Antimicrobial Susceptibility Testing

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Antimicrobial Susceptibility Testing

 

Robert Lo, Ph.D, D.V.M

 

Antimicrobial compounds, including both naturally and chemically synthesized compounds, have been one of the most important inventions to combat infections. The first commercial antibiotic, penicillin, was accidentally identified of by Alexander Fleming in 1928. Lots of antibiotics with different mechanisms of antimicrobial activity have been discovered or synthesized after the discovery of penicillin. Currently, antibiotics are classified into different groups based on their mechanism of antimicrobial activity. The main groups are: inhibit cell wall synthesis (β-lactams and glycopeptides), depolarize cell membrane (lipopeptides), inhibit protein synthesis (aminoglycosamides, chloramphenicol, lincosamides, macrolides, oxazolidinones, streptogramins, and tetracyclines), inhibit nucleic acid synthesis (Quinolones), inhibit fatty acid synthesis (platensimycin), and inhibit metabolic pathway (sulfonamides and trimethoprim).

 

Antibiotics have saved millions of lives worldwide from diseases and infections once considered life threatening and fatal. With so many groups of antibiotics to treat pathogens, it seems that antibiotics would win the battles against the infection. In fact, antibiotics were also widely used not only in the healthcare industry but also in food and animal industries because of their versatile nature. However, bacterial pathogens have their own ways, antibiotic resistance, to fight with antibiotics and even win the battles. Currently, antibiotic-resistant bacterial pathogens are a global health epidemic, spreading at a rapid rate. A recent report on the casualties related to antibiotic resistance by the world health organization (WHO) depicted an alarming 700,000 lives per year currently, and predicts a disturbing 10 million/year by 2050, ensuring that antibiotic resistance will be the most prevalent cause of death (Brogan and Mossialos, 2016). This epidemic is accelerated by widespread misuse of antibiotics in clinics and agriculture over the last few decades, allowing bacteria to evolve and develop means of resistance (Laxminarayan et al, 2013; Van Boeckel et al, 2014).

 

The clinical microbiology laboratory serves as a valuable ally to clinicians in the diagnosis and treatment of infectious diseases via the isolation of bacteria to confirm susceptibility to chosen empirical antimicrobial agents, or to detect resistance in individual bacterial isolates. Through the use of in vitro antimicrobial susceptibility testing (AST), the laboratory can specifically determine which antibiotics effectively inhibit the growth of a given bacterial isolate, allowing for targeted therapy. Antimicrobial resistance is a growing concern in both community and health care settings; as such, decisions surrounding empirical antibiotic treatment are becoming more complicated, and the importance of routine antimicrobial susceptibility testing to guide therapeutic decisions has increased.

 

Currently, AST is usually performed in a clinical microbiology lab, which necessitates transportation of the patient samples from the healthcare provider to the lab. Susceptibility testing requires a pure culture of the offending pathogen, a process which may take several days. This delay prolongs the time to diagnosis of resistant bacteria and decisions for appropriate and effective antibiotic therapy. Delays in timely administration of appropriate therapeutics lead to increased patient mortality, poor clinical outcomes (Daniels, 2011), and use of broad-spectrum antibiotics, the latter of which promotes antibiotic resistance. Every hour of delay in administrating the targeted antibiotics to septic shock patients, decreases their chances of survival by 7.6% (Puskarich et al, 2011). To survive this evolutionary war against bacteria, obtaining rapid AST results to determine the Minimum inhibitory concentration (MIC) values are of high priority in any clinical setting.

 

MICs of various antimicrobial susceptibility testing (AST) are categorized by various international agencies. These MIC guidelines determine whether an antibiotic is susceptible or not. MIC is defined as the lowest concentration of antibiotic required preventing visible growth of a microorganism in a agar or broth dilution susceptibility test and is used to determine if the infected pathogen is susceptible or resistant to an antibiotic (Bauer et al, 2014; Jorgensen and Ferraro, 2009; Wiegand et al, 2008). A breakpoint is defined as the concentration of an antibiotic that enables interpretation of AST to define isolates as susceptible, intermediate, or resistant (Humphrieset al, 2016; Wiegand et al, 2008). The Clinical and Laboratory Standards Institute (CLSI) provides the most popular guidelines, which are based on pharmacokinetic–pharmacodynamic (PK-PD) properties and mechanisms of resistance [9]. Most European countries follow the MIC cut-offs based on PK-PD properties, and the epidemiological MIC cut-offs (ECOFFS) as determined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (Wiegand et al, 2008). These numbers provide valuable information to physicians to determine the appropriate targeted antibiotic to be administered to the patient. It is important to note here that just having either bacterial identification or AST alone, will not yield clinically significant reports for patient treatment. The combined results from bacterial identification and AST are imperative to meaningfully determine the right antibiotic choice for that particular pathogen (Marschal et al, 2017).

 

Here, we summarize some of the current phenotypic methods, discuss the emerging technologies, and provide scientific opinions on future AST technologies.

 

Disk Diffusion

Disc diffusion or the Kirby–Bauer test is one of the classic microbiology techniques, and it is still very commonly used (Bauer et al, 1966; Clinical and Laboratory Standards Institute, 2009; Jorgensen and Turnidge, 2007). Because of convenience, efficiency, and cost, the disc diffusion method is probably the most widely used method for determining antimicrobial resistance around the world. Commercially-prepared, fixed concentration, paper antibiotic disks are placed on the inoculated agar surface (Figure 1). Plates are incubated for 16–24 h at 35°C prior to determination of results. The diameter of the zone of clearance around the disc is measured and compared to the CLSI reference table to determine if the organism is susceptible, intermediate or resistant against the antibiotic agents tested (Reller et al, 2009). This method can test multiple drugs or concentrations on a single agar plate but only yields qualitative results since it doesn’t determine the MIC values which is of high clinical significance for effective patient treatment.

 

Figure 1. Disk diffusion, demonstrating of inhibition zones.

 

Broth Dilution

One of the earliest antimicrobial susceptibility testing methods was the macrobroth or tube-dilution method (Ericsson and Sherris, 1971). This procedure involved preparing two-fold dilutions of antibiotics (eg, 1, 2, 4, 8, and 16 µg/mL) in a liquid growth medium dispensed in test tubes (Ericsson and Sherris, 1971; Jorgensen and Turnidge, 2007). The bacteria of interest are isolated to obtain single colonies on an agar plate, suspended in media, diluted appropriately and added to each tube to obtain a final concentration of ~5×105 CFU/ml, as per the CLSI recommendation. Following overnight incubation at 35°C, the tubes were examined for visible bacterial growth as evidenced by turbidity. The lowest concentration of antibiotic that prevented growth represented the minimal inhibitory concentration (MIC). This method is easy to interpret and accurate in identifying the MIC for a given bacteria-antibiotic combination but is labor and material intensive and also time consuming due to the difficulty in running multiple samples simultaneously to test a wide panel of pathogens.

 

The miniaturization and mechanization of the test by use of small, disposable, plastic “microdilution” trays has made broth dilution testing practical and popular. Standard trays contain 96 wells, each containing a volume of 0.1 mL that allows approximately 12 antibiotics to be tested in a range of 8 two-fold dilutions in a single tray (Clinical and Laboratory Standards Institute, 2009; Jorgensen and Turnidge, 2007). The samples are dispensed into micro-titer well plates, typically containing 96 wells (12×8) as an array with each row containing a given antibiotic at doubling concentrations and every row having a different antibiotic of interest. Due to its miniaturization and small volumes, multiple drugs and/or bacteria can be tested simultaneously on a single micro well plate. Such a method can handle multiple samples or test multiple antibiotics simultaneously using small volumes. The panels used can be customized, pre-made and hence enable ready utilization in labs without extensive capability to run other AST methodologies. However, the time taken to obtain the results is similar in comparison to microdilution method.

 

Etest

This is one of the commonly used gradient diffusion methods developed by bioMérieux, where a plastic strip impregnated with gradually decreasing concentrations of a given antibiotic is placed on the surface of an agar plate pre-inoculated with bacteria to be tested. The strip has an interpretive scale on the other side, which aids in reading the zone of inhibition. The plates are incubated for ~24 hours, at the end of which the inhibition zone is identified and the corresponding MIC value is determined. The MIC is interpreted as a point on the scale of the strip where the inhibition zone intersects the strip (White et al, 1996). Generally, Etest results have correlated well with MICs generated by broth or agar dilution methods (Baker et al, 1991; Citron et al, 1991; Huang et al, 1992; Jorgensen et al, 1994). However, there are some systematic biases toward higher or lower MICs determined by the Etest when testing certain organism-antimicrobial agent combinations (Jorgensen et al, 1994; Prakash et al, 2008). This can represent a potential shortcoming when standard MIC interpretive criteria derived from broth dilution testing (Prakash et al, 2008) are applied to Etest MICs that may not be identical.

 

Automated instrument systems

Since the dawn of automated technologies in the 1980s, antibiotic susceptibility tests have been perpetually improvised and, hence, have superseded conventional phenotypic methods (Reller et al, 2009). Automation, simplicity, and compactness are the major reasons for their widespread acceptance in diagnostics.

 

VITEK®system: This system was originally developed in the1970s by bioMérieux for determining bacterial identification (ID) and AST profiles simultaneously from isolated patient samples. The current systems in the market are VITEK® 2 compact and VITEK® 2 systems both of which utilize broth microdilution technique. The system uses “AST cards” which contain micro-wells with fluidic connections to automatically fill the samples into multiple wells simultaneously.

 

BD PhoenixTM automated identification and susceptibility testing system: This is another automated microdilution-based system employed in clinical microbiology labs and approved by the Food and Drug Administration (FDA) for AST determination. The assay uses redox indicator for detection of growth of organisms in the micro-well panels (Carroll et al, 2006). Each micro-well contains an antibiotic at a particular concentration which is rehydrated with addition of bacterial suspension. These panels are incubated over a time period and scanned for microbial growth using chromogenic or fluorogenic substrates. Each panel contains an ID and an AST section, each with multiple micro-wells.

 

SensititreTM: This is a commercially available product by Thermo Fisher Scientific based on microdilution method similar to VITEK® and the PhoenixTM systems. The actual detection of growth/no growth of bacteria is customizable for the results to be read manually, semi-automated or fully automated. For manual interpretation, the wells are observed for visual turbidity, while for automated detection, fluorescence technology is used to monitor activity of specific enzymes produced by the organism over the incubation time. The enzymes produced cleave the bond between the fluorophore and the quencher substrate, releasing the fluorophore to emit fluorescence (Belak, 2001).

 

Micro-scan walk away®: This is an automated system by Beckman-Coulter for bacterial ID and AST based on broth microdilution method. This system is available in 40 and 96panel modules, for medium and large-scale operations. It utilizes colorimetric readings based on usage of photosensors and color wheel/lamp assembly for optical detection of bacteria in the wells.

 

Emerging and Future Technologies

Newer AST techniques, which are currently and actively being pursued by commercial entities for clinical translation, are considered as emerging technologies for the purpose of this review. With the increasing clinical demand for rapid AST, various new AST techniques based on optical imaging (Choi et al, 2014; Metzger et al, 2014; Mohan et al, 2014), micro-channel resonators (Etayash  et al, 2016; Godin et al, 2013; Longo et al, 2013) and other biosensors (Hayden et al, 2016; Sinn et al, 2016) have been pursued.

 

The emerging technologies, being actively pursued by commercial entities discussed above, promise rapid AST within a few hours. Furthermore, some of the technologies can be directly applied to patient samples without any sample pretreatment. However, further shortening the test time and applying them to slowly growing organisms will require innovative approaches.

 

Conclusion

While all AST methods offer qualitative assessments using susceptible, intermediate, or resistant categories, certain methods specify qualitative and effective antibiotic dosage (e.g., minimum inhibitory concentration) and formulate a profile of empirical therapy for the proper management of individual patients’ health against deadly infections. The concern of antibiotic resistance is a certainty, therefore improve the turn-around times for sample processing in the labs, reduce the burden on technicians, provide rapid reporting of AST, with the ultimate goal of faster treatment of patients, reduced load on use of broad-spectrum antibiotics and better clinical outcomes.

 

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