TABLE 5.
Methodological considerations for detection and characterization of drug efflux pumps
| Method | Consideration(s) | References |
|---|---|---|
| Microbiological | ||
| Antimicrobial susceptibility testing ± EPI | Can be a routine assay | 60, 346, 550, 553, 630, 989 |
| Used for development of novel antibacterials | ||
| Requires appropriate EPI and needs to rule out nonefflux inhibitory effects of the EPI | ||
| Unable to provide identity of the pumps | ||
| Genetic and molecular | ||
| PCR | Readily carried out and widely used | 528, 1022, 1023 |
| Can largely screen the distribution of efflux genes | ||
| Multiplex PCR can be used for identifying multiple resistance determinants | ||
| Requires sequences of the pump genes | ||
| RT-PCR | Readily carried out (qualitative and quantitative) and widely used | 199, 200, 380, 381, 383, 385, 413, 417, 420–422, 451, 502, 531, 572, 573, 628, 668, 1024–1027 |
| Can link gene expression with resistance phenotype (without or with an EPI) | ||
| Can assess the impact of factors (e.g., induction) on pump expression | ||
| Requires purification of RNA and that there is no DNA contamination | ||
| Requires sequences of the pump genes | ||
| Requires appropriate controls (e.g., a housekeeping gene) for comparison | ||
| Cloning and expression in native and/or exogenous host and mutational analysis of efflux components | Can be used for determining the function and substrate specificity (including identification of important residues of pump components) | 82, 306, 489, 560, 623 |
| Drug efflux pump-deficient hypersusceptible E. coli can often be used as a host | ||
| Requires appropriate expression vector and host | ||
| Overexpression of a pump may be toxic to the host | ||
| Genetic inactivation | Can be used to assess the role of a specific pump in intrinsic and acquired resistance when combined with susceptibility testing | 10, 13, 27, 28, 30, 164, 560, 574, 623 |
| Can be used to assess the role of pumps beyond drug resistance (e.g., biofilm formation, stress response, fitness, and virulence) | ||
| Can be used to study pump regulation | ||
| Requires appropriate methods to construct mutants | ||
| Genomic/proteomic analysis including a microarray assay | Used to determine the distribution of various classes of pumps, including putative drug pumps and other resistance determinants | 496, 500, 534, 570, 615 |
| Microarray assay may compare a large no. of efflux pump genes and nonefflux genes | ||
| May not reveal a function and needs experimental approaches for confirmation | ||
| Requires certain instrument facilities | ||
| Biochemical | ||
| Cell-based drug accumulation or uptake assay | Can be readily carried out | 9, 11–13, 60–63, 465, 563 |
| Can be developed for high-throughput screening methods for searching for novel antimicrobials and EPIs | ||
| May be used to measure steady-state drug levels | ||
| May be used for transport kinetic studies | ||
| Requires the substrates to be traceable, such as radiolabeled or fluorescent substrates | ||
| An ionophore proton conductor, CCCP, has often been used | ||
| Membrane vesicles | Can be used to demonstrate the efflux process | 133, 1028 |
| Require delicate experimental conditions (e.g., French cell press and radiolabeled substrates) | ||
| Not widely used and mostly demonstrated in E. coli with certain pumps | ||
| Liposome reconstitution transport | Can be used to demonstrate the efflux process | 23, 57, 142, 1028 |
| Requires expression and purification of efflux protein components | ||
| Immunoblot assay | Confirms the presence of pumps | 483, 628, 1027 |
| Quantifies pump expression | ||
| Used to study pump component interactions | ||
| Requires pump component-specific antibodies | ||
| Structural studies | Determines molecular and biochemical basis of efflux pumps and drug-pump interactions | 66–68, 70, 74, 75, 91, 93, 114, 166 |
| Used to search for novel antimicrobials and EPIs | ||
| Requires delicate biochemical experimental conditions for studying crystal structures | ||
| Computer simulations can also be used |