**5. Removal of biofilms**

Another anti-biofilm approach is the dissociation of the biofilm matrix which accounts for around 90% of biofilm dry mass. This dissociation will ultimately expose the sessile bacteria to the antibiotics as well as host immune defense. The enzymes majorly employed for biofilm matrix-degradation can be divided into three categories Proteases, nucleases and polysaccharide degrading enzymes [33].

Moreover, the surfactants also possess the antibiofilm activities as the cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Tween 20 have been found to promote the detachment or dispersal of biofilms. Surfactin; a biosurfactant produced by the *Bacillus subtilis* was shown to inhibit the biofilm dispersal in *E. coli*, *Proteus mirabilis,* and *S. typhimurium* [33].

### **6. Bacteriophages as antibiofilm agents**

Bacteriophages are considered as the largest creature in the biosphere, because of antibiotic resistance development, bacteriophages play an important role in the destruction of microbes. Use of bacteriophages is now considered as an alternative strategy to antibiotics, particularly for disruption or biofilm inhibition. Bacteriophages are beneficial than chemical agents and antibiotics. The isolation of bacteriophage is simple and fast, furthermore, its production is also cheap, and these are very distinct against a host or either host range, therefore, do not disrupt the normal flora. Bacteriophages are ecologically friendly, so with the persistence of host bacteria, they can replicate at the target site and have no adverse effects.

Bacteriophages also considered as potent antibiofilm mediators, e.g., phage T4 can cause infection and replicates within *E. coli* biofilms and by destroying microbial cells it can disturb the biofilm matrix. Doolittle and colleagues reported a study and demonstrated the interaction of phages with biofilms. The interaction among biofilm and phage is a dynamic as well as a sequential process. Phage adsorption with the target bacterial receptors is the significant phase in phage infection. The EPS matrix suggests a potent challenge for bacteriophages as EPS must be enough penetrated so that bacteriophages can attach with and reach to the particular host receptors. Furthermore, the EPS matrix also helps in the protection of bacteria in the biofilm. Moreover, by diffusion or through phage derived enzymes, for example, polysaccharide depolymerase can easily penetrate the EPS layer because these enzymes have the ability to destroy the structure of biofilm so that these phages can readily anchor to outer membrane receptors, lipopolysaccharides, or other proteins that are essential for replication process [34]. It is surely suggested that these phages induced depolymerizes can easily disrupt biofilms. Now genetically engineering for phages have been introduced that explicit biofilm degrading enzymes during infection. The scientist has engineered a gene namely "dispersion" (dspB) into an *E. coli* specific T7 phage to yield an engineered enzymatic phage, which shows more efficacy for the removal of biofilms as compared to non-cloned phages.

Despite the several benefits of phage use, there are some disadvantages also, for example, the release of a considerable amount of bacterial membrane-bound endotoxins, decreased number of phages encoding toxins, insufficient pharmacokinetic data and conversion of lytic phages to prophages is also a big concern. Some of the above-mentioned problems have been well determined through different processes like designing a recombinant phage from *Pseudomonas aeruginosa* filamentous phage to minimize the mortality rate in experimental animals and release of membrane-bound endotoxins to report the endotoxin release issue is major advances to overcome the above-mentioned concerns [35]. It has been observed that bacteriophages and antibiotics have a big potential to control biofilms such as phage PhilBB-PF 7A plays role in the removal of *Pseudomonas fluorescens* biomass and has shown almost 63–91% activity.

Different studies show some of the strongest inhibitions, for example, the existence of biofilm EPS matrix hindering the control of biofilm via antibacterial agents and higher antibiotic resistance can be controlled through phage use. Furthermore, there are many limitations of phage use such as microbial resistance to phages, virulence genes that are phage-encoded can incorporate inside the host bacterial genome and the narrow host range. Phage efficacy can also be reduced by the immune system, and phage preparations that are improperly obtained can also contain endotoxin. To control these obstacles engineered phages or phage mixtures can be an effective alternative. Moreover, after proper selection and several studies phages has become one of the most useful anti-biofilm agents.
