Table 1 Common antimicrobial sensitivity testing methods
Test | Definition | Advantages | Disadvantages |
|---|---|---|---|
Phenotypic Antimicrobial Sensitivity Testing (AST) | |||
Minimum Inhibitory Concentration (MIC) | • Lowest concentration of an antibiotic that causes visible inhibition of bacterial growth12 • The broth microdilution method is the gold standard in the clinics116,117 | • Easy, low cost and high-throughput methodology118,119 | • Fails to discriminate between bacteriostatic and bactericidal120,121 • Static assay: cannot replicate complex dynamic interactions of human infections122 • Does not recapitulate infection microenvironment (host-pathogen interactions)123 • Requires previous bacterial isolation step, and bacterial growth required to read out (24h-48h)124 • Cannot distinguish if any resistance stems from planktonic or biofilm growth16,95 |
Minimum Bactericidal Concentration (MBC) | • Lowest concentration of antibiotic that causes a killing >99.9% of the bacterial population125 • Typically determined by colony forming unit (CFU) enumeration | • Low cost methodology118,119 • Easy to standardise16 • Discriminates between bacteriostatic and bactericidal120 • MBC/MIC ratios historically used to measure tolerance120 | • Static system: cannot replicate complex dynamic interactions of human infections124 • Does not recapitulate infection microenvironment (host-pathogen interactions)123 • Requires previous bacterial isolation step and bacterial growth. and an additional step for CFU determination (2-3 days)124 • Cannot distinguish if any resistance stems from planktonic or biofilm growth16,95 |
Time-killing assay | • Bacterial population dynamics over time in the presence of antibiotic concentrations126 | • Provides dynamics of microbial killing over time121 • Discriminates between bacteriostatic and bactericidal effects121 • Can be used to determine tolerance14 • Killing mechanism can be classified as time- or concentration-dependent126 | • Requires multiple time points, each involving sampling, dilution and plating127 • Does not recapitulate infection microenvironment (host-pathogen interactions)123 • Does not directly identify mechanisms of resistance nor how bacteria may evolve to resist the antimicrobial agent • Only planktonic bacteria are tested, which may overestimate efficacy of antimicrobial agent compared with biofilms16,95 |
Pharmaco-kinetics/ Pharmaco-dynamics (PK/PD) | • Dynamic method allowing the addition of oxygen/ nutrient or drugs in a specific rate (dosing) to mimic an in vivo scenario128 • Typically involves a system of containers, tubing and a pump e.g. the hollow fibre bioreactor129 | • Provides dynamics of microbial killing over time130 • Controls critical parameters (multiple drug doses over time)130 • Mimic some conditions found in human body, so a more accurate representation of antimicrobial effect130,131 | • Requires multiple time points, each involving sampling, dilution and plating130 • While US FDA has approved hollow fibre bioreactor for in vivo-like studies, it does not fully capture the complexities of the human body130 |
Omics based-AST methods | |||
Nucleic acid amplification tests (NAATs) | • Allow the identification of bacterial species and known antibiotic resistance genes • PCR and quantitative PCR (qPCR) are the most common methods used132 | • Rapidness and automation124,133 • Leads to an earlier diagnosis | • DNA isolation and purification required • Only known resistance mechanism can be detected124 • Elevated cost of specialist equipment • The identified AMR genes may not be directly associated with the pathogen responsible for the disease, or the detected resistance gene might not be functional124 |
Whole genome sequencing (WGS) | • Identifies pathogens (strain level) and predicts antimicrobial susceptibility from bacterial DNA sequences119 | • Samples can be obtained directly from bodily fluids/ tissues (no bacterial culture required, significantly reducing readout time)134 • Allows study of bacterial evolution and data surveillance135 • Provides extensive characterization of resistance markers, plasmid replicons and virulence factors135 | • Elevated cost of specialist equipment124,135 • Complex-associated bioinformatics increases turnaround time124 • No guarantee that flagged resistance genes expressed in all situations, nor that bacteria was alive136 • Resistance gene must be known to detect it137. • 2015 European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee concluded that evidence for WGS in establishing antimicrobial susceptibility was poor; absence of standardized protocols and guidelines confounds accurate/ consistent interpretation of results in the clinic124,138 |
Mass spectrometry time-of-flight (MALDI-TOF) | • Analyses molecular composition of proteins/ peptides by measuring their mass-to-charge ratio, can identify certain biomarkers at molecular level. When combined with a reference database, can be used to identify a pathogen and its resistance profile139,140 | • Has transformed microorganism identification in routine clinical microbiology141 | • Elevated cost of specialist equipment139,140 • Requires prior growth of a colony (additional time required)139,140 • Method usually coupled with an MIC assay, so certain limitations inherent in MICs are extrapolated to this methodology142 |
