**Acknowledgements**

*Bacterial Biofilms*

biofilm formation and bacteria host interactions. Using a null NanA mutant and DANA, a substrate analog acting as competitive inhibitor, it was demonstrated that the downregulation of NanA or inhibition of its enzymatic activity affects biofilm

Another important protein target is GlmU, a bifunctional enzyme with acetyltransferase activity involved in the biosynthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a key precursor of β-1,6-N-acetyl-D-

then be an efficient method to disable the bacterium defense mechanism.

*S. aureus* is a common pathogen responsible for nosocomial and community infections being able to colonize the squamous epithelium of the anterior nares. One of the adhesins likely to be responsible for this ability is the *S. aureus* surface protein G (SasG), which promotes cellular aggregation leading to biofilm formation [53, 54]. SasG comprises an N-terminal A domain and repeated B domains with only the B domain required for the accumulation of biofilm. Expression of SasG does not increase the adherence of bacteria, and it is not involved in primary attachment but plays a role in the accumulation phase of biofilm formation [55]. For different aspects and playing different roles, NanA, GlmU, and SasG may all represent interesting targets to address the inhibition of biofilm production.

Currently, biofilm infections constitute a serious medical problem, and their treatment is far from being satisfactory. Biofilm formation inhibitors have several potential therapeutic applications as coatings in medical devices or in the prophylaxis of implanted surgery. In this respect, the identification of new strategies to counteract biofilm formation is a broad subject of study. The antibiofilm activity of many molecules such as proteins, peptides, and small organic molecules is currently under investigation. Each of these molecules is endowed with specific characteristics and can exert its ability to inhibit bacterial biofilm formation with different mechanisms. Antibiofilm agents are able to act both at the initial stages of biofilm formation, such as bacterial adhesion to the host surface, and on preformed biofilm, leading to the disruption of the EPS architecture. Many small organic molecules are able to interfere with the bacterial QS system, but their lack of activity in *in vivo* models and the high toxicity make these molecules of limited use in clinical

As antimicrobial peptides show a broad spectrum of action, exploiting different activities including antibiofilm capabilities, these molecules might be considered as new promising factors to impair biofilm formation that exploit different mecha-

glucosamine polysaccharide adhesin required for biofilm formation [50, 51]. GlmU is a possible factor involved in biofilm production in *M. smegmatis*, a nonpathogenic bacterium homologous to the pathogenic *M. tuberculosis*. The response of *M. smegmatis* to alkylating stress is different from *E. coli*, resulting in an increase in biofilm formation possibly due to a very strong defense mechanism. In this contest, GlmU has an important role in the process of biofilm production in *M. smegmatis*, being its expression highly upregulated when the bacterium needs to activate defense mechanisms [52]. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that the downregulation of GlmU decreased *M. smegmatis* capabilities to produce biofilm, whereas the overexpression of enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the *M. smegmatis* defense mechanisms. Focusing on the inhibition of GlmU might

formation and adhesion properties of *E. coli* [48, 49].

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applications.

nisms to hamper biofilms at different stages.

**5. Conclusions**

This work was supported in part by MIUR grants ARS01\_00597 Project "NAOCON" and PRIN 2017 "Identification and characterization of novel antitumoral/antimicrobial insect-derived peptides: a multidisciplinary, integrated approach from in silico to *in vivo*."
