Biofilm Formation and Bacterial Dispersal Mechanisms: can we prevent their formation or influence the structures?

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Francesco Centorrino

Bacterial biofilm formation

Bacteria can create structures called biofilms. Sometimes, these are very difficult to eradicate and can also encapsulate a serious public health threat when considering those consisting of organisms that are pathogenic to humans. Bacteria can also move away from the original biofilm in case of limiting conditions, so they can disperse and colonize new environments. The mechanisms of biofilm dispersion are known for only a few types of bacteria. These include the formation of spores in Bacillus subtilis in which a special phenomenon related to the change of characteristics in the morphology of the bacterial biofilm and the dispersion process according to the change of calcium ion concentration has been noted.

Biofilm formation and function

Bacteria can anchor themselves to a surface and give rise to multicellular communities: biofilms (Fig. 1). The development of such structures involves several steps: adhesion of a planktonic cell to a surface, formation and growth of the biofilm then removal of the cells (“biofilm dispersal“).

[Figure 1 - Phases characterizing biofilm formation - Credits: H. Vlamakis et al., 2013]
Figure 1 – Phases characterizing biofilm formation [Credits: H. Vlamakis et al., 2013]

The ultimate goal of the community is to create a useful microenvironment for the resident bacteria to retain water, nutrients, and extracellular enzymes but also to have protection against antibiotics and environmental stress phenomena.

Increased cell numbers in the colony, reduced resources, oxygen limitation or waste accumulation can trigger the escape of some cells from the structure. This phenomenon, called dispersal, involves the secretion of substances (such as polysaccharide-degrading enzymes, proteases, nucleases, and biosurfactants) useful for disassembling matrix components and the diffusion of planktonic cells to new locations so that new bacterial biofilms are formed in environments with better conditions.

The analysis conducted on Bacillus subtilis

Bacillus subtilis is considered a model organism for many studies; in fact, many microbiological phenomena have been analyzed by studying its characteristics and/or behaviors. This Gram-positive bacterium forms spores. These spores can disperse in the environment and allow colonies to develop wherever possible, thus new robust biofilms (solid colonies) arise or float on the surface of liquid media.

The structures arise due to exopolysaccharides, TasA amyloid fiber protein polymers, and BslA hydrophobins. These components are synthesized by the 15-gene operon epsA-epsO, the operon tapA-sipW-tasA and bslA, respectively. These biofilm matrix genes are repressed by the transcriptional repressors AbrB and SinR. In addition, multiple transcriptional activators are required to achieve the expression of these matrix genes.

The analysis on the dispersal of B. subtilis has not been thorough but preliminary studies report activation of the alternative stress-responsive sigma factor σB induces dispersal by increasing transcription of sinR.

Two mechanisms associated with dispersion

Analyses were conducted by exploiting transcriptional fusions of green fluorescent protein (GFP) inserted into the chromosome of a precise strain and with the observation by flow cytometry (Fig. 2). One strain, called wild-type (without GFP reporter), was used as a negative control.

The observations obtained were reported in the previous section where the expression of some biofilm matrix genes that can cause bacterial biofilm dispersal was discussed. In addition, overexpression or decreased expression of the epsA operon alone partially prevented biofilm dispersal.

[Figure 2 - Diagram of flow cytometry analysis- Credits: sigmaaldrich]
Figure 2 – Diagram of flow cytometry analysis [Credits: sigmaaldrich]

Next, the phenomenon of sporulation was evaluated (Fig. 3) to see whether this may also be involved in dispersal. Mutants were grown with deletions of genes encoding sporulation-specific sigma factors: σF, σE, σG, and σK.

These sigma factors are activated in the order indicated during sporulation, and activation of σK requires the action of the preceding factors. In such mutants, a milder biofilm dispersal than in the wild-type is noted and certainly, a sporulation-associated, σK-dependent gene is involved in biofilm dispersal.

[Figure 3 - Spore formation for B. subtilis - Credits: University of Groningen]
Figure 3 – Spore formation for B. subtilis [Credits: University of Groningen]

It was therefore hypothesized that a sporulation-dependent mechanism could cause biofilm dispersal. These results, taken together, are useful to conclude that two mechanisms, thus decreased expression of the epsA operon and an unknown σK-dependent mechanism, cause biofilm dispersal.

The influence of calcium on biofilm formation

To determine which metal influences biofilm dispersion, elements were gradually removed from the culture medium. Removal of calcium (Fig. 4) did not affect biofilm formation but induced dispersion. In contrast, removal of manganese impaired biofilm formation while removal of magnesium or iron had no effect on biofilm formation/dispersion.

[Figure 4 - Chemical and physical properties of calcium]
Figure 4 – Chemical and physical properties of calcium

Beyond the reactions triggered by reducing concentrations of certain elements, it was observed that calcium addition did not alter the expression of biofilm matrix and sporulation genes but not those involved in dispersal, including the epsA operon.

Notably, the latter results indicate that calcium stabilizes biofilm architecture, although in parallel the expression of biofilm matrix genes decreases and sporulation is triggered. In this regard, we speculate that calcium could stabilize biofilms, not prevent their formation but positively change the morphology of the biofilm by strengthening the matrix structure.

Calcium and colony architecture

The addition of calcium to the medium prevented biofilm dispersion. In the presence of calcium, bacterial biofilms remained undisturbed for at least five days after inoculation, while the expression of biofilm matrix genes decreased dramatically by the second day. This reduction changed the architecture of the wrinkles on the surface of the biofilms, which appeared few in number and were partially visible.

Normally these wrinkles arise because bundles of cell chains cannot elongate freely because of physical interactions with other bundles, and cell growth produces a distortion within the bundles. Part of the bundles are pushed into the air and these surface wrinkles are formed.

The attenuated wrinkles in the absence of calcium suggest that the bundles of cell chains are not structurally strong enough to support upward growth when this element is absent. Therefore, the morphology of the bacterial biofilm is strongly related to the concentration of calcium, which, varying in its concentration, can strengthen or weaken the overall structure resulting in the formation of a smoother or less wrinkled surface.

The relationship between Ca-DPA and the dispersion phenomenon

The spoVF operon, in B. subtilis, encodes DPA synthetase. Dipicolinic acid, also called DPA (Fig. 5), contains small soluble alpha/beta-type proteins that protect DNA. This acid is accumulated together with calcium, forming calcium salt and comes in handy for the bacterium to protect and stabilize the hereditary material.

Chemical structure dipicolinic acid (DPA; a) and role of calcium in the formation of DPA molecules (b)
Figure 5 – Chemical structure dipicolinic acid (DPA; a) and role of calcium in the formation of DPA molecules (b)

Referring to the presence or absence of calcium and the huge amount of Ca-DPA that is synthesized and stored in spores during sporulation, it can be said that the processes of biofilm formation and sporulation compete with each other in case of low concentration of this element.

Due to the occurrence of the phenomenon of low-level calcium dispersion by spore formation and subsequent detachment, most of the calcium ions bound to the components of the biofilm matrix can be transported within the spores as Ca-DPA during sporulation (Fig. 6).

[Figure 6 - Calcium accumulation in spores during the sporulation phase and formation of the Ca-DPA complex - Credits: Nishikawa et al., 2021]
Figure 6 – Calcium accumulation in spores during the sporulation phase and formation of the Ca-DPA complex [Credits: Nishikawa et al., 2021]

In this scenario, when the extracellular calcium level is low, the calcium ions bound to the components of the biofilm matrix function as a reservoir from which the cells can import ions useful to support spore formation and be able to continue with dispersion-detachment processes. In addition, it has been verified that this phenomenon, referred to as “calcium biomineralization,” within the medium occurs most when the pH becomes alkaline (pH 8.3).

Conclusions

The results of studies conducted on dispersal in biofilms, as far as B. subtilis is concerned, reveal that two mechanisms cause this phenomenon. What has been reported suggests that further investigations into this aspect may be crucial in identifying useful techniques to limit the development, on specific surfaces/environments, of microbial communities that are hazardous to human health.

Furthermore, the particular insight regarding calcium levels in the medium allowed us to observe that this element counteracted dispersal mechanisms, did not cause alterations in inherent gene expression, and positively affected biofilm stabilization.

Consequently, the analysis of response mechanisms linked with the reduction of calcium concentration in environments can also be an important step in defining methods that can limit the formation of spores and the growth/diffusion of potentially pathogenic biofilms.

Gennaro Velotto

Original article: Formazione di biofilm e Meccanismi di dispersione batterica: possiamo impedirne la formazione o influenzare la loro struttura? – Gennaro Velotto

References

Foto dell'autore

Francesco Centorrino

Sono Francesco Centorrino e scrivo per Microbiologia Italia. Mi sono laureato a Messina in Biologia con il massimo dei voti ed attualmente lavoro come microbiologo in un laboratorio scientifico. Amo scrivere articoli inerenti alla salute, medicina, scienza, nutrizione e tanto altro.

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