Drug Susceptibility Testing Definition and Description

The field of DST is rife with technical jargon that merits a brief overview. Box 10.1 defines com­mon DST vocabulary found throughout the chapter.

AST is the cornerstone of the practice of infectious diseases and the foundation of many multidisciplinary laboratories, namely clinical laboratories, national reference centres, hospi­tal laboratories and basic research groups.

‘Corresponding author: jaryd.sullivan@mail.mcgill.ca © CAB International 2020. Paratuberculosis: Organism, Disease, Control, 2nd Edition (eds M.A. Behr et al.)

Box 10.1. Vocabulary.

Antibiotic: A chemical compound within the subset of antimicrobials that inhibits the growth of bacteria.

Antibiotic resistance: The innate or acquired ability of bacteria to resist the effects of an antibi­otic. Resistance is typically concerning at clinically relevant antibiotic concentrations. This concept is largely determined in vitro and is distinct from therapeutic failure, wherein an antibiotic fails to eliminate the infection.

Antimicrobial: A synthetic or naturally derived compound that inhibits the growth of bacteria, vi­ruses or fungi.

Antimicrobial susceptibility testing (AST): Laboratory testing on microorganisms to determine resistance and susceptibility levels to antimicrobials.

CLSI: The Clinical and Laboratory Standards Institute is a US-based organization that develops clinical and laboratory standards, practices and guidelines.

Critical concentration: The lowest concentration of drug that inhibits 95% of wild strains that have never been exposed to drugs.

Drug susceptibility testing (DST): Subset of AST used in the field of mycobacteriology.

EUCAST: The European Committee on Antimicrobial Testing is a European-based society that de­velops laboratory standards for clinical microbiology and infectious diseases.

Inoculum: Volume/concentration of microorganism used in an AST assay.

Indeterminate (I): A microorganism that is deemed neither susceptible nor resistant according to established minimum inhibitory concentration (MIC) boundaries. These MICs do not suggest with­holding the drug but emphasize appropriate adjustment of the dose. NB Some use ‘I’ to indicate Intermediate, but this use of I is discouraged by the CLSI because it implies a strength phenotype only, and excludes technical errors.

Isolate: A microbe isolated from a patient sample.

Microorganism: Categorical term encompassing bacteria, fungi and viruses. Commonly shortened to microbe.

Minimum inhibitory concentration (MIC): The lowest concentration of an antimicrobial that inhib­its microbial growth. Typically denoted as MIC₉₀ for inhibition of 90% of growth.

Resistance pattern: Summary of antimicrobial testing results for an isolate.

Resistant (R): A patient isolate should not be susceptible to dosage normally recommended to treat that type of infection and species.

Susceptible (S): A microorganism that is not resistant to antimicrobials at clinically relevant con­centrations. A patient isolate should be susceptible to dosage normally recommended to treat that type of infection and species.

terms, a microorganism is considered to be resist­ant if greater than 0.1% of the inoculum grows through the critical concentration of the drug. In accordance, EUCAST defines resistance with a similar definition but with greater emphasis on standard dosing regimen. For example, EUCAST categorizes a microorganism as resistant when ‘there is high likelihood of therapeutic failure even when there is increased exposure' (Kahlmeter, 2019, p.

Mycobacteria DST is a variant of AST where resistance is established if the antimicrobial critical concentration permits more than 1.0% of inocu­lum growth compared with drug-naive wild strains (Desmond et al., 2011). Mycobacterium tuberculosis DST has been shown to predict clinical outcomes in trials and meta-analyses, so it would appear that the 99% threshold is useful (Kim, 2005; Lew et al., 2008). Whether these modified standards apply to MAP and other non-tuberculous mycobacteria (NTM) - is unknown. Determining the resistance pattern could help guide appropriate treatment options, identify a possible mechanism for failed antimicrobial therapy and maintain surveillance for emerging drug resistance in populations (see Box 10.2, for example).

Traditionally, DST examines whether a par­ticular drug can inhibit the growth or metabo­lism of a bacterium in vitro. Classical DST relies on macroscopic observation of growth in drug and drug-free media through colony-forming unit (CFU) counts on agar or optical density

Box 10.2. S vs R vs I.

If organisms are highly resistant to an antibiotic, it is unlikely that the antibiotic can provide clinical benefit, as seen in Panel A (Fig. 10.1). However, the MIC distribution may follow a distribution closer to that seen in Panel B, where organisms that are not S have an MIC that is increased, but to a lesser extent. In the example of M. tuberculosis, resistance to isoniazid is a heterogeneous phenotype. Resistance due to katG deletions cannot be overcome, as isoniazid is a pro-drug, for which bacterial KatG is required for activation. Resistance attributed to promoter mutations in inhA leads to so-called ‘low-level resistance'. Here, the provision of an MIC result that is one to two dilutions higher than the standard threshold, coupled with a genetic test detecting the mutation, can inform the clinician that the resistance can be overcome with a higher dose of the antibiotic.

(OD_600nm) readout in liquid culture. Indeed, these methods are slow and dated for modern medi­cine, however, they remain the gold standard ac­cording to the CLSI guidelines.

Researchers and clinical labs have de­veloped methodologies for DST that meas­ure the metabolism of bacteria rather than visible growth as a means to circumvent the issue of slow-growing mycobacteria. Resazurin Microtiter Assay (REMA) and Bactiter Glo™ are two assays that monitor metabolism through intracellular redox potential and ATP synthe­sis, respectively (Niles et al., 2008; Necchi et al., 2018). Additionally, the Mycobacteria Growth Indicator Tube (MGIT) design was modified from the original BACTEC™ blood culture system from BD Diagnostic Systems to culture bacteria from patient samples (Automated Blood Culture System, 2014). Each MGIT™ tube contains an inorganic dye, which fluoresces upon exposure to CO₂ produced from metabolically active my­cobacteria. MGIT™ culture systems could be described as the backbone of clinical mycobacte- riology labs in many countries due to the ease of use and automated system.

The gold standard for M. tuberculosis front­line drug DST remains the agar proportion method for isoniazid (INH), rifampicin (RIF) and ethambutol (EMB), and the radiometric propor­tion method for pyrazinamide (PZA) - despite the arrival of new methods with shorter incu­bation times. In light of these standards, the CLSI recognizes growth on solid media is time consuming and recommends broth culture with a shorter incubation time for industrial­ized countries. The Centres for Disease Control and Prevention also recommends broth culture for DST. The goal is to report results of primary DST within 15-30 days of receiving the patient sample. Unfortunately, countries lacking the fa­cilities for automated culture systems must rely on standard agar growth.

Fig. 10.1. Histogram of hypothetical minimum inhibitory concentration (MIC) distribution for a microorganism. (A) Categorical distribution. (B) Graded distribution.

10.3