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Antibiotic sensitivity testing

Antibiotic sensitivity testing, also known as antimicrobial susceptibility testing (AST), is a laboratory procedure that determines the in vitro activity of antibiotics against isolated bacteria, categorizing them as susceptible, intermediate, or resistant to guide targeted antimicrobial therapy for infections.^[1] This testing is essential in clinical microbiology to identify effective treatments, prevent the overuse of broad-spectrum antibiotics, and support antimicrobial stewardship programs aimed at combating rising bacterial resistance.^[2] By providing results such as the minimum inhibitory concentration (MIC)—the lowest antibiotic concentration that inhibits visible bacterial growth—AST enables personalized patient care and reduces the risk of treatment failure.^[3] Common methods for AST include phenotypic approaches like disk diffusion and dilution techniques, as well as automated systems and emerging molecular methods such as PCR and whole-genome sequencing. These are standardized by organizations such as the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST).^[1]^[3] The importance of AST extends beyond individual treatment to public health surveillance, as it monitors local and global resistance patterns, informs outbreak investigations, and evaluates new antibiotics like aztreonam-avibactam for hard-to-treat infections through programs such as the CDC's Expanded AST initiative.^[4] As of 2021, bacterial antimicrobial resistance was directly responsible for 1.14 million deaths worldwide and associated with 4.71 million deaths, underscoring AST's role in reducing morbidity by enabling rapid, evidence-based decisions that curb the spread of resistant pathogens.^[5] Quality control, guided by CLSI and EUCAST breakpoints, ensures reliable results, with ongoing advancements focusing on speed and accuracy to address the global challenge of antibiotic resistance.^[1]

Overview

Definition and Principles

Antibiotic sensitivity testing (AST), also known as antimicrobial susceptibility testing, is a laboratory procedure used to determine the in vitro susceptibility of microorganisms, particularly bacteria, to specific antimicrobial agents. This testing evaluates how effectively an antibiotic inhibits or kills a bacterial isolate obtained from a clinical specimen, providing essential data for selecting appropriate therapies.^[1] The foundational principles of AST center on quantifying the antimicrobial's effect on microbial growth under controlled conditions. A central concept is the minimum inhibitory concentration (MIC), defined as the lowest concentration of an antimicrobial agent that prevents visible growth of the microorganism after standardized incubation, typically 18-24 hours.^[6] MIC testing distinguishes between susceptible, intermediate, and resistant categories based on pharmacokinetic and pharmacodynamic data, guiding clinical decisions.^[1] AST also differentiates between bacteriostatic and bactericidal effects of antimicrobials. Bacteriostatic agents inhibit bacterial replication and growth without directly killing the cells, allowing the host immune response to eradicate the infection. In contrast, bactericidal agents actively kill bacteria by interfering with vital cellular processes, such as cell wall synthesis or protein production. While MIC primarily measures bacteriostatic activity, the minimum bactericidal concentration (MBC) extends this by identifying the lowest concentration that reduces the bacterial population by 99.9% or more, determined through subculture from MIC-negative wells.^[7] In diffusion methods, susceptibility is assessed via the zone of inhibition, the diameter of the clear area surrounding an antibiotic disk on an agar plate where bacterial growth is absent, with larger zones indicating higher sensitivity due to greater drug diffusion and activity.^[8] Dilution methods, conversely, determine endpoints by observing the first dilution tube or well showing no turbidity or growth, corresponding to the MIC value.^[9] These principles enable AST to inform the refinement of empirical antibiotic therapy, shifting from broad-spectrum to targeted treatments to enhance efficacy and curb resistance.^[8]

Importance in Clinical Microbiology

Antibiotic sensitivity testing (AST) plays a pivotal role in combating antimicrobial resistance (AMR) by enabling the selection of targeted antibiotic therapies, thereby minimizing the overuse of broad-spectrum agents that contribute to resistance development. By identifying specific susceptibility patterns of bacterial isolates, AST facilitates precise treatment regimens that preserve the efficacy of existing antibiotics and slow the emergence of multidrug-resistant pathogens. For instance, routine AST supports global surveillance efforts, such as the World Health Organization's (WHO) Global Antimicrobial Resistance and Use Surveillance System (GLASS), which relies on standardized susceptibility data from clinical samples to track resistance trends across countries and inform public health policies. In 2019, bacterial antimicrobial resistance was directly attributable to 1.27 million deaths worldwide and associated with nearly 5 million deaths; by 2021, attributable deaths were estimated at 1.14 million, underscoring the persistent need for such targeted interventions to mitigate its spread.^[3]^[10]^[5]^[11]^[12] In terms of patient outcomes, AST significantly enhances recovery rates and reduces mortality in severe infections such as sepsis and pneumonia by ensuring the prompt administration of effective antibiotics. In sepsis, where delayed appropriate therapy can increase mortality by up to 7.6% per hour, AST results guide clinicians to de-escalate from empirical broad-spectrum coverage to narrower agents, shortening hospital stays and lowering complication risks. Similarly, for community-acquired pneumonia, susceptibility data helps avoid ineffective treatments, leading to faster resolution of symptoms and decreased intensive care needs. Studies highlight that infections caused by multidrug-resistant bacteria, such as Pseudomonas aeruginosa in pneumonia, are associated with higher mortality rates compared to susceptible strains, emphasizing AST's direct impact on survival.^[13]^[3] AST contributes substantially to antimicrobial stewardship programs by providing essential data for hospital formularies, de-escalation protocols, and local antibiograms that optimize antibiotic prescribing. These programs leverage AST to promote selective reporting of susceptibility results, which has been shown to reduce the use of agents like ciprofloxacin by encouraging narrower-spectrum alternatives, thereby enhancing patient safety and curbing resistance. In institutional settings, integrating AST with stewardship initiatives improves overall antibiotic utilization, reducing unnecessary exposure and supporting evidence-based guidelines for therapy adjustment. Globally, aggregated AST data from stewardship efforts feeds into surveillance networks, aiding in the formulation of national and international strategies to address AMR hotspots.^[3]^[13]^[11]

Applications

Uses in Diagnosis and Treatment

Antibiotic sensitivity testing (AST), also known as antimicrobial susceptibility testing, plays a pivotal role in guiding the selection of antibiotics for treating bacterial infections by identifying which antimicrobial agents are effective against a specific isolate. In clinical practice, AST results enable clinicians to shift from broad-spectrum empiric therapy to targeted treatment, reducing the risk of treatment failure and minimizing the emergence of resistance. For instance, by determining the minimum inhibitory concentration (MIC), AST provides quantitative data that informs optimal dosing and drug choice, ensuring efficacy while limiting unnecessary exposure to ineffective agents. A core function of AST is to differentiate bacterial strains into susceptible, intermediate, or resistant categories based on standardized breakpoints established by organizations such as the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Susceptible strains indicate that the infection is likely to respond to the antibiotic at standard doses, while resistant strains necessitate alternative therapies to avoid poor outcomes. The intermediate category suggests potential efficacy with higher doses or specific conditions, allowing for nuanced therapeutic adjustments that personalize patient care. This categorization directly influences diagnostic confirmation of the infection's etiology and supports de-escalation of antibiotics once susceptibility is confirmed. In urinary tract infections (UTIs), AST is routinely used to tailor therapy for common uropathogens like Escherichia coli, where initial empiric treatment with agents such as nitrofurantoin or trimethoprim-sulfamethoxazole can be adjusted based on results to improve cure rates and reduce recurrence. Similarly, for bloodstream infections, rapid AST performed directly from positive blood cultures facilitates timely intervention in sepsis cases, enabling the replacement of empiric broad-spectrum antibiotics with narrower, effective options within hours, which has been shown to decrease mortality and hospital stay duration. These applications highlight AST's value in high-stakes scenarios where delayed or inappropriate therapy can lead to severe complications. AST integrates seamlessly with other diagnostic tools, such as microbial culture and identification methods like matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), to provide a comprehensive profile of the pathogen. This combined approach accelerates the overall diagnostic process, from isolate identification to susceptibility reporting, ensuring that treatment decisions are based on both the organism's identity and its response to antibiotics. By linking phenotypic or molecular AST data with culture results, clinicians achieve a holistic understanding that enhances diagnostic accuracy and therapeutic precision in individual patient management.

Role in Infection Control and Surveillance

Antibiotic susceptibility testing (AST) plays a pivotal role in outbreak investigations by enabling the rapid identification of resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), in hospital settings, allowing for targeted interventions to contain transmission.^[14] During outbreaks, AST results help confirm the causative organism and its resistance profile, facilitating epidemiological tracing and implementation of control measures like contact precautions.^[1] For instance, in ICU settings, AST-guided screening of healthcare workers and patients has been used to detect and isolate MRSA carriers, preventing further spread.^[15] AST contributes significantly to surveillance networks by aggregating susceptibility data from clinical isolates, which supports the monitoring of antimicrobial resistance (AMR) patterns at national and international levels. The U.S. Centers for Disease Control and Prevention's National Antimicrobial Resistance Monitoring System (NARMS) performs AST on bacteria like Salmonella and Campylobacter from human, animal, and food sources to track trends, link resistant infections to sources, and guide outbreak responses.^[16] Similarly, the World Health Organization's Global Antimicrobial Resistance and Use Surveillance System (GLASS) relies on standardized AST protocols to collect data on bacterial susceptibility, enabling countries to detect emerging resistance and inform global AMR strategies.^[11] These networks emphasize laboratory-based AST to ensure comparable data across regions, with WHO recommending surveillance at local, intermediate, and national levels to assess resistance burdens.^[17] In infection control, AST informs measures such as patient isolation for multidrug-resistant organisms (MDROs), reducing nosocomial transmission by identifying colonization or infection early.^[1] Hospitals use AST results to screen high-risk patients, implementing isolation protocols that have proven effective in preventing outbreaks of resistant pathogens like carbapenem-resistant Enterobacteriaceae.^[3] This surveillance-driven approach integrates AST with infection prevention programs, ensuring timely decolonization or cohorting to limit spread.^[18] AST data also impacts policy by providing evidence for antibiotic stewardship guidelines and restriction policies, helping to curb overuse of broad-spectrum agents and preserve treatment options.^[19] Organizations like the Clinical and Laboratory Standards Institute (CLSI) update breakpoints based on aggregated AST surveillance, influencing national policies such as those from the Infectious Diseases Society of America (IDSA) that promote targeted therapy to combat resistance.^[1] For example, NARMS findings have shaped U.S. public health responses, including food safety regulations to mitigate resistant foodborne pathogens.^[16]

Methods

Phenotypic Methods

Phenotypic methods for antibiotic sensitivity testing rely on observing the direct effects of antibiotics on bacterial growth in culture, providing essential data on susceptibility by measuring inhibition of visible growth. These techniques, standardized by organizations such as the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), form the cornerstone of routine laboratory assessments for guiding antimicrobial therapy.^[20] The disk diffusion method, also known as the Kirby-Bauer test, involves inoculating a Mueller-Hinton agar plate with a standardized bacterial suspension equivalent to a 0.5 McFarland standard, then placing antibiotic-impregnated paper disks on the surface. After incubation at 35 ± 2°C for 16-18 hours, the diameter of the clear zone of inhibition around each disk is measured in millimeters using a ruler or caliper. Interpretation categorizes results as susceptible, intermediate, or resistant based on CLSI or EUCAST breakpoint tables, which correlate zone sizes with clinical efficacy; for example, larger zones indicate greater susceptibility.^[20]^[21] Broth dilution methods determine the minimum inhibitory concentration (MIC), defined as the lowest antibiotic concentration preventing visible bacterial growth. In macrodilution, serial two-fold dilutions of antibiotics are prepared in tubes with a standardized inoculum (approximately 5 × 10^5 CFU/mL) in broth, incubated at 35 ± 2°C for 16-20 hours, and read visually for turbidity or via spectrophotometry. Microdilution adapts this to 96-well plates for higher throughput, using the same inoculum and incubation conditions, with endpoints read similarly to identify the MIC. These techniques adhere to CLSI M07 guidelines for reproducibility.^[22]^[22] The gradient diffusion method, exemplified by the Etest, employs a plastic strip coated with a predefined exponential gradient of antibiotic concentrations (typically 0.016-256 μg/mL) placed on an inoculated agar plate. Following incubation at 35 ± 2°C for 16-24 hours, the MIC is estimated by reading the point where bacterial growth intersects the strip's scale, offering a semi-quantitative alternative to full dilution methods with high agreement to broth microdilution results.^[23]^[24] Phenotypic methods are considered the gold standard for correlating in vitro results with clinical outcomes due to their direct assessment of bacterial response, but they are time-consuming, requiring 18-48 hours for incubation and readout, which can delay therapy initiation.^[3]^[25]

Molecular Methods

Molecular methods for antibiotic sensitivity testing involve genetic approaches that detect resistance mechanisms by targeting DNA or RNA sequences directly from clinical samples, enabling prediction of susceptibility without relying on bacterial growth. These techniques focus on identifying specific genes or mutations associated with resistance, providing a genotypic assessment that complements traditional phenotypic testing.^[3] PCR-based assays are widely used for rapid detection of key resistance genes. Real-time PCR targets the mecA gene, which encodes penicillin-binding protein 2a responsible for methicillin resistance in Staphylococcus aureus (MRSA), allowing identification within 1-2 hours from blood or other samples. Similarly, real-time PCR assays detect the blaKPC gene, which produces Klebsiella pneumoniae carbapenemase enzymes conferring resistance to carbapenem antibiotics in Enterobacteriaceae, with high sensitivity and specificity in clinical isolates. Multiplex PCR formats enable simultaneous screening for multiple genes, such as those for extended-spectrum beta-lactamases (e.g., blaCTX-M), enhancing efficiency in outbreak settings.^[26]^[27]^[3] Whole-genome sequencing (WGS) offers a comprehensive analysis by sequencing the entire bacterial genome to identify resistance-conferring mutations, plasmids, and acquired genes. Tools like ResFinder and CARD databases annotate sequences to predict resistance profiles for a broad range of antibiotics, including beta-lactams and aminoglycosides, with applications in surveillance of multidrug-resistant pathogens. WGS has demonstrated high concordance (over 95%) with phenotypic results for common resistance determinants in species like Escherichia coli and Pseudomonas aeruginosa.^[28]^[3] These molecular methods provide key advantages, including rapid turnaround times of hours compared to days for culture-based approaches, and applicability to unculturable or low-viability pathogens by directly analyzing nucleic acids from samples. However, they do not confirm phenotypic expression of resistance, as the presence of a gene does not always correlate with functional protein activity or clinical resistance. Limitations include the need for sophisticated bioinformatics pipelines for WGS data interpretation and potential overestimation of resistance due to silent or non-expressed genes, often necessitating phenotypic confirmation for treatment decisions.^[3]^[29]^[30]

Mass Spectrometry-Based Methods

Mass spectrometry-based methods, particularly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), enable rapid antibiotic susceptibility testing (AST) by analyzing proteomic profiles of bacteria following antibiotic exposure. The principle involves laser-induced ionization of bacterial proteins in a matrix, generating ions that are separated by their mass-to-charge ratio in the time-of-flight analyzer to produce a characteristic spectral fingerprint. After brief incubation with antibiotics, spectral differences arise from metabolic changes or enzymatic degradation; for instance, β-lactamase activity hydrolyzes antibiotics like ertapenem, producing detectable peaks (e.g., at m/z 370 and 414 for cefotaxime hydrolysis) in resistant strains, allowing differentiation from susceptible ones within 90-150 minutes.^[31]^[32] This approach contrasts with traditional growth inhibition by focusing on biochemical signatures rather than visible colony formation.^[33] Applications of MALDI-TOF MS in AST include direct identification and susceptibility assessment from clinical samples such as positive blood cultures or urine, bypassing lengthy subculturing. Methods like the Bruker MBT-ASTRA system expose bacteria to antibiotics for 1.5-4 hours, followed by spectral comparison to reference databases, achieving results for Gram-negative bacilli in as little as 75 minutes post-incubation. For example, it detects carbapenem resistance in Enterobacteriaceae with 98% sensitivity and 100% specificity by monitoring growth via area under the curve (AUC) of spectral peaks. This enables same-day reporting, critical for timely therapy in sepsis cases.^[31]^[34] Key advantages of MALDI-TOF MS-based AST include its speed—delivering results in 1-4 hours versus 18-24 hours for conventional phenotypic tests—and low operational cost per test (approximately €0.50-1.50), making it suitable for high-volume labs. It requires minimal sample preparation and integrates well with existing identification workflows. However, disadvantages encompass the need for expensive specialized equipment (initial costs exceeding €150,000) and trained personnel, limiting adoption in resource-constrained settings. Additionally, while validated for common pathogens, broader AST applications remain emerging, with ongoing needs for standardization and database expansion to ensure reproducibility across diverse resistance mechanisms.^[35]^[36] Recent developments focus on hybrid AST integrating MALDI-TOF MS with phenotypic readouts, enhancing accuracy by combining spectral biomarkers with growth assays. Systems like BIOMIC V3 with MALDI-Link automate transfer of MALDI identification results to disk diffusion testing, reducing workflow time and errors while adhering to CLSI M100 guidelines for breakpoint interpretation. As of 2024, machine learning-augmented MALDI-TOF models have improved resistance prediction (e.g., AUROC >0.95 for Staphylococcus epidermidis), supporting hybrid validation in CLSI-compliant protocols. These advances address limitations in direct susceptibility, with FDA clearances for related rapid systems in 2024 signaling growing clinical adoption.^[37]^[36]

Interpretation and Reporting

Susceptibility Categories and Breakpoints

Antibiotic susceptibility testing (AST) results are interpreted using standardized categories that classify bacterial isolates based on their response to antimicrobial agents, primarily through the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines.^[38]^[39] CLSI defines four susceptibility categories: Susceptible (S), indicating the isolate is inhibited by achievable concentrations of the antimicrobial with standard dosing; Susceptible dose-dependent (SDD), where susceptibility relies on higher-than-standard doses; Intermediate (I), signifying that the isolate may be inhibited at concentrations higher than usual or with alternative routes; and Resistant (R), where the isolate is not inhibited even at maximum achievable concentrations.^[40] EUCAST employs three categories: Susceptible (S), where standard dosing is expected to achieve therapeutic concentrations; Susceptible, increased exposure (I), requiring higher doses or more frequent administration; and Resistant (R), where the isolate cannot be inhibited by achievable concentrations regardless of dosing.^[41] These categories guide clinical decision-making by linking minimum inhibitory concentration (MIC) values to expected treatment outcomes. Breakpoints, the specific MIC or zone diameter thresholds defining these categories, are determined by integrating data from pharmacokinetics/pharmacodynamics (PK/PD), MIC distributions of wild-type and resistant populations, and clinical trial outcomes.^[42]^[43] PK/PD indices, such as the free area under the curve to MIC ratio (fAUC/MIC) for concentration-dependent agents or the percentage of time above MIC (%fT>MIC) for time-dependent agents, establish pharmacodynamic targets (e.g., fAUC/MIC of 30-50 for beta-lactams) that predict efficacy, often using Monte Carlo simulations to ensure target attainment in at least 90% of patients.^[44] MIC distributions help set breakpoints to separate susceptible wild-type populations from resistant ones, while clinical trial data correlate MICs with success rates, adjusting thresholds to reflect real-world outcomes.^[43]^[8] Several factors influence breakpoint selection, including drug dosing regimens, which affect PK variability across patients; the site of infection, altering local drug penetration and efficacy requirements; and host factors such as immune status or organ function, which impact overall exposure.^[42]^[44] For instance, breakpoints for urinary tract infections may differ from those for pneumonia due to higher antibiotic concentrations in urine.^[43] Recent updates reflect evolving data for novel antibiotics; for example, EUCAST revised area of technical uncertainty (ATU) breakpoints for cefiderocol in 2024 to better address its siderophore-based mechanism against multidrug-resistant Gram-negative bacteria.^[45] These revisions ensure breakpoints align with updated PK/PD models and clinical evidence from trials involving complex infections.^[46]

Reporting Standards and Guidelines

Antimicrobial susceptibility testing (AST) results are typically reported in formats that include minimum inhibitory concentration (MIC) values, inhibition zone diameters from disk diffusion tests, or categorical interpretations such as susceptible (S), susceptible with increased exposure (I), or resistant (R), depending on the method used and the clinical context.^[47] Laboratories often prioritize categorical results for direct clinical guidance, while MIC or zone sizes provide quantitative data for nuanced cases, such as pharmacokinetic/pharmacodynamic considerations. The Clinical and Laboratory Standards Institute (CLSI) M100, in its 35th edition (2025), outlines standardized reporting protocols, recommending that laboratories report only antimicrobials tested and appropriate for the isolate and infection site, with suppression of broader-spectrum agents if narrower-spectrum options are susceptible to promote stewardship.^[48] Similarly, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) provides harmonized recommendations through its breakpoint tables (version 15.0, 2025) and expert rules, emphasizing selective reporting to avoid misleading results, such as suppressing intrinsically resistant combinations or inferring susceptibility from related agents without retesting.^[47] Both organizations advocate including interpretive comments for unusual resistance patterns, such as multidrug resistance or discrepancies in zone sizes near breakpoints, to alert clinicians without altering the primary result.^[49] Special considerations in reporting include cascade or selective suppression to prevent misuse, for instance, withholding results for non-formulary drugs or those inappropriate for specific infections like aminoglycosides in meningitis. EUCAST expert rules further specify suppression for agents like rifampicin in certain Enterococcus isolates due to limited efficacy evidence, while CLSI tables guide routine suppression of agents like carbapenems for Enterobacterales susceptible to third-generation cephalosporins.^[50] In digital reporting, AST results are increasingly integrated with electronic health records (EHRs) to enable antimicrobial stewardship alerts, such as flagging discordant therapies or prompting de-escalation based on susceptibility categories.^[51] This integration supports real-time decision-making, with systems automating comments or suppressing irrelevant results to enhance clinical utility.^[52]

Clinical Implementation

Laboratory Workflow and Automation

The laboratory workflow for antibiotic susceptibility testing (AST) begins with sample collection, typically involving the acquisition of clinical specimens such as blood, urine, or tissue from patients suspected of bacterial infections. These samples are transported to the microbiology laboratory under appropriate conditions to preserve viability, followed by primary culture on selective or non-selective media to isolate the pathogen.^[1] Once growth is observed, subculturing is performed to obtain a pure isolate, which is essential for accurate testing.^[3] The next stages involve isolate identification, often using phenotypic or molecular methods to confirm the bacterial species, and preparation for AST setup. In the setup phase, a standardized bacterial inoculum—equivalent to a 0.5 McFarland standard—is prepared and applied to testing platforms, such as agar diffusion disks or broth microdilution panels containing antibiotics at varying concentrations. Incubation follows under controlled conditions (e.g., 35–37°C for 16–18 hours for most bacteria), allowing visible growth inhibition to be assessed during the reading stage, where zones of inhibition or minimum inhibitory concentrations (MICs) are measured and interpreted against clinical breakpoints.^[1]^[53] Automation has streamlined this workflow, enabling high-throughput processing in clinical laboratories. Systems like the VITEK 2 (bioMérieux) use advanced colorimetry to automate identification and AST simultaneously, loading inoculated cards into the instrument for continuous monitoring of growth kinetics without manual intervention. Similarly, the BD Phoenix system (Becton Dickinson) employs broth microdilution panels in an automated reader that detects turbidity changes, reducing hands-on time and integrating data output directly to laboratory information systems. These platforms support both phenotypic methods and brief incorporation of molecular identification steps for faster isolate confirmation. Emerging rapid systems, such as VITEK REVEAL (as of 2025), can deliver AST results in as little as 3 hours using advanced detection technologies.^[54]^[55]^[56]^[57] Traditional manual workflows typically require 24–72 hours from isolate to final AST results due to sequential culturing and incubation steps. Automated systems optimize turnaround time to as little as 6–13 hours for complete ID and susceptibility profiles, enhancing efficiency in busy laboratories by minimizing labor and error-prone manual readings.^[53]^[58]^[56] Quality assurance is embedded throughout the workflow to ensure reliability. Internal controls, such as testing reference strains (e.g., Escherichia coli ATCC 25922) with each batch of patient samples, verify the performance of media, antibiotics, and incubation conditions per CLSI guidelines. Proficiency testing programs, like those outlined in CLSI QMS24, involve periodic external challenges to assess laboratory accuracy and maintain accreditation, with results reviewed to calibrate instruments and retrain staff.^[38]^[59]^[60]

Challenges and Quality Control

Antibiotic sensitivity testing (AST) faces several inherent challenges that can compromise its accuracy and clinical utility. Mixed cultures, where multiple bacterial species are present in a sample, often lead to underdetection of resistant subpopulations, as susceptible strains may dominate growth and mask resistance in polymicrobial infections.^[61] Fastidious organisms, such as Haemophilus influenzae or Neisseria gonorrhoeae, pose difficulties due to their slow growth or specific nutritional requirements, which can delay or prevent reliable susceptibility determination.^[62] Heteroresistance, characterized by a small subpopulation of resistant cells within a largely susceptible bacterial population, is particularly insidious, as standard AST methods may fail to detect it, resulting in misclassification and treatment failure.^[63] Error sources in AST further exacerbate these issues, stemming from both technical and interpretive factors. Technical errors frequently arise from improper inoculum preparation, such as deviations in bacterial density (e.g., using a McFarland 0.5 standard), which can alter minimum inhibitory concentration (MIC) values and zone sizes in disk diffusion tests, leading to false susceptible or resistant categorizations.^[3] Interpretive discrepancies between major guidelines, like those from the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), arise from differing clinical breakpoints. To mitigate these challenges, robust quality control (QC) measures are essential in AST laboratories. Reference strains from the American Type Culture Collection (ATCC), such as Escherichia coli ATCC 25922 for disk diffusion validation, are routinely used to monitor test performance, ensuring reproducibility across batches of media, antibiotics, and instruments.^[64] Validation of new AST methods involves parallel testing against established reference procedures, including at least 20 consecutive days of QC with ATCC strains to establish acceptable ranges before clinical implementation.^[65] Solutions to address these limitations emphasize standardization and oversight. Laboratory staff training programs, such as those offered by CLSI, focus on proper inoculum preparation, guideline adherence, and error recognition to reduce operator variability.^[66] Accreditation by bodies like the College of American Pathologists (CAP) mandates comprehensive QC protocols, including annual proficiency testing and breakpoint updates, to maintain high standards in microbiology labs.^[67] Ongoing harmonization efforts between CLSI and EUCAST, intensified through joint reviews in 2024 and 2025, aim to align breakpoints for key antibiotics, reducing interpretive errors and promoting global consistency in AST reporting.^[68]

Historical Development

Early Methods and Discoveries

The origins of antibiotic sensitivity testing trace back to Alexander Fleming's serendipitous observation in 1928, when he noted a clear zone devoid of bacterial growth surrounding a contaminating mold, Penicillium notatum, on a staphylococcal culture plate.^[69] This phenomenon, which Fleming described as evidence of an antibacterial substance he named penicillin, represented the earliest conceptual basis for assessing microbial susceptibility to antimicrobial agents and was detailed in his 1929 publication. Although Fleming did not explicitly term it a "zone of inhibition," this idea of measurable bacterial growth suppression became foundational, with the concept formalized and quantified in susceptibility assays during the 1950s.^[70] The penicillin era of the 1940s spurred practical innovations in testing methods amid urgent wartime needs for evaluating antibiotic efficacy. Early disk diffusion techniques involved impregnating filter paper disks with penicillin and placing them on agar plates inoculated with bacteria, allowing diffusion of the antibiotic to create observable inhibition zones whose diameters indicated susceptibility.^[71] These approaches were pioneered by researchers such as C.G. Pope in 1940 at the Wellcome Physiological Research Laboratories and refined by Norman Heatley in 1944, who adapted cylinder and disk placements for more consistent measurements during penicillin production scaling.^[72] By the mid-1950s, William M. Kirby and Arthur W. Bauer developed a more rigorous version, standardizing variables like agar depth, inoculum density, and disk potency to correlate zone sizes with clinical outcomes, marking a significant advancement in reproducibility. Despite these developments, early antibiotic sensitivity testing faced substantial limitations, including inconsistent results due to unstandardized protocols for media composition, incubation temperatures, and bacterial inoculum preparation, which hindered inter-laboratory comparisons.^[73] Testing was largely confined to single agents like penicillin, reflecting the era's focus on a limited antibiotic repertoire and overlooking the growing diversity of pathogens and emerging resistance patterns.^[72] A pivotal milestone in the 1960s was the advent of broth microdilution techniques for determining the minimum inhibitory concentration (MIC), which provided quantitative precision by serially diluting antibiotics in microtiter wells and observing the lowest concentration preventing visible growth. This method addressed the imprecision of diffusion-based assays and the cumbersome volumes of earlier macro-dilution approaches introduced in the 1940s, enabling more efficient testing of multiple antibiotics against clinical isolates.^[71]

Standardization and Modern Advances

The Clinical and Laboratory Standards Institute (CLSI), originally established in 1967 as the National Committee for Clinical Laboratory Standards (NCCLS), was formed to develop consensus-based standards for clinical laboratory practices, including the establishment of interpretive breakpoints for antibiotic susceptibility testing (AST) to ensure consistent and reliable results across laboratories.^[74] These breakpoints define susceptibility, intermediate, and resistance categories based on minimum inhibitory concentrations (MICs) or zone diameters, addressing the need for standardized antimicrobial testing amid growing antibiotic use in the mid-20th century.^[75] In the 1990s, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was founded in 1997 by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and national breakpoint committees; it is now jointly organized with the European Centre for Disease Prevention and Control (ECDC).^[39] EUCAST's development of evidence-based breakpoints complemented CLSI's work, focusing on integrating epidemiological data and promoting uniformity in reporting to combat rising antimicrobial resistance (AMR).^[76] This era also saw the emergence of automated AST systems in the 1980s, such as the VITEK AutoMicrobic System introduced in 1979 by bioMérieux, which streamlined identification and susceptibility testing through card-based incubation and photometric reading, reducing manual labor and turnaround time compared to traditional disk diffusion methods.^[77] By the 1990s, molecular integration advanced AST, with techniques like PCR-based detection of resistance genes (e.g., mecA for methicillin resistance, cloned in 1992) enabling genotypic identification of mechanisms, often within hours, as exemplified in early commercial assays for rapid AMR profiling.^[78] Key events in the 1990s, including widespread vancomycin-resistant Enterococcus (VRE) outbreaks in hospitals, prompted urgent responses that accelerated standardization and innovation in AST; for instance, U.S. Centers for Disease Control and Prevention (CDC) guidelines in 1995 emphasized improved detection methods, spurring development of rapid phenotypic and molecular assays to identify VRE and guide therapy amid limited treatment options. In the 21st century, particularly the 2010s, both CLSI and EUCAST shifted toward incorporating pharmacokinetic/pharmacodynamic (PK/PD) data into breakpoint revisions, using Monte Carlo simulations to predict clinical efficacy based on drug exposure and MIC distributions, as seen in CLSI's 2015 updates for beta-lactams and EUCAST's 2011 PK/PD framework for cephalosporins.^[44] This integration enhanced breakpoint accuracy by aligning laboratory results with patient outcomes, reducing discrepancies in susceptibility reporting for evolving resistance patterns.^[79]

Future Directions

Emerging Technologies

Emerging technologies in antibiotic sensitivity testing (AST) are transforming the field by enabling faster, more precise results compared to traditional culture-based methods, often achieving turnaround times under two hours through innovative platforms that integrate advanced analytics and miniaturization.^[80] These advancements address the critical need for rapid diagnostics in combating antimicrobial resistance, with phenotypic approaches leading the way by directly observing bacterial responses to antibiotics without relying on genomic predictions alone.^[81] Rapid phenotypic AST systems leverage microfluidics to miniaturize assays, allowing automated dilution and exposure of bacteria to antibiotics in controlled microchannels, yielding susceptibility results in as little as 30 to 90 minutes. For instance, the Self Dilution for Faster Antimicrobial Susceptibility Testing (SDFAST) device uses self-diluting microfluidic channels to perform phenotypic testing on clinical isolates, demonstrating high accuracy for Gram-positive and Gram-negative bacteria with minimal sample volumes.^[82] Similarly, portable intelligent digital microfluidic systems combine temperature control, illumination, and image analysis to monitor bacterial growth inhibition in real-time, reducing assay times to under two hours while maintaining concordance rates above 90% with standard methods.^[83] Digital microscopy complements these by employing time-lapse imaging or nanomotion detection to track individual bacterial responses, such as motility changes or vibrations indicative of antibiotic effects, enabling susceptibility determination in 1 to 1.5 hours for urinary tract infection samples.^[84] These technologies build on established mass spectrometry methods like MALDI-TOF as precursors for initial identification but extend to direct susceptibility profiling.^[80] Integration of artificial intelligence (AI) and machine learning (ML) with spectral data from MALDI-TOF mass spectrometry is enhancing predictive AST by analyzing subtle proteomic signatures associated with resistance patterns. ML algorithms trained on mass spectra can classify antibiotic resistance in pathogens like Pseudomonas aeruginosa with sensitivities exceeding 95%, providing results in under 30 minutes post-identification without additional culturing.^[85] Recent models, such as those combining support vector machines or neural networks with MALDI-TOF data, achieve over 90% accuracy in predicting resistance to multiple drug classes across clinical isolates, facilitating real-time decision-making in hospital settings.^[86] These AI-driven approaches are particularly valuable for high-throughput labs, where they process spectral datasets to forecast susceptibilities for priority pathogens, outperforming traditional phenotypic tests in speed while correlating strongly with genotypic results.^[87] Point-of-care (POC) devices are bringing AST closer to the bedside through portable PCR-based systems and biosensor kits that detect resistance markers or phenotypic changes directly from samples like urine or blood. For example, Sysmex Europe's POC AST system delivers susceptibility results from positive urine cultures in approximately 30 minutes using automated phenotypic analysis, enabling immediate therapy adjustments in outpatient settings.^[88] Biosensor kits, often incorporating electrochemical or optical detection, monitor bacterial metabolic shifts in response to antibiotics via handheld platforms, achieving detection limits suitable for low-burden infections with results in 45 to 60 minutes.^[89] These devices emphasize user-friendly designs for non-specialist use, such as in emergency departments, and support multiplex testing for common resistance genes alongside phenotypic confirmation.^[90] Regulatory validation is advancing these technologies, with several systems receiving FDA clearance between 2023 and 2025 to ensure clinical reliability. The Accelerate Arc system, cleared in 2024, automates sample preparation from positive blood cultures for rapid microbial identification using MALDI-TOF MS in about 90 minutes, aiding workflows for Gram-positive organisms.^[91] Ongoing submissions like the WAVE system in 2025, which remains pending FDA review as of November 2025, promise even shorter times for Gram-negative testing.^[92]

Research Priorities and Innovations

Research priorities in antibiotic susceptibility testing (AST) emphasize tackling antimicrobial resistance (AMR) in low-resource settings, where limited access to diagnostics hinders timely treatment and surveillance. The World Health Organization (WHO) has identified inadequate bacteriology testing infrastructure in these areas as a critical barrier, advocating for scalable, low-cost AST methods to support patient management and outbreak detection.^[81]^[93] Expanding AST to fungal and viral pathogens represents another key priority, driven by rising antifungal and antiviral resistance. WHO research agendas highlight the need for improved detection and susceptibility testing of priority fungal pathogens, such as Candida auris, to track resistance trends and inform therapy. For viral infections, efforts focus on enhancing antiviral susceptibility testing to address emerging resistance, particularly in high-burden settings.^[94]^[95]^[96] Innovations in nanotechnology enable direct-from-blood AST, bypassing traditional culture steps for faster results. Nanomotion detection platforms measure bacterial vibrations to assess susceptibility within hours, offering high accuracy for bloodstream infections. Integrating multi-omics approaches, such as genomics and transcriptomics, with machine learning enhances AMR prediction by identifying resistance biomarkers beyond phenotypic testing.^[84]^[97] Funding and collaborations have intensified post-2024 through NIH and WHO initiatives aligned with global AMR action plans, including the WHO's October 2025 global call to action emphasizing diagnostics integration and securing funding.^[98]^[99]^[100]^[101] The WHO's 2024-2025 efforts promote evidence-based national action plans, while the U.S. National Action Plan for Combating Antibiotic-Resistant Bacteria prioritizes surveillance and innovation funding. These programs foster international partnerships to accelerate AST advancements. Persistent gaps include standardizing rapid AST methods and generating clinical trial data for new breakpoints. While rapid phenotypic assays show promise, variability in performance metrics like categorical agreement requires harmonized protocols for widespread adoption. Updating breakpoints often lacks robust clinical outcome data, creating delays in implementation and potential mismatches in susceptibility reporting.^[102]^[103]^[104]

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