Testing Antimicrobials Using Minimum Biofilm Eradication Concentration (MBEC)

Figure 1. Biofilm growth on rocks in a stream (USGS) and within a kitchen pipe (MSU Center for Biofilm Engineering).

Biofilms are complex communities of microorganisms that adhere to surfaces and are encased in a protective extracellular matrix. They can develop in diverse environments, including natural settings like riverbeds and geysers, as well as man-made structures such as kitchen sinks. Critically, biofilms are also known to form on medical devices and implants, including urinary and central venous catheters, pacemakers, artificial heart valves, silicone breast implants, and cochlear devices.

Figure 2. Biofilm formation on a voice prosthesis implant.

Traditional antimicrobial susceptibility testing methods, like the Minimum Inhibitory Concentration (MIC) assay, are effective for assessing antibiotics against planktonic (free-floating) bacteria and fungi. However, these methods are inadequate for evaluating the efficacy of antimicrobials against biofilm-associated microorganisms. Biofilms act as a barrier, impeding the penetration of antibiotics and contributing to increased resistance.

To address this challenge, Emery Pharma employs the Minimum Biofilm Eradication Concentration (MBEC) assay, utilizing the Calgary Biofilm Device (CBD). This device consists of a 96-well plate with pegs that facilitate the adherence and growth of biofilms. The MBEC assay is a time-efficient and accurate method for testing the efficacy of antimicrobial agents against biofilms.

Figure 3. Calgary Biofilm Device (CBD)

In the MBEC assay, biofilms of the target microorganism are cultivated on the pegs using appropriate growth media and incubation conditions. Motile microorganisms, such as Pseudomonas aeruginosa and Escherichia coli, tend to form denser biofilms due to their flagella-mediated motility. In contrast, non-motile organisms like Staphylococcus aureus form less dense biofilms. To support the growth of fastidious microorganisms, pegs can be coated with supplemental nutrients or hydroxyapatite.

Table 1 shows experimental biofilm densities of various organisms grown at Emery Pharma. Different species and strains of microorganisms vary significantly in their ability to form biofilm. For instance, Staphylococcus aureus ATCC 29213 has been observed to develop denser biofilms than methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591, even under identical growth conditions. Emery Pharma can cultivate biofilm from a wide variety of bacterial and fungal strains to meet your study’s specific needs.

Table 1: Organism Biofilm Densities

Once the biofilm has matured, the MBEC assay is conducted by rinsing the Calgary Biofilm Device to remove planktonic microorganisms. The pegged lid is then exposed to serial dilutions of the antimicrobial agents. After treatment, the pegs are rinsed to eliminate residual antimicrobial agents and transferred to fresh media. The MBEC is determined as the lowest concentration of the antimicrobial that eradicates the biofilm, indicated by the absence of growth in the corresponding well. Alternatively, a spectrophotometer can be used to measure the optical density (OD) at 650 nm; an OD650 reading of less than 0.1 signifies biofilm eradication.

Figure 4. Schematic of Biofilm Formation on a Calgary Device Peg.

Emery Pharma also offers the determination of the Minimum Biofilm Inhibitory Concentration (MBIC), which is the lowest concentration of an antimicrobial agent that prevents the initial formation of a biofilm. Typically, the MBIC is lower than the MBEC, as preventing biofilm formation requires less antimicrobial activity than eradicating an established biofilm.

With extensive experience in biofilm-related studies, Emery Pharma provides MBEC and MBIC assay services, along with other models such as the CDC reactor, drip-flow reactor, and capillary biofilm systems. Our team can also assist with custom biofilm study needs. Contact us today if you are interested in our biofilm services!

References:

1) Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms. Journal of Clinical Microbiology 1999; Vol 37, No. 6: p. 1771-1776.