**3.2 EPS**

*Bacterial Biofilms*

**Figure 1.**

*Stages of biofilm development.*

form the majority in most of the environments and single species biofilms host the

be capable of attaching itself to and moving on the surface, detecting their cell density and ultimately to form a 3-D mesh of cells enclosed by exo-polysaccharide [3]. There is also an important role of cell membrane proteins, extracellular polysac-

**Step1. Attachment:** Conditioning layer is formed which have a loose collection of carbohydrates and proteins which gets unite with minerals in hard water. It

**Step2. Irreversible attachment:** As soon as conditioning layer formed, electrical charge accumulates on the surface which attracts the bacteria having opposite charge that result in irreversible attachment of microbial cells. The charges are sufficiently weak that microorganisms could be easily removed by the mild cleanser

**Step3. Proliferation:** In this phase, bacteria get attached to the surface as well as with each other by secreting EPS (an extracellular polymeric substance) that

**Step4. Maturation:** The biofilm environment consists of the nutrient-rich layer which supports the rapid growth of microorganisms. Complex diffusion channels are present in a mature biofilm to transport nutrients, oxygen and other components required for bacterial growth and removes waste products and dead

**Step5. Dispersion:** It is the process of dispersal of biofilm in which actively growing cells gradually sheds daughter cells [1]. Because as long as fresh nutrients are kept providing, biofilm continues to grow and when they get nutrient deprived, they return to their planktonic mode by detaching themselves from the surface [3]. This process probably happens to allow bacterial cells to get sufficient nutrients [2]. There is also a possibility of the detachment process to be species-specific as *Pseudomonas fluorescence* recolonizes surface after approx. 5 hours, *Vibrio harveyi*

The initiation of biofilm formation have some requirements as the bacteria must

surface of medical implants and hence being the reason of infections.

charides and signaling molecules [2] (**Figure 1**).

entraps the cells within a glue-like matrix.

attracts the microbial cells to get attached with the surface.

after 2 hours and *Vibrio parahaemolyticus* after 4 hours [1].

**2.1 Biofilm formation steps**

and sanitizers.

cells [4, 5].

**150**

Exopolysaccharide which is produced by the bacteria, are the major component of a biofilm. It constitutes about 50–90% of the total organic matter in a biofilm [6]. It is mainly composed of polysaccharides, some of which may neutral or polyanionic in case of Gram-negative bacteria or cationic as in case of Gram-positive bacteria. The anionic property of polysaccharide is confirmed by the presence of uronic acids (such as D-glucuronic, D-galacturonic, and mannuronic acids) or ketal-linked pyruvate. This anionic property plays an important role in the association of divalent cations like calcium and magnesium that have been shown to provide greater binding force in developed biofilm by cross-linking with polymer strands [1]. Along with the polysaccharide (which constitutes 1–2% of EPS), EPS also contains proteins [<1–2% (including enzymes)], DNA (<1%), RNA (<1%) as well as some lipids and humic substances [7]*.*

### **3.3 eDNA**

The microbial genetics and the environment in which bacteria grows are the determining factors for the composition of a biofilm. *Pseudomonas aeruginosa, Streptococcus intermedius*, *Enterococcus faecalis* and *Staphylococcus* are the species in which eDNA was initially observed.

One of the common mechanism by which eDNA is released is Autolysis. Released eDNA plays an important role in the development of the biofilm, biofilm structure stabilization as well as in gene transfer mechanisms. This genetic transfer is responsible for spreading of virulence and antibiotic resistance genes in circulating strains exposed to the selective pressure of medical treatment. *Streptococcus pneumonia* and related *Streptococci* are a good example of this [8].
