Phenotypic Methods for Antibiotic Susceptibility Testing

Antibiotic-resistant bacterial pathogens are a global health epidemic, spreading at a rapid rate. The widespread misuse of antibiotics in clinics and agriculture over the past few decades has accelerated the spread of the epidemic by causing bacteria to evolve and develop resistance. The development and use of rapid tests for the identification of resistant bacteria have been identified as one of the priorities to combat antibiotic resistance. Microorganisms are the main content of live biotherapeutic products (LBP) and it is necessary to perform antibiotic susceptibility tests (AST). Creative Biolabs is an outstanding, independent USA company offering first-class AST service to leading pharma, biotech, contract research organizations, and universities worldwide.

Overview of Phenotypic AST

Phenotypic AST is usually based on the detection of bacterial growth differences with or without antibiotics in liquid culture or on solid agar plates. The disk diffusion method is used to identify inhibition zones on solid agar plates, whereas in liquid tests, detection is based on changes in OD values. These methods are generally reliable for detecting resistance and determining concentrations of antibiotics that block bacterial growth. Phenotypic approaches, through their fundamental interactions with the physical environment, offer more avenues for emerging engineering techniques to improve their efficiency and versatility.

Phenotypic AST Methods at Creative Biolabs

Disk diffusion is the gold standard for determining bacterial susceptibility. It is a common method for antibiotic resistance detection because of its simplicity and low cost.

Dilution was one of the earliest tools in microbiological practice and it allows the growth and identification of bacterial populations in suspension. The two basic dilution methods are microdilution and macrodilution, of which broth and agar are the most commonly used media. In agar dilution, antibiotics are diluted into an agar medium, then plates are formed and bacterial cells are applied to the surface of the agar plate.

Etest is another significant development for the routine analysis of widespread antibiotic resistance in bacteria. The simplicity, accuracy, and reliability of the Etest make it suitable and convenient for commercialization approved by the Food and Drug Administration (FDA). The ability of convenient interpretations of MIC under diverse physical conditions made the Etest a preferential method over standardized disk diffusion and dilution techniques in clinical laboratories for AST.

Representation of various conventional culture-based AST approaches.Fig.1 Representation of various conventional culture-based AST approaches. (Song, 2021)

MALDI-TOF MS is another sensitive method for bacterial identification. Many studies have shown that this method is of great significance in distinguishing MRSA, MSSA, and other sensitive bacteria from drug-resistant bacteria. The newly developed MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) is a more straightforward and cost-effective modulation of MALDI-TOF MS used for both AST and MIC determination.

Microfluidic AST platforms for phenotypic screening have emerged, with superior performance in certain aspects compared to the conventional methods. The advantages are relatively lower cost, fewer resources, shorter turnaround time, possible automation, faster evaluation, and higher sensitivity.

The discovery of antibiotics is paradigm-altering, as it is not only an effective tool against chronic infections but also open new avenues for drug industries. Because of the huge social impact of multidrug resistance and the significant reduction in drug development, it is imperative to identify which microbes require more attention in drug development than others.

Creative Biolabs has unique R&D expertise to provide the highest quality custom LBP development services of different sizes and at reasonable prices in the industry. We have been chosen by big pharma and biotech customers. If you are interested in our phenotypic AST services, please contact us for more.


  1. Song, D.; Lei, Y. Mini-review: Recent advances in imaging-based rapid antibiotic susceptibility testing. Sensors and Actuators Reports. 2021, 3: 100053.

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