Determining Biofilm Antibiotic Tolerance Using Alginate Beads
Microbial Solutions
Clio Andreae

Determining Biofilm Antibiotic Tolerance Using Alginate Beads

Understanding the tolerance of Pseudomonas aeruginosa biofilms to antimicrobial therapy will aid in the treatment of chronic infections.

Bacterial biofilms are matrix associated communities of bacteria that can form on plastic surfaces, indwelling medical devices and during chronic infections, e.g. within the lungs. Biofilms, unlike their free-floating planktonic cell counterpart, are known to form mushroom-like structures in the lab, which are known to display high tolerance to antimicrobial therapy (Fig. 1). Biofilms exhibit distinct microenvironments, displaying low oxygen tensions deeper within the structure, causing the bacteria to exhibit metabolic changes resulting in increased tolerance to antimicrobial therapy. 

Figure 1: Graphic representation of the formation of a surface associated biofilm. Image shows biofilm formation on plastic surfaces, from the attachment of planktonic cells (antibiotic susceptible) to a mature biofilm which exhibits high tolerance to antimicrobials. Mature biofilms are known to exhibit different microenvironments within their structure, with microaerobic/anaerobic environments seen deeper within the biofilm matrix.

The resilience of these bacterial biofilms makes them difficult to control and has led to many instances of infections in clinical settings. No study to date has successfully observed the classical mushroom-like biofilm structure seen on plastic surfaces, which brings into question the validity of in vitro model systems.

Research over the past 15 years has allowed us to gain further insight into the mechanisms behind the high antimicrobial tolerance exhibited by biofilms and determine the bacterial biofilm structure seen during chronic and recurrent infections. In 2013 Bjarnsholt et al.  showed that biofilm structures found in clinical settings, or in vivo, were different from structures observed in the lab. They discovered that Pseudomonas aeruginosa forms small aggregate structures during chronic infections that are composed of the host cell matrix (e.g.the lung mucus of CF patients), immune cells, and bacterial cells (Fig. 2) Although smaller than the biofilms observed in the lab, the biofilm aggregates were shown to display oxygen-depleted zones in vivo and were associated with a respiratory burst produced by the immune cells.

Research groups in Denmark have recently developed an alginate bead model system that mimics the P. aeruginosa aggregate structure seen in vivo during chronic infections (Fig. 2 and Fig. 3) (Sønderholm et al., 2017, Sønderholm et al. 2018). This system has been used for Gram-negative (e.g. E. coli, P. aeruginosa, and K. pneumonia) and Gram-positive (e.g. S. aureus and Enterococcus faecalis) biofilms (Dall et al., 2017), P. aeruginosa biofilm-aggregates (Sønderholm et al 2017, Dall et al. 2017), and biofilm pharmo-kinetics (Cao et al. 2016).

Figure 2: The in vivo biofilm. Image shows a Pseudomonas aeruginosa biofilm aggregate seen during a chronic infection within an infected cystic fibrosis lung. Image taken from Bjarnsholt et al. (2013).
Figure 3 – Alginate encapsulated P. aeruginosa forms aggregates of comparable size to those seen in vivo. Images show growth of P. aeruginosa aggregates (20 µm in size, comparable to that seen in Fig. 2) within alginate beads when grown without nitrate (A) or with nitrate (B). Image has been taken from Sønderholm et al. (2017). Addition of nitrate (B) allows for P. aeruginosa to grow further into the bead structure, by the use of nitrate as an alternate electron acceptor for anaerobic growth via denitrification

An important factor causing high antimicrobial tolerance seen in bacterial biofilms has been attributed to the presence of a microaerobic/anaerobic environment. The lack of oxygen is thought to induce a ‘dormant/persistent’ metabolic state, which affects antimicrobial action and uptake into the bacterial cell. Several antimicrobials are thought to display limited activity with limited oxygen, and anaerobic microenvironments are thought to account for an increase in tolerance to ceftazidime, ciprofloxacin and tobramycin, which are used for treatment of P. aeruginosa infections.

Use of alginate beads allows for rapid reproducible growth and enumeration of bacteria, and can be adapted to allow for bacterial encapsulation (biofilm-aggregate model) or used as a dissolvable surface for biofilm formation. The bead system has been developed as a cost effective, high throughput model system by Charles River Laboratories (CRL) to better mimic tolerance levels that are seen during a chronic infection. In this system CRL have shown P. aeruginosa to display a 10 x higher tolerance to ciprofloxacin and 100 x higher tolerance to tobramycin, when compared to the MIC determined by CSLI standard methods, giving comparable results to those seen with Sønderholm et al. (2017). We envisage data from these alginate bead biofilm studies to not only provide data on the potential antimicrobial tolerance levels seen in vivo but could also help inform treatment regimens to ensure effective antimicrobial therapy for chronic infections.


Bjarnsholt T., et al. (2013) The in vivo biofilm.

Sønderholm et al (2017) – The consequences of being in an infectious biofilm: Microenvironmental conditions governing antibiotic tolerance

Sønderholm et al (2017) - Pseudomonas aeruginosa aggregate formation in an alginate bead model system exhibits in vivo-like characteristics

Sønderholm et al (2018) – Tools for studying growth patterns and chemical dynamics of aggregated Pseudomonas aeruginosa exposed to different electron acceptors in an alginate bead model

Cao et al. (2015) - Antibiotic penetration and bacterial killing in a Pseudomonas aeruginosa biofilm model

Cao et al. (2016) - Diffusion Retardation by Binding of Tobramycin in an Alginate Biofilm Model