Microstructural characteristics of thin biofilms through optical and scanning electron microscopy

The combination of a conventional optical microscope with a specially designed glass flow cell was used to visualize in situ biofilms formed on opaque thin biomaterials through a simple non-invasive way (optical microscopy of thin biofilms, OMTB). Comparisons of OMTB with scanning electron microscopy (SEM) images were made. Thin metallic dental biomaterials were used as substrata. They were immersed in a synthetic saliva and in a modified Mitis–Salivarius medium inoculated with a consortium of oral microorganisms. To study the effect of bacterial motility, Pseudomonas fluorescens cultures were also used. The processes which give rise to the formation of the biofilm were monitored through OMTB. Biofilm microstructures like pores, water channels, streamers and chains of Streptococci, attached to the surface or floating in the viscous interfacial environment, could be distinguished. Thickness and roughness of the biofilms formed on thin substrata could also be evaluated. Distortions introduced by pretreatments carried out to prepare biological materials for SEM observations could be detected by comparing OMTB and SEM images. SEM images (obtained at high magnification but ex situ, not in real time and with pretreatment of the samples) and OMTB images (obtained in situ, without pretreatments, in real time but at low magnification) in combination provided complementary information to study biofilm processes on thin substrata.     

Retention of bacteria on a substratum surface with micro-patterned hydrophobicity

Bacteria adhere to almost any surface, despite continuing arguments about the importance of physico-chemical properties of substratum surfaces, such as hydrophobicity and charge in biofilm formation. Nevertheless, in vivo biofilm formation on teeth and also on voice prostheses in laryngectomized patients is less on hydrophobic than on hydrophilic surfaces. With the aid of micro-patterned surfaces consisting of 10-microm wide hydrophobic lines separated by 20-microm wide hydrophilic spacings, we demonstrate here, for the first time in one and the same experiment, that bacteria do not have a strong preference for adhesion to hydrophobic or hydrophilic surfaces. Upon challenging the adhering bacteria, after deposition in a parallel plate flow chamber, with a high detachment force, however, bacteria were easily wiped-off hydrophobic lines, most notably when these lines were oriented parallel to the direction of flow. Adhering bacteria detached slightly less from the hydrophilic spacings in between, but preferentially accumulated adhering on the hydrophilic regions close to the interface between the hydrophilic spacings and hydrophobic lines. It is concluded that substratum hydrophobicity is a major determinant of bacterial retention while it hardly influences bacterial adhesion.     

A different path: Revealing the function of staphylococcal proteins in biofilm formation

Staphylococcus aureus and Staphylococcus epidermidis cause dangerous and difficult to treat medical device-related infections through their ability to form biofilms. Extracellular poly-N-acetylglucosamine (PNAG) facilitates biofilm formation and is a vaccination target, yet details of its biosynthesis by the icaADBC gene products is limited. IcaC is the proposed transporter for PNAG export, however a comparison of the Ica proteins to homologous exo-polysaccharide synthases suggests that the common IcaAD protein components both synthesise and transport the PNAG. The limited distribution of icaC to the Staphylococcaceae and its membership of a family of membrane-bound acyltransferases, leads us to suggest that IcaC is responsible for the known O-succinylation of PNAG that occurs in staphylococci, identifying a potentially new therapeutic target specific for these bacteria.     

Formation and dimerization of the phosphodiesterase active site of the Pseudomonas aeruginosa MorA,a bi-functional c-di-GMP regulator

Diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively synthesise and hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), and both activities are often found in a single protein. Intracellular c-di-GMP levels in turn regulate bacterial motility, virulence and biofilm formation. We report the first structure of a tandem DGC–PDE fragment, in which the catalytic domains are shown to be active. Two phosphodiesterase states are distinguished by active site formation. The structures, in the presence or absence of c-di-GMP, suggest that dimerisation and binding pocket formation are linked, with dimerisation being required for catalytic activity. An understanding of PDE activation is important, as biofilm dispersal via c-di-GMP hydrolysis has therapeutic effects on chronic infections.     

In vitro and in vivo model systems to study microbial biofilm formation

Biofilm formation is often considered the underlying reason why treatment with an antimicrobial agent fails and as an estimated 65-80% of all human infections is thought to be biofilm-related, this presents a serious challenge. Biofilm model systems are essential to gain a better understanding of the mechanisms involved in biofilm formation and resistance. In this review a comprehensive overview of various in vitro and in vivo systems is presented, and their advantages and disadvantages are discussed.     

The C-terminal amphipathic α-helix of Pseudomonas aeruginosa PelC outer membrane protein is required for its function

Pseudomonas aeruginosa is an opportunistic pathogen, which causes numerous infections and can adopt a versatile lifestyle. During chronic infection, P. aeruginosa becomes established as a bacterial community known as a biofilm. Biofilm formation results from the production of a matrix mainly comprised of exopolysaccharides. P. aeruginosa possesses several gene clusters which contribute to the formation of the matrix, including the pel genes. Among the pel genes, pelC encodes an outer membrane protein, which may serve as a transporter of polysaccharide to the bacterial cell surface. Whereas outer membrane proteins usually display an amphipathic β-barrel fold, we show that PelC requires a C-terminal amphipathic α-helix for outer membrane insertion and function. Such a structural feature has only previously been reported for the Wza outer membrane protein of Escherichia coli, and our data suggest that this characteristic may be found in a large family of proteins, particularly outer membrane proteins specialized in polysaccharide transport.     

Biofilm-induced modifications in the proteome of Pseudomonas aeruginosa planktonic cells

While recent studies focused on Quorum Sensing (QS) role in the cell-to-cell communication in free or biofilm cultures, no work has been devoted up to now to investigate the communication between sessile and planktonic bacteria. In this aim, we elaborated an original two-chambered bioreactor and used a proteomic approach to study the alterations induced by Pseudomonas aeruginosa biofilm cells on protein expression in planktonic counterparts (named SIPs for Surface-Influenced Planktonics). Proteomic analyses revealed the existence of 31 proteins whose amount varied in SIPs, among which five corresponded to hypothetic proteins and two (the Fur and BCP proteins) are involved in bacterial response to oxidative stress. An increase in the concentration of C₄-HSL (rhlR–rhlI-dependent QS) and 3-oxo-C₁₂-HSL (lasR–lasI-dependent QS) autoinducer molecules was shown in the planktonic compartment. Interestingly, among proteins that were accumulated by SIPs was 3-oxoacyl-[acyl-carrier-protein] reductase, a protein involved in the production of the autoinducer 3-oxo-C₁₂-HSL. These results demonstrate that planktonic organisms are able to detect the presence of a biofilm in their close environment and to modify their gene expression in consequence.     

Study of the initial phase of biofilm formation using a biofomic approach

Aims

The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test® and the Crystal violet staining technique.

Methods and results

Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test® and after 6 h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility.

Conclusions

Our results show that the BioFilm Ring Test® is a method specially suited for the study of the initial phase of the formation of a biofilm.

Significance and impact of study

The BioFilm Ring Test® is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.