €1.5 billion. Globally, the toll exceeds 500,000 deaths per year, disproportionately affecting infants. The slow turnaround time (TAT) of conventional diagnostic methods, often spanning several days, compels clinicians to initiate broad-spectrum antibiotic therapy before definitive AMR diagnosis. This practice contributes to microbiome dysbiosis and exacerbates the AMR problem. Therefore, the development and deployment of rapid, sensitive, and affordable platforms for AMR diagnosis are urgently needed to guide targeted antibiotic use and improve patient outcomes. Rapid diagnostic tests (RDTs) are recognized as essential tools in antimicrobial stewardship programs, demonstrating potential to reduce mortality, shorten hospital stays, and lower healthcare costs by optimizing antibiotic administration.
This review provides a comprehensive overview of current and emerging methodologies in AMR diagnosis, highlighting their advantages and limitations. We critically evaluate established techniques, such as phenotypic and molecular methods, alongside innovative approaches including next-generation sequencing (NGS), whole genome sequencing (WGS), whole genome metasequencing (WGM), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and infrared (IR) spectroscopy. Particular emphasis is placed on microfluidics and lab-on-a-chip technologies, which offer significant promise for transforming AMR diagnosis. Finally, we summarize commercially available platforms for AST, providing a perspective on the current landscape of AMR diagnosis solutions. Figure 1 offers a visual summary of the diagnostic methods and technologies discussed.
Figure 1. Summarizing chart of conventional and advanced methods and technologies for AMR diagnosis.
2. Conventional AMR Diagnostic Methods
Despite the advent of newer technologies, conventional methods remain fundamental in AMR diagnosis. These primarily encompass culture-based and molecular-based approaches, complemented by microscopy and spectrometry techniques.
2.1. Phenotypic Methods
Phenotypic methods for AMR diagnosis rely on observing bacterial growth in the presence of antibiotics. These methods are categorized as manual or automated. Manual methods include agar dilution, gradient tests (E-tests), disk diffusion, and broth microdilution. Automated platforms like VITEK® 2 COMPACT, Sensititre™ ARIS™ 2X, and Alfred 60AST system automate variations of these core phenotypic assays. Broth dilution platforms often utilize pre-prepared cartridges or plates with antibiotic gradients and controls. Sensititre panels, for example, are microdilution plates pre-loaded with dried antimicrobials, used to assess carbapenem-resistant Klebsiella pneumoniae susceptibility to polymyxins. These platforms offer real-time growth monitoring and minimum inhibitory concentration (MIC) analysis, leveraging extensive databases for organism identification and AMR diagnosis.
Phenotypic methods yield both qualitative and quantitative data. Dilution methods and E-tests provide quantitative MIC values, representing the lowest antibiotic concentration preventing visible bacterial growth. Disk diffusion assays indicate resistance through zones of inhibition. E-tests are particularly useful for fastidious microorganisms like Campylobacter spp. Standardized interpretation of AST results is crucial, with organizations like EUCAST and CLSI providing guidelines. However, discrepancies exist in breakpoint interpretations for different bacterial species, such as amikacin resistance in Escherichia coli, where EUCAST provides a more stringent susceptibility breakpoint than CLSI. Despite these variations, phenotypic methods remain a cornerstone of AMR diagnosis in clinical microbiology labs globally.
2.2. Molecular-Based Methods
Molecular methods in AMR diagnosis offer advantages over phenotypic assays by directly detecting antimicrobial resistance genes (ARGs). These assays enable multiplexing, precise characterization of resistance mechanisms, and can be applied to non-purified polymicrobial samples, eliminating isolate purification steps. Molecular methods also facilitate rapid adaptation to detect newly emerging resistance factors. However, they have limitations. They cannot determine MIC values and may miss novel or uncharacterized ARGs. The vast diversity of ARGs also poses a challenge for comprehensive assay development, potentially increasing costs compared to phenotypic methods. Nevertheless, advances in molecular techniques are increasingly integrating them into routine AMR diagnosis.
Molecular techniques for AMR diagnosis leverage nucleic acid amplification and hybridization to detect ARGs and their expression. These techniques provide rapid and sensitive ARG detection. The decreasing cost of next-generation sequencing (NGS) and whole genome sequencing (WGS) has significantly expanded ARG databases, enhancing the targets available for molecular AMR diagnosis. PCR-based and isothermal amplification methods, along with DNA microarrays, are key molecular approaches detailed below.
2.2.1. PCR-Based Methods
Polymerase chain reaction (PCR) is the most widely used nucleic acid amplification technique for AMR diagnosis. Real-time, quantitative, digital, and multiplex PCR assays have further enhanced the clinical utility of genetic testing in AMR diagnosis. The evolution of NGS and WGS has expanded ARG target availability, driving the development of high-throughput quantitative PCR (HT-qPCR). HT-qPCR is a cost-effective and rapid method for simultaneously analyzing numerous ARGs across various sample types. For example, HT-qPCR has been used to profile ARGs in park soils and to screen antimicrobial susceptibility of Orientia tsutsugamushi clinical isolates. Chemically synthesized double-stranded (ds) DNA can serve as qPCR standards for ARGs, offering comparable performance to natural DNA and enabling robust AMR diagnosis across diverse sample matrices. Multiplex real-time PCR assays are used for AMR characterization in Neisseria gonorrhoeae, targeting resistance to multiple antibiotics. While accurate in detecting resistance-conferring mutations, sensitivity limitations may restrict direct clinical specimen application, but it remains valuable for screening AMR in gonococcal isolates faster than culture-based methods. Multiplex real-time PCR assays have also been developed for rapid and reliable detection and differentiation of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in pediatric samples. Ligation mediated PCR (LM-PCR) offers a method for strain characterization and differentiation based on specific melting-profile DNA patterns, applicable to both fungal and bacterial isolates, and has been used for epidemiological typing of various pathogens, including extended-spectrum-beta-lactamase-producing Escherichia coli.
2.2.2. Isothermal Amplification Methods
Isothermal DNA amplification is a significant advancement in molecular AMR diagnosis, eliminating the need for thermocycling required in traditional PCR. Methods like strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HDA) facilitate rapid, next-generation molecular diagnostics for AMR diagnosis.
Isothermal methods offer advantages over PCR, including eliminating thermocycling, which reduces power consumption and analysis time. A simple water bath or hotplate can replace thermocyclers. Isothermal amplification is generally faster and more sensitive than PCR, achieving rapid amplification and detection within minutes due to continuous amplification. Methods like LAMP, RCA, and HDA also eliminate template denaturation and, in the case of LAMP and HDA, tolerate biological components, simplifying sample preparation for AMR diagnosis. While some isothermal methods, like LAMP, require complex primer design, they often offer greater specificity than PCR. LAMP and RPA are particularly promising for point-of-need (PON) diagnostics in resource-limited settings due to their simplicity, sensitivity, cost-effectiveness, and minimal DNA template requirement. Isothermal methods are well-suited for microfluidic platforms for AMR diagnosis. LAMP amplicons can be detected visually through turbidity or color changes, further simplifying AMR diagnosis in resource-constrained environments. However, multiplexing isothermal methods can be challenging, and some methods, like LAMP and NASBA, have complex reaction mechanisms and primer requirements.
Commercial in vitro diagnostic (IVD) products based on PCR and isothermal nucleic acid amplification technology (NAAT) have emerged due to advancements in engineering, reagent formulations, and automation. Integrated and automated platforms combine nucleic acid extraction, purification, amplification, and detection with sophisticated data analysis software, providing accurate and streamlined AMR diagnosis.
2.2.3. DNA Microarrays
DNA microarrays are tools for assessing bacterial genomic diversity and detecting antimicrobial resistance genes for AMR diagnosis. These arrays initially used glass slides spotted with DNA probes targeting genes of interest. Comparative genomic hybridization was performed, followed by hybridization analysis. Early microarray systems were costly and time-consuming due to glass slides and fluorescent dyes. However, significant advancements have simplified and improved DNA microarray technology. A rapid DNA labeling system using biotinylated primers has been developed for disposable microarrays. Microarrays for simultaneous ARG detection in Staphylococcus isolates using fluorescently labeled PCR products have also been created. A rapid cartridge-based melting curve assay for pyrazinamide-resistant Mycobacterium tuberculosis has been developed for point-of-care AMR diagnosis in resource-limited settings, utilizing a closed cartridge and battery-powered analyzer.
3. Non-Conventional AST Methods
This section reviews promising non-conventional methods for AST and AMR diagnosis, including sequencing technologies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and Fourier transform infrared (FTIR) spectroscopy.
3.1. Genome Sequencing and Metagenomics in AMR Diagnostics
DNA sequencing has revolutionized AMR diagnosis. Early Sanger sequencing methods, developed in the 1970s, could sequence hundreds of bases per day. Automated sequencers based on Sanger sequencing, introduced in 1995, enabled the sequencing of the first complete bacterial genome. Next-generation sequencing (NGS) technologies, developed in the 2000s, dramatically increased throughput through multiplexing, allowing simultaneous analysis of thousands of samples for AMR diagnosis and research. A typical NGS workflow involves DNA extraction, fragmentation, adaptor ligation, DNA amplification, and sequencing.
Second-generation sequencing, or short-read sequencing, includes platforms like 454 pyrosequencing and Illumina. Pyrosequencing detects pyrophosphate release during nucleotide incorporation, while Illumina platforms use reversible terminator nucleotides with fluorescence labeling. Third-generation sequencing, exemplified by Pacific Biosciences and Oxford Nanopore Technologies, offers real-time, single-molecule long-read sequencing. Pacific Biosciences uses an optical approach with zero-mode waveguides, and Oxford Nanopore measures electrical signal changes as DNA molecules pass through nanopores. These advanced sequencing approaches enable whole genome sequencing (WGS) and whole metagenome sequencing (WMS), facilitating comprehensive AMR diagnosis by characterizing complex microbial communities and identifying antibiotic resistance determinants directly from patient samples without prior isolation.
The availability of bacterial sequence data has surged due to sequencing technology advancements and decreasing costs. Improved bioinformatics tools and databases have made sequencing a viable tool for ARG identification, characterization, and surveillance for AMR diagnosis. Numerous bioinformatic tools and databases (Table 1) have been developed for detecting ARGs from WGS and WMS data, complementing traditional culture-based methods and providing rapid resistance determination in both cultivable and uncultivable bacteria. Sequencing data organization is crucial for ARG analysis. Short reads from Illumina platforms can be analyzed using assembly-based methods (assembling reads into contigs and comparing to reference databases) or read-based methods (mapping reads directly to reference databases to predict resistance).
Table 1. Bioinformatic tools and databases for AMR gene detection from sequencing data.
A major advancement is the ability to predict AMR directly from genomic data. Studies have shown high concordance (>96%) between known mutations or ARGs and MIC values for various antimicrobials. Machine learning techniques applied to genome sequencing data are increasingly used to predict AMR and MIC values, further enhancing the utility of sequencing in AMR diagnosis.
While long-read sequencing offers comprehensive genome capture, it requires significant investment in equipment and expertise. These systems also typically need larger DNA quantities and longer preparation times and may have higher error rates than short-read sequencing platforms. Nanopore sequencing, however, offers advantages like portability, affordability, and on-site sequencing. The MinION nanopore system is a portable, real-time device for DNA and RNA sequencing, detecting ionic current changes as nucleic acids pass through nanopores, making it a valuable tool for rapid AMR diagnosis in diverse settings.
3.1.1. Pyrosequencing
Pyrosequencing was proposed as a rapid tool for Yersinia pestis detection and for AMR diagnosis in 2012. An assay based on pyrosequencing for ARG profiling was developed. Pyrosequencing was also evaluated for detecting drug-resistant Mycobacterium tuberculosis, demonstrating high specificity (96–100%) in detecting resistance-associated mutations. It was further assessed for rapid detection of resistance to fluoroquinolones, rifampicin, kanamycin, and capreomycin in M. tuberculosis isolates, showing high sensitivity for rifampicin and fluoroquinolones resistance detection. While effective, pyrosequencing has largely been superseded by more advanced sequencing technologies for AMR diagnosis.
3.1.2. WGS
WGS has been evaluated for predicting AMR in non-typhoidal Salmonella isolates from human and food sources. Data indicated a strong correlation between acquired resistance and known resistance determinants, useful for risk assessment related to antibiotic use in food animal production. WGS was also used to determine ARG occurrence in Streptococcus uberis and Streptococcus dysgalactiae from dairy cows, revealing associations between unique ARG sequences and phenotypic AMR profiles. Studies on Campylobacter showed a high correlation (99.2%) between resistance genotypes and phenotypes, suggesting WGS as a reliable indicator for resistance to various antibiotics in this organism. These findings and subsequent studies highlight WGS as a powerful tool for AMR diagnosis and surveillance programs. Recent WGS studies have revealed the co-existence of antibiotic resistance and virulence factors in carbapenem-resistant Klebsiella pneumoniae, emphasizing the importance of genomic surveillance in monitoring evolving AMR threats.
3.1.3. Combination of Short and Long Read WGS Sequencing
Plasmids play a significant role in ARG transfer among bacteria, but are challenging to assemble from short-read WGS data alone. Combining short- and long-read WGS sequencing enables comprehensive characterization of ARGs on plasmids and their genomic context, crucial for understanding AMR spread and conducting effective risk assessments. This combined approach overcomes limitations in plasmid reconstruction, providing valuable insights for AMR diagnosis and control.
3.1.4. Nanopore Sequencing
Nanopore sequencing has found broad applications in virus and yeast research, de novo bacterial assembly, viral pathogen identification, metagenomics studies, and AMR diagnosis. The MinION nanopore sequencer has been used to resolve the structure and chromosomal insertion site of antibiotic resistance islands in Salmonella Typhi and to identify AMR determinants in multidrug-resistant E. coli. Long-read analysis facilitated the identification of mobile genetic elements carrying ARGs and revealed co-location of multiple resistance determinants on the same mobile element, enhancing understanding of AMR transmission in E. coli. MinION has also been shown to rapidly identify bacterial pathogens and acquired resistance genes directly from urine samples within 4 hours, highlighting the potential of WMS-based AMR diagnosis to guide antimicrobial therapy adjustments. Nanopore sequencing has been utilized to detect ARGs, assess their taxonomic origins, and decode their genetic organization, informing targeted interventions to mitigate AMR spread in hospitals and other settings. It has also been applied for rapid plasmid, virulence marker, phage, and ARG determination in Shiga toxin-producing E. coli. Recent studies demonstrate MinION’s capability for rapid pathogen, plasmid, and ARG identification in bacterial DNA extracted from positive blood cultures, achieving pathogen identification within 10 minutes and ARG/plasmid detection within 1 hour using raw nanopore sequencing data. MinION sequencing data for Streptococcus suis accurately predicted multilocus sequence types and AMR profiles. Ultra-long read Nanopore sequencing has been successfully used for AMR detection in Mannheimia haemolytica, enabling complete or near-complete genome assembly and ARG detection with minimal reads.
Unlike phenotypic tests, NGS provides the molecular basis of AMR, informing monitoring schemes and facilitating the understanding of resistance acquisition mechanisms. NGS can also characterize novel resistance mechanisms by sequencing phenotypically resistant isolates, offering significant value compared to nucleic acid-based techniques like PCR in advancing AMR diagnosis.
3.2. MALDI-TOF Mass Spectrometry in AMR Diagnostics
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an alternative to traditional genotypic and phenotypic methods for AMR diagnosis. MALDI-TOF MS analyzes the cellular proteome, profiling proteins (primarily ribosomal, 2–20 kD) from whole bacterial cell extracts to create unique spectral fingerprints for microorganism identification at genus, species, and subspecies levels.
Sample preparation involves mixing the sample with an energy-absorbent matrix solution. Laser ionization of the sample within the matrix generates protonated ions, which are separated based on their mass-to-charge (m/z) ratio in a time-of-flight (TOF) analyzer. The resulting Peptide Mass Fingerprint (PMF), a distinctive mass spectrum, is compared to reference spectra databases for microorganism identification. MALDI-TOF MS has also been applied to detect antibiotic resistance mechanisms, such as carbapenemases, contributing to rapid AMR diagnosis. Standardization is crucial for reproducible results. MALDI-TOF MS is considered rapid (minutes), accurate, easy to use, cost-effective, and environmentally friendly for bacterial identification and AMR diagnosis. However, high equipment and maintenance costs and system size limit its use in low-resource settings or as a point-of-care (POC) AMR diagnosis platform. It is also not suitable for mixed samples without prior purification and cultivation. Databases with spectra differentiating susceptible and resistant strains are essential for effective AMR diagnosis. Table 2 lists recent applications of MALDI-TOF MS in AMR diagnosis. Commercially available MALDI-TOF MS systems include MALDI Biotyper (Bruker Daltonik) and VITEK MS (bioMérieux).
Table 2. Applications of MALDI-TOF MS in AMR detection.
3.3. Fourier Transform Infrared (FTIR) Spectroscopy in AMR Diagnostics
Fourier transform infrared (FTIR) spectroscopy is an emerging phenotypic method for AMR diagnosis, leveraging recent advancements in optical technologies. FTIR provides detailed biochemical information at the molecular level by quantifying infrared (IR) light absorption by cellular components like lipids, lipopolysaccharides, carbohydrates, proteins, and nucleic acids. This generates a characteristic FTIR spectrum reflecting the sample’s complete biochemical composition. FTIR spectroscopy can differentiate molecular changes associated with AMR development in prokaryotes, offering a tool for rapid AMR diagnosis.
Combining FTIR spectroscopy with artificial neural networks (ANNs) has enabled the detection of cephalothin-susceptible uropathogenic E. coli strains with high accuracy (95%). FTIR microscopy and computational classification methods have been used to determine E. coli susceptibility to various antibiotics, achieving 85% success in classifying sensitive and resistant strains. FTIR microscopy coupled with statistical classification has also demonstrated the detection of structural molecular changes linked to AMR. Atomic force microscopy-infrared spectroscopy (AFM-IR), a nanoscale technique, combined with chemometric analysis, has identified chemical composition changes in S. aureus associated with vancomycin and daptomycin resistance, advancing single-cell AMR diagnosis. AFM-IR combines IR and scanning probe microscopy for improved resolution and cellular mapping at the atomic scale.
FTIR offers advantages such as speed, cost-effectiveness, and environmental friendliness in AMR diagnosis studies. However, like MALDI-TOF MS, high equipment and maintenance costs and system size limit its application in low-resource settings or as a POC AMR diagnosis platform. Sample purification, cultivation, and databases with spectra differentiating susceptible and resistant strains are also required.
4. Microfluidics and Lab-on-a-Chip Technologies towards Rapid Diagnostics
Microfluidics-based lab-on-a-chip (LoC) devices offer promising tools for rapid AMR diagnosis, clinical diagnostics, food safety, and environmental monitoring. LoC technology offers advantages over macro-scale methods, including rapid and high-throughput analysis, precise fluid manipulation, low cost, low reagent and power consumption, small sample volumes, automation, integration, compactness, and portability, making them ideal for advanced AMR diagnosis. Microfluidic detection methods for AMR diagnosis fall into genotypic and phenotypic assays. Genotypic microfluidic assays (e.g., PCR, LAMP) target genetic markers like ARGs, bypassing bacterial growth and reducing TAT to several hours. Combining microfluidics with isothermal DNA amplification further enhances these methods by eliminating thermocycling, offering cost-effective and efficient diagnostic tools for various applications. Phenotypic microfluidic assays monitor bacterial growth in the presence of antibiotics for accurate AST results. Bacteria are confined in small volumes (chambers, channels, droplets), captured using antibodies on magnetic beads or membranes, or encapsulated in agarose or using hydrodynamic trapping. Hydrodynamic trapping immobilizes bacteria effectively in microfluidic devices. Antibody-based capture is costly and strain-specific. Droplet-based methods often require sophisticated readout systems. Agarose-based methods, while adaptable to multi-well plates, lack straightforward arraying for automated detection and analysis. Further research and development are needed for these systems to become commercially viable for widespread AMR diagnosis. Spectroscopy-based, colorimetric-based, pH-based, and quartz-crystal microbalance (QCM)-based microfluidic approaches are being developed for point-of-care (POC), multiplexed, single-cell, or single-molecule AMR diagnosis.
4.1. Spectroscopy-Based Approaches
Surface-enhanced Raman spectroscopy (SERS) is a biochemical fingerprinting technique reflecting macromolecular profiles and changes within bacterial cells due to antibiotic action. SERS can investigate bacterial resistance or susceptibility and antibiotic mechanisms using whole-cell spectral fingerprints. SERS enables rapid, accurate, and ultra-sensitive detection of resistant bacteria with minimal sample preparation, making it highly valuable for advanced AMR diagnosis. SERS is also integrated into LoC platforms. Microfluidic chips combined with SERS have been developed for rapid MSSA and MRSA detection and differentiation. Integrated multimodal microfluidic systems can perform on-chip bacterial enrichment, metabolite collection, and in situ SERS measurements for AST with a low detection limit. Microfluidic platforms integrating SERS with microwells enable low-concentration bacterial encapsulation and label-free detection for in situ AST.
However, SERS has limitations. Sample drying prior to analysis can cause reproducibility issues. Liquid-phase bacterial detection, while ideal for interrogating cells in their natural environment, is challenging due to Raman laser scattering. SERS is often applied to single-species samples under controlled conditions. Comprehensive SERS spectral databases and advanced spectral data analysis methods are still needed for complex sample AMR diagnosis. Ideally, bacterial SERS biosensors should simultaneously detect multiple strains from complex samples to maximize their impact on AMR diagnosis.
4.2. Colorimetric-Based Approaches
Colorimetric-based microfluidic platforms are being developed for pathogen identification and AST, simplifying AMR diagnosis. Integrated, automated microfluidic platforms can perform AST for multiple antibiotic combinations against bacterial pathogens, providing on-chip MIC determination using pH-dependent colorimetric broths with a TAT of 16–24 hours. Automated fluid control is achieved using pneumatically controlled modules. Polymer-based microfluidic devices for Campylobacter spp. identification and AST utilize incubation micro-chamber arrays with chromogenic media and antibiotics. Bacterial growth is visualized by color changes, enabling rapid on-chip identification and AST within 24 hours with low detection limits, although performance can vary with food matrices.
4.3. pH-Based Approaches
Microfluidic devices integrating pH-sensitive chitosan hydrogels and Fourier transform reflectometric interference spectroscopy (FT-RIFS) enable rapid AST by detecting small pH changes. Whole bacterial growth detection TAT is less than 2 hours, significantly accelerating AMR diagnosis. Real-time, ultra-fast electronic detection microdevices for ARG detection use RPA isothermal amplification coupled with thin-film transistor pH sensors, achieving detection in under 3 minutes with high sensitivity. Polymer/paper hybrid microfluidic chips facilitate one-step multiplexed uropathogen identification and AST using paper substrates within cell culture microchambers for versatile antimicrobial and chromogenic media combinations. Assays are completed within 15 hours with camera-based chromogenic reaction monitoring. Laser-patterned paper-based microfluidic devices for E. coli identification and susceptibility testing use visual color change observation, suitable for low-resource settings and minimally trained personnel, expanding access to AMR diagnosis.
4.4. Quartz-Crystal Microbalance (QCM)-Based Approaches
Quartz-crystal microbalance (QCM) is a nanogram-sensitive piezoelectric sensor for real-time, rapid, on-site AMR diagnosis. Highly sensitive, accurate, and dynamic QCM systems with magnesium zinc oxide (MZO) nanostructures can monitor antimicrobial effects on E. coli and Saccharomyces cerevisiae and detect ARGs. Low cost, small clinical sample volume requirements, and rapid detection (within 10 minutes) are key advantages for point-of-care AMR diagnosis.
4.5. POC Approaches
Point-of-care (POC) systems for AMR diagnosis are crucial for rapid clinical decision-making. POC systems for bacteriuria and urinary tract infection (UTI) AMR diagnosis have been developed with a 2-hour TAT and detection ranges from 50 to 105 CFU/mL. Detection is based on portable particle-counting instruments with miniature confocal microscopes and real-time data analysis software, enabling growth curve measurements of fluorescently stained bacteria in control and antibiotic-treated samples. These POC systems eliminate pre-processing steps and offer high sensitivity for AMR diagnosis at the point of care. Rapid diagnostic platforms integrating single-step blood droplet digital PCR assays with high-throughput 3D particle counters can perform bacterial identification and AST directly from whole blood samples, eliminating culture and sample processing steps, achieving high sensitivity (10 CFU/mL) and fast TAT (1 hour) for transformative POC AMR diagnosis.
4.6. Multiplex Approaches
Multiplex microfluidic chips enable high-throughput rapid phenotypic AST. An 8-sample multiplex chip arrays bacterial isolates and agarose in microchambers, monitoring bacterial colony growth under antibiotic gradients using dark-field microscopy and automated image analysis. TAT is 5 hours, achieving stable MIC values with 100% agreement with reference methods. This system allows simultaneous and rapid analysis of multiple samples or parallel testing of several antibiotics, enhancing efficiency in AMR diagnosis.
4.7. Single-Cell or Single-Molecule Approaches
Rapid AST systems based on microfluidic agarose channels immobilize bacteria for single-cell growth monitoring by microscopy. MIC values are determined by analyzing time-lapse images of single cells cultured under varying antibiotic concentrations, achieving TATs under 4 hours. Single-cell imaging-based rapid AST systems using microfluidic chips with cell traps have been developed for UTIs caused by resistant bacteria, with a 30-minute TAT even with low CFU urine samples, significantly accelerating AMR diagnosis. Versatile microfluidic systems for fast bacterial classification (3 minutes) and phenotypic AST at the single-cell level integrate tunable microfluidic valves and real-time visual detection for cell entrapment and classification based on size and shape. Susceptibility determination, by monitoring single-cell growth in the presence of antibiotics, takes only 30 minutes. These systems can be applied broadly for bacterial detection and complex polymicrobial sample analysis, offering transformative potential for AMR diagnosis. Table 3 summarizes key microfluidic platforms for AMR diagnosis.
Table 3. Summary of microfluidic platforms for AMR diagnosis.
5. Overview of Commercially Available AST Platforms
This section describes commercially available systems for AST and AMR diagnosis.
Adagio™ Antimicrobial Susceptibility Testing System (Bio-Rad Laboratories) is an automated system using imaging to measure inhibition zone sizes around antibiotic discs, coupled with data management software for rapid and accurate result generation and automated AST interpretation for streamlined AMR diagnosis. Evaluation showed good agreement with visual validation.
VITEK® 2 COMPACT (bioMérieux) is a compact, automated system for microbial identification and AST, reducing hands-on time and providing rapid reporting with a 2–18 hour TAT, although primary organism isolation is required. It utilizes fluorogenic methodology for identification and turbidimetric methods for AST, offering a cost-effective and space-saving solution for AMR diagnosis.
Accelerate Pheno™ (Accelerated Diagnostics) is a fully automated system performing identification in approximately 2 hours and AST within approximately 7 hours directly from samples without culturing. It uses gel electrofiltration for sample cleanup and fluorescence in situ hybridization for pathogen detection, species identification, and quantitation. Automated digital microscopy with morphokinetic cellular analysis (MCA) tracks phenotypic features and extrapolates MIC values. User-friendliness is a key advantage, but it lacks intervention flexibility and is limited to fresh blood culture processing for rapid AMR diagnosis.
Alfred 60AST system (Alifax) is a fully automated system for bacterial culture, residual antimicrobial activity (RAA), and susceptibility testing, including sample inoculation, reading, and result transmission. Using light scattering, it detects live bacteria, growth curves, bacterial counts, and drug resistance in a few hours (4–6 hours) with high sensitivity and specificity. Coupled with MALDI-TOF MS for direct identification, it offers rapid AST. Its plasticity allows user interventions, which can be both an advantage and a drawback, requiring skilled personnel for result interpretation in AMR diagnosis.
MicroScan WalkAway plus System (Beckman Coulter) (40 or 96-panel capacity models) provides automated microorganism identification and AST results efficiently with minimal labor from isolate inoculums within 4 hours (or overnight).
BD Phoenix™ (Becton, Dickinson and Company) is an AST system providing rapid, reliable, and accurate results from colony inoculums, using an oxidation/reduction indicator and turbidimetric growth detector, processing up to 200 ID/AST sets in under 4.5 hours for efficient AMR diagnosis.
Sensititre™ ARIS™ 2X (Thermo Fisher) provides bacterial pathogen identification and emerging antibiotic resistance detection using broth microdilution with automation, improving patient care and lab efficiency. Growth measurements and endpoint MIC determinations are based on fluorogenic substrate hydrolysis by bacterial isolates for comprehensive AMR diagnosis.
GeneFluidics offers automated platforms for research, including ProMax®, UtiMax®, and BsiMax® platforms providing identification and AST results from isolates, urine, and whole blood samples, respectively. These platforms use molecular-based, PCR-less identification of species-specific phenotypic markers of resistance and susceptibility, with electrochemical sensor array detection technology for innovative AMR diagnosis.
Comparison of Platforms
Alfred 60AST coupled with MALDI-TOF MS offers faster TAT and is more cost-effective than Accelerate Pheno™. However, Accelerate Pheno™ provides identification and MIC determination in a single cartridge, making it suitable for smaller laboratories without MALDI-TOF MS. VITEK2, BD Phoenix, MicroScan WalkAway, and Sensititre ARIS 2X are FDA-cleared IVD diagnostics, providing fast results (2–18 hours) but requiring standardized microbial inoculum preparation, which necessitates a 1–2 day culturing step prior to AST platform use, representing a limitation in achieving truly rapid AMR diagnosis directly from clinical samples. Table 4 summarizes commonly used commercial AST platforms.
Table 4. Commercially available AST platforms for AMR diagnosis.
6. Conclusions and Future Perspectives
The AMR crisis necessitates a collaborative response from academia, risk managers, governments, and industry to enhance diagnostic and treatment methodologies. Developing novel tools to overcome limitations of current gold standards and AST methods is crucial for improved AMR diagnosis. Current limitations include sample pre-treatment needs, low sensitivity, microorganism identification challenges, and lack of integration, automation, and portability. Advancements in new testing platforms with superior performance characteristics are needed for rapid approval and market availability. Improving existing methods and platforms also remains vital to enhance AMR diagnosis capabilities.
The AST market is projected to reach USD $4.2 billion by 2025, highlighting the growing need for effective AMR diagnosis solutions. Despite automated AST platforms reducing incubation and detection times, high prices hinder widespread adoption, particularly in smaller institutions. Manual AST products and disk diffusion methods remain prevalent due to lower costs. Hospitals and diagnostic laboratories are the largest end-users of AST technologies.
While methods and technologies reviewed show great potential in addressing the AMR challenge, several questions remain. General applicability, validation against reference methods, and timelines for commercialization are critical considerations for widespread adoption and impact on AMR diagnosis. Many methods claiming rapid AMR detection (minutes to hours) often overlook time-consuming pre-treatment steps like culture enrichment and isolation, which are essential considerations for real-world AMR diagnosis workflows.
Standard cultivation tests for AST have 18–36 hour TATs and provide MIC but are unsuitable for non-culturable pathogens. Automated platforms offer 2–24 hour TATs, some providing MIC, but also are not compatible with non-culturable pathogens. MALDI-TOF MS has a 2–4 hour TAT and can sometimes determine MIC, but shares limitations and lacks standardized AST protocols and data analysis software. NAAT-based approaches have 0.5–4 hour TATs, suitable for non-culturable pathogens and emerging ARGs, but do not determine MIC and require ongoing validation and standardization for each diagnostic update. WGS is a relatively new but powerful tool for rapid AST, with bioinformatics and universal databases being key challenges. Microfluidics offers significant potential and versatility, with miniaturized biosensing schemes promising integration, automation, portability, and rapid TAT, but requiring upscaling for mass production, integration of pre-treatment steps, and user-friendly interfaces for broader adoption in AMR diagnosis.
No single technology is currently optimal for rapid AMR diagnosis, but several hold promise for the future market, which can be divided into central lab-based and PON-based categories. Central labs can integrate WGS, WGM, PCR, MALDI-TOF MS, FTIR, and automated AST platforms. PON settings are better suited for portable, affordable microfluidic-based AST platforms with fast TATs. Table 5 summarizes the advantages and disadvantages of various AMR diagnosis methods and technologies.
Table 5. Advantages and disadvantages of AMR diagnostic methods and technologies.
In conclusion, developing reliable, sensitive, and affordable diagnostics is critical to combating AMR. Rapid diagnostic technologies, especially in primary care settings, can enhance targeted treatment. Advanced monitoring systems and surveillance programs are essential for tracking antimicrobial consumption. Emerging approaches like machine learning and data mining, combined with automation, will play a crucial role in next-generation AMR diagnosis. Epidemiological surveillance is vital for informing therapy guidelines, antibiotic stewardship programs, public health interventions, and the development of novel antimicrobials and vaccines. Advancements in cutting-edge methods and technologies for AMR and AST, coupled with enhanced data transmission through surveillance programs, are crucial steps in minimizing the detrimental effects of the AMR threat and improving global AMR diagnosis capabilities.
Acknowledgments
This work was supported by funding from the Spanish Ministry of Science, Innovation and Universities (grant number AGL2016-78085-P). G.K. and I.B. acknowledge the European Food Risk Assessment (EU-FORA) Fellowship Programme 2019–2020, (European Food Safety Authority, EFSA).
Abbreviations
| AFM-IR | Atomic Force Microscopy-Infrared Spectroscopy |
|---|---|
| AMR | Antimicrobial Resistance |
| ANN | Artificial Neural Networks |
| ARG | Antimicrobial Resistance Gene |
| AST | Antimicrobial Susceptibility Testing |
| CLSI | Clinical and Laboratory Standards Institute |
| EFSA | European Food Safety Authority |
| ESBL | Extended-spectrum β-lactamase |
| EU | European Union |
| EUCAST | European Committee on Antimicrobial Susceptibility Testing |
| FDA | Food and Drug Administration |
| FTIR | Fourier Transform Infrared |
| FT-RIFS | Fourier Transform Reflectometric Interference Spectroscopy |
| GMO | Genetically Modified Organism |
| HAD | Helicase-Dependent Amplification |
| HT-qPCR | High Throughput Quantitative PCR |
| IR | Infrared |
| IVD | in vitro Diagnostic |
| KPC | Klebsiella pneumoniae Carbapenemase |
| LAMP | Loop-Mediated Isothermal Amplification |
| LoC | Lab-on-a-Chip |
| LoD | limit of detection |
| MALDI-TOF MS | Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry |
| MCA | Morphokinetic Cellular Analysis |
| MDR | Multidrug-Resistant |
| MIC | Minimum Inhibitory Concentration |
| MLST | multi-locus sequence typing |
| MRSA | methicillin resistant S. aureus |
| MSSA | methicillin susceptible S. aureus |
| MZO | Magnesium Zinc Oxide |
| NAAT | Nucleic Acid Amplification Technology |
| NASBA | Nucleic Acid Sequence-Based Amplification |
| NGS | Next-Generation Sequencing |
| PCR | Polymerase Chain Reaction |
| PMF | Peptide Mass Fingerprint |
| POC | Point-of-Care |
| QCM | Quartz-Crystal Microbalance |
| RAA | Residual Antimicrobial Activity |
| RCA | Rolling Circle Amplification |
| RDT | Rapid Diagnostic Test |
| RPA | Recombinase Polymerase Amplification |
| SDA | Strand Displacement Amplification |
| SERS | Surface Enhanced Raman Spectroscopy |
| TAT | Turnaround Time |
| TMA | Transcription Mediated Amplification |
| UTI | Urinary Tract Infection |
| WGS | Whole Genomic Sequencing |
| WHO | World Health Organization |
| WMS | Whole Metagenome Sequencing |
Author Contributions
Conceptualization, G.D.K., M.P., A.A.-O.; methodology, G.D.K.; validation G.D.K.; E.A.A., M.P., I.B., A.A.-O.; formal analysis, investigation, resources, and data curation, G.D.K.; writing—original draft preparation, G.D.K.; writing—review and editing, G.D.K., M.P.; visualization, G.D.K.; supervision, M.P.; project administration, E.A.A.; funding acquisition, G.D.K., I.B., A.A.-O. All authors have read and agreed to the published version of the manuscript.
Funding
European Food Safety Authority: EU-FORA Fellowship Programme 2019–2020, Ministerio de Ciencia, Innovación y Universidades: AGL2016-78085-P.
Conflicts of Interest
The authors declare no conflict of interest.
Footnotes
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