The minimum bactericidal concentration of antibiotics

Introduction

The minimal bactericidal concentration (MBC) is a quantitative metric that can be used to assess the effect of antibacterial agents on microorganisms. Whereas the minimum inhibitory concentration (MIC) indicates the lowest concentration needed to inhibit bacterial growth, the MBC indicates the lowest concentration needed to kill bacteria. Here we look at how microplate readers are used to assess MBC values and what this can bring to studies of antibacterial resistance and microbiology. 

What is the minimum bactericidal concentration?

The minimum bactericidal concentration (MBC) is the lowest concentration of an antibiotic (expressed in mg/L) that under defined in vitro conditions reduces by 99.9% (3 logarithms), the number of organisms in a medium containing a defined inoculum of bacteria, within a defined period of time.^1,2 The reduction is usually expressed as the  proportion of the inoculum (number of living colony-forming units or cfu introduced) that is rendered incapable of reproduction on subculture within that period. The effects can be presented as a time/kill curve in which an inoculum is incubated with the antibiotic and samples are tested for numbers of surviving cfu at defined time intervals.

MBC values provide scientists with a metric for killing bacteria and as such they have many applications in microbiology, studies of the environment, biotechnology, drug discovery and other areas of research. MBCs are often cited as metrics in studies of antimicrobial resistance.

Antimicrobial resistance or AMR is a burgeoning global challenge facing society.³ It is ranked as one of the top ten threats to public health worldwide by the World Health Organization. The measurement of MBC values provides scientists with a way to quantitate the impact of the different bactericidal agents against microbial growth and the effectiveness of new approaches designed to ameliorate drug resistance in microbial communities. It is also useful for quantifying the effects of antimicrobials on biofilms, communities or arrangements of microorganisms that may also contribute to recalcitrant infections.⁴

Early work leading to the concept of minimum bactericidal concentration   

Alexander Fleming discovered penicillin in 1928. However, it was not until the early 1940s through the work of scientists including Ernst Chain, Edward Abraham and Howard Florey that penicillin was purified and mass produced (Fig. 1). These initial studies on the efficacy of penicillin included not only experiments to determine its impact on bacterial growth (MIC) but also measurements of the antibiotics ability to kill bacteria. The early studies by these researchers were instrumental in establishing dosage guidelines and understanding how to use penicillin as a therapeutic intervention. This work served as a foundation to contemporary methods to determine MBCs.

Methods to determine minimum bactericidal concentration

The determination of the MBC of a particular antibiotic relies first on working out the MIC value. MIC is the lowest concentration of an antimicrobial agent that shows no visible growth compared to the control. The use of serial dilutions to determine MICs is described in detail in the BMG LABTECH blog The minimum inhibitory concentration of antibiotics. In this approach, a series of decreasing concentrations of antimicrobial agents is tested for its ability to inhibit the growth of a microorganism (Fig. 3). The decreasing concentrations are prepared by serial dilution of the sample with the highest concentration of antimicrobial agent. After incubation under standard conditions, each tube is visually examined for microbial growth. The lowest concentration of the antimicrobial agent that shows no visible growth is taken as the MIC. As we will see later, MIC values can also be determined from microplates.

Once the MIC value is known, the MBC value can be determined by subculture from tubes at or above the MIC. For this purpose, aliquots are inoculated onto antibiotic-free agar plates. A small volume is administered onto the agar plate which is incubated under standard conditions for a known period of time (typically 24-48 hours). After incubation, the agar plates are checked for bacterial growth.

What factors affect MBC?

Like the determination of MIC, it is essential to perform MBC tests under standard conditions. Careful attention must be given to conditions like pH, temperature and the composition of agar media used for plates. Factors like nutrients, aeration and oxygen may also need to be controlled to ensure precise measurements of MBCs. Similar considerations may need to be given to moisture and osmolarity.

At the same time, the properties of the antibiotic should also be borne in mind to ensure the fidelity of measurements. For example, molecular structures may inform the way different antibiotics should be solubilized for use in assays. Small molecules like ampicillin are readily soluble in water or phosphate buffers. Others may require different conditions. Knowledge of the mode of action of different antibiotics can also inform the way experiments are set up for optimal measurements of MIC values.

Another important consideration to ensure reliable MBC measurements is to be aware of variability due to different bacterial strains. Different bacterial strains exhibit genetic differences and may employ multiple types of resistance mechanisms. 

How do you measure MBC? 

The first step in determining MBC is to find the MIC value. Microplate readers offer streamlined solutions that enable a modern approach to monitor MIC of multiple samples (6–1536 wells) in real-time with high accuracy and with no manual intervention. Microplate readers can be used to determine the MIC by measuring the light signal (absorbance, fluorescence or luminescence) of samples in a microplate containing different concentrations of an antibacterial compound. The ability of a microplate reader to measure thousands of samples in a matter of minutes allows for quick and precise determination of the lowest concentration of inhibitor needed. A typical set up for a MIC assay on a 96-well plate is shown in Fig.4 for a test compound. Once the MIC values are known, MBC values can be determined by subculture of samples from wells at or above the MIC. As in the tube dilution experiments, aliquots are inoculated onto antibiotic-free agar plates.

Advantages of microplate-based minimum bactericidal concentration determination

Microplate readers offer several advantages to automate and streamline the measurement of MIC values. High throughput measurements are possible on 96-, 384- and 1536-well microplates to determine the MIC which subsequently facilitates the determination of MBCs after agar plating. This allows for testing of many different antimicrobial concentrations as well as multiple microbial strains in parallel.

Microplate readers automate the measurements of OD600 which is indicative of bacterial growth. You can read more about measuring microbial growth using OD₆₀₀ in this blog from BMG LABTECH. 

The use of microplate readers reduces the potential impact of human error and its associated variability of measurements and leads to more reliable results. Measurements can be made at regular intervals (e.g., minutes or hours) without manual interventions. Real time data can be readily collected which accelerates analysis. In addition to high precision and sensitivity of measurements, microplate readers come equipped with data collection and software analysis options. MIC values can therefore be determined based on defined criteria and graphs plotted to assist in calculations. 

Overall, microplate readers help to standardize the process for determining MIC values and ensure that conditions are consistent across all wells in the microplate. Improvements to MIC determination have a knock-on effect for working out MBC values. Faster, more efficient determination of MIC values accelerates the steps that precede agar plating which in turn speeds up the determination of MBCs.

Additional features like incubation, shaking and an Atmospheric Control Unit provide distinct benefits for the support of consistent growth conditions for microorganisms that have more demanding parameters for growth. This type of consistency is essential for accurate determination of MIC values for more fastidious microbes. The Atmospheric Control Unit from BMG LABTECH provides researchers with a system that enables control of both the oxygen and carbon dioxide concentrations in an independent manner. Consistent stirring options also deliver benefits for MIC experiments.

In the paper “Biological characterization and metabolic variations among cell-free supernatants produced by selected plant-based lactic acid bacteria” a team of researchers used MBC values to assess the antibacterial and antioxidant properties as well as the variation in metabolites of the cell-free supernatant produced by lactic acid bacteria.⁵ Lactic acid bacteria have gained attention as probiotics due to their recognized safety and ability to promote health. The MIC and MBC of lactic acid bacteria cell-free supernatants were determined using a dilution method in 96-well polystyrene microtiter plates (Eppendorf, Germany) along with colony forming unit counting. The MBC endpoint was defined as the lowest concentration of cell-free supernatant that resulted in the elimination of over 99.9% of the pathogens, as evidenced by the absence of visible bacterial growth on the Muller Hinton agar plates after incubation for 24 h at 37 °C.

The MIC and MBC values against the tested foodborne pathogens fell within the range of 3.12–12.5 mg/mL and 6.25–25.00 mg/mL for MIC and MBC, respectively. These values are consistent with effective probiotic activities against various harmful bacteria including Bacillus cereus, Salmonella typhimurium and Escherichia coli. The results were obtained on a SPECTROstar^® Nano microplate reader. 

The type of approach used to determine MIC and MBC values will also depend on the growth characteristics of the bacterium under study. Mycobacterium tuberculosis has a relatively slow growth rate but is an important target for drug discovery. To circumvent time-consuming assays rapid, non-invasive luminescence-based assays have been widely used in anti-tuberculosis drug discovery and development. In the paper “In vitro profiling of antitubercular compounds by rapid, efficient, and nondestructive assays using autoluminescent Mycobacterium tuberculosis” a group of researchers describe an autoluminescent-based platform suitable for both MIC and MBC determination.⁶ Typically, agar plating is required for MBC determination. However, in this case, the traditional method of MBC determination by plating onto agar plates was used to validate a rapid, high throughput luminescence-based assay. This approach was shown to be suitable for profiling tuberculosis drug leads in an efficient manner. The optimized luminescent-based assays were useful in profiling the biological activities of two ClpC1 modulators, ecumicin and rufomycin. The postantibiotic effects of these modulators were comparable to the first-line tuberculosis drug, rifampin. The measurements were made on a CLARIOstar^® microplate reader.

A growing need for MBC measurements

The need for the rapid determination of MBC values is increasing due to several factors. First MBC values are crucial in evaluating the efficacy of new antimicrobials and other drugs during research and development. Second, the global increase in antimicrobial resistance necessitates precise and effective use of both new and existing antibiotics. MBC values help in selecting the most effective antibiotic options in specific scenarios. They also help in deciding the optimal doses of antimicrobial agent to use versus resistant strains. Collectively, this can assist in slowing the spread of antimicrobial resistance.

While BMG LABTECH microplate readers are not for use in environments that require in vitro diagnostic approved devices and processes, they may provide researchers with many solutions to support fundamental research in non-clinical settings where scientists are looking to use MIC and MBC values as part of their research efforts.

Many areas of investigation require robust research into antimicrobials and their modes of action. Overall, the growing threat of antimicrobial resistance and the need for new and more effective treatments underscore the increasing importance of MIC and MBC values in research settings across the globe.

BMG LABTECH solutions

What is the preferred BMG LABTECH microplate reader for specific needs and applications related to the determination of MIC and MBC values? Absorbance detection for the measurement of OD₆₀₀ is available on BMG LABTECH’s complete portfolio of microplate readers with the ultra-fast spectrometer. The exception is the NEPHELOstar Plus which is a dedicated laser-based nephelometer for light scattering and turbidity measurements.

BMG LABTECH also offers a range of multi-mode detection devices for sensitive fluorescence and luminescence measurements.

Bacteria require specific temperatures and aeration for maximum growth rates. To ensure optimal growth conditions, all BMG LABTECH readers offer accurate temperature regulation up to 45°C (optionally up to 65°C). Three shaking modes with adjustable speed up to 700 rpm (optionally to 1100 rpm) provide optimum aeration settings for your strain. Additionally, the VANTAstar, CLARIOstar Plus, the Omega series and the SPECTROstar Nano can be equipped with an extraordinary robust transport system for shaking 24/7 where required.

The VANTAstar, the CLARIOstar Plus, the Omega series and NEPHELOstar Plus can be combined with the Atmospheric Control Unit making them the preferred choice for different kinds of live cell assays including bacterial growth assays.

Both the VANTAstar and CLARIOstar Plus further allow for wavelength flexibility and include Enhanced Dynamic Range technology for superior performance in a single luminescence or fluorescence run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators^TM and different filter options.

All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstar Plus,  and PHERAstar® FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection. The VANTAstar can be equipped with a modular injection unit. The SPECTROstar Nano comes with a dedicated cuvette-port which can also be used to study bacterial growth over time in a cuvette-based approach.

Collectively, BMG LABTECH multi-mode readers combine high-quality measurements with miniaturised assays, short measurement times, and offer considerable savings on materials and other resources.

The NEPHELOstar Plus offers turbidimetric measurements for the determination of bacterial growth at very high sensitivity. It can be used for example to study the early stages of bacterial growth.

BMG LABTECH microplate readers are available for research purposes only.

References