Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

SUMMARY: Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function.

The cell wall (CW) of bacteria is an intricate arrangement of macromolecules, at least constituted of peptidoglycan (PG) but also of (lipo)teichoic acids, various polysaccharides, polyglutamate and/or proteins. During bacterial growth and division, there is a constant balance between CW degradation and biosynthesis. The CW is remodeled by bacterial hydrolases, whose activities are carefully regulated to maintain cell integrity or lead to bacterial death. Each cell wall hydrolase (CWH) has a specific role regarding the PG: (i) cell wall amidase (CWA) cleaves the amide bond between N-acetylmuramic acid and L-alanine residue at the N-terminal of the stem peptide, (ii) cell wall glycosidase (CWG) catalyses the hydrolysis of the glycosidic linkages, whereas (iii) cell wall peptidase (CWP) cleaves amide bonds between amino acids within the PG chain. After an exhaustive overview of all known conserved catalytic domains responsible for CWA, CWG, and CWP activities, this review stresses that the CWHs frequently display a modular architecture combining multiple and/or different catalytic domains, including some lytic transglycosylases as well as CW binding domains. From there, direct physiological and collateral roles of CWHs in bacterial cells are further discussed.

A community-based sessile life style is the normal mode of growth and survival for many bacterial species. Under such conditions, cell-to-cell interactions are inevitable and ultimately lead to the establishment of dense, complex and highly structured biofilm populations encapsulated in a self-produced extracellular matrix and capable of coordinated and collective behavior. Remarkably, in food processing environments, a variety of different bacteria may attach to surfaces, survive, grow and form biofilms. Salmonella enterica, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus are important bacterial pathogens commonly implicated in outbreaks of foodborne diseases, while all are known to be able to create biofilms on both abiotic and biotic surfaces. Particularly challenging is the attempt to understand the complexity of inter-bacterial interactions that can be encountered in such unwanted consortia, such as competitive and cooperative ones, together with their impact on the final outcome of these communities (e.g. maturation, physiology, antimicrobial resistance, virulence, dispersal). In this review, up-to-date data on both the intra- and inter-species interactions encountered in biofilms of these pathogens are presented. A better understanding of these interactions, both at molecular and biophysical levels, could lead to novel intervention strategies for controlling pathogenic biofilm formation in food processing environments and thus improve food safety.