Romain Briandet

Resistance of bacterial biofilms to disinfectants: a review

A biofilm can be defined as a community of microorganisms adhering to a surface and surrounded by a complex matrix of extrapolymeric substances. It is now generally accepted that the biofilm growth mode induces microbial resistance to disinfection that can lead to substantial economic and health concerns. Although the precise origin of such resistance remains unclear, different studies have shown that it is a multifactorial process involving the spatial organization of the biofilm. This review will discuss the mechanisms identified as playing a role in biofilm resistance to disinfectants, as well as novel anti-biofilm strategies that have recently been explored.

Biofilm-associated persistence of food-borne pathogens

Microbial life abounds on surfaces in both natural and industrial environments, one of which is the food industry. A solid substrate, water and some nutrients are sufficient to allow the construction of a microbial fortress, a so-called biofilm. Survival strategies developed by these surface-associated ecosystems are beginning to be deciphered in the context of rudimentary laboratory biofilms. Gelatinous organic matrices consisting of complex mixtures of self-produced biopolymers ensure the cohesion of these biological structures and contribute to their resistance and persistence. Moreover, far from being just simple three-dimensional assemblies of identical cells, biofilms are composed of heterogeneous sub-populations with distinctive behaviours that contribute to their global ecological success. In the clinical field, biofilm-associated infections (BAI) are known to trigger chronic infections that require dedicated therapies. A similar belief emerging in the food industry, where biofilm tolerance to environmental stresses, including cleaning and disinfection/sanitation, can result in the persistence of bacterial pathogens and the recurrent cross-contamination of food products. The present review focuses on the principal mechanisms involved in the formation of biofilms of food-borne pathogens, where biofilm behaviour is driven by its three-dimensional heterogeneity and by species interactions within these biostructures, and we look at some emergent control strategies.

Involvement of motility and flagella in Bacillus cereus biofilm formation.

Bacillus cereus is a food-borne pathogen and a frequent contaminant of food production plants. The persistence of this pathogen in various environments results from the formation of spores and of biofilms. To investigate the role of the B. cereus flagellar apparatus in biofilm formation, we constructed a non-flagellated mutant and a flagellated but non-motile mutant. Unexpectedly, we found that the presence of flagella decreased the adhesion of the bacterium to glass surfaces. We hypothesize that this decrease is a consequence of the flagella hindering a direct interaction between the bacterial cell wall and the surface. In contrast, in specific conditions, motility promotes biofilm formation. Our results suggest that motility could influence biofilm formation by three mechanisms. Motility is necessary for the bacteria to reach surfaces suitable for biofilm formation. In static conditions, reaching the air-liquid interface, where the biofilm forms, is a strong requirement, whereas in flow cells bacteria can have access to the bottom glass slide by sedimentation. Therefore, motility is important for biofilm formation in glass tubes and in microtitre plates, but not in flow cells. Motility also promotes recruitment of planktonic cells within the biofilm by allowing motile bacteria to invade the whole biofilm. Finally, motility is involved in the spreading of the biofilm on glass surfaces.

Listeria monocytogenes EGD-e Biofilms: No Mushrooms but a Network of Knitted Chains

ABSTRACT Listeria monocytogenes is a food pathogen that can attach on most of the surfaces encountered in the food industry. Biofilms are three-dimensional microbial structures that facilitate the persistence of pathogens on surfaces, their resistance toward antimicrobials, and the final contamination of processed goods. So far, little is known about the structural dynamics of L. monocytogenes biofilm formation and its regulation. The aims of this study were, by combining genetics and time-lapse laser-scanning confocal microscopy (LSCM), (i) to characterize the structural dynamics of L. monocytogenes EGD-e sessile growth in two nutritional environments (with or without a nutrient flow), and (ii) to evaluate the possible role of the L. monocytogenes agr system during biofilm formation by tracking the spatiotemporal fluorescence expression of a green fluorescent protein (GFP) reporter system. In the absence of nutrient flow (static conditions), unstructured biofilms composed of a few layers of cells that covered the substratum were observed. In contrast, when grown under dynamic conditions, L. monocytogenes EGD-e biofilms were highly organized. Indeed, ball-shaped microcolonies were surrounded by a network of knitted chains. The spatiotemporal tracking of fluorescence emitted by the GFP reporter system revealed that agr expression was barely detectable under static conditions, but it progressively increased during 40 h under dynamic conditions. Moreover, spatial analysis revealed that agr was expressed preferentially in cells located outside the microcolonies. Finally, the in-frame deletion of agrA, which encodes a transcriptional regulator, resulted in a decrease in initial adherence without affecting the subsequent biofilm development.

The biofilm architecture of sixty opportunistic pathogens deciphered using a high throughput CLSM method.

This study proposes a high throughput method based on Confocal Laser Scanning Microscopy (CLSM) combined with the use of 96-wells microtiter plates compatible with high resolution imaging for the study of biofilm formation and structure. As an illustration, the three-dimensional structures of biofilms formed by 60 opportunistic pathogens were thus observed and quantified. The results revealed the diversity of biofilm architectures. Specific spatial arrangement such as the mushroom-like structures already described for Pseudomonas aeruginosa was observed. Other features, such as hollow voids in microcolonies of Salmonella enterica strain Agona, were identified for the first time. The combined use of microplates and confocal imaging proved to be a good alternative to the other high throughput methods commonly used as it enables the direct, insitu, qualitative and quantitative characterization of biofilm architecture. This high content method should lead to a clearer understanding of the structure-function relationships implicated in biofilms traits.

Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms.

ABSTRACT In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as “active” phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.

Effects of the Growth Procedure on the Surface Hydrophobicity of Listeria monocytogenes Cells and Their Adhesion to Stainless Steel

The aim of this study was to examine the physicochemical surface properties and the ability to adhere to stainless steel of three strains of Listeria monocytogenes after different cultivation procedures. To this end, bacteria were cultivated at 37 degrees C after storage at two frequently used temperatures (4 degrees C or -80 degrees C) and were then transferred into the liquid medium (trypticase soy broth supplemented with 6 g liter(-1) of yeast extract, pH 7.3) between one and four times. In addition, the influence of supplementing the growth medium with lactic acid was explored, this organic acid being representative of both the dairy and cured meat industries. The hydrophobic/hydrophilic and electron-acceptor/electron-donor characteristics of the strains were evaluated by the microbial adhesion to solvents method. Using this technique, we recorded an increase in the hydrophobic properties of one strain stored at 4 degrees C, with an increasing number of transfers in the media (P < 0.05). Another plant-isolated strain appeared more hydrophobic and stuck better to stainless steel when cells were stored at 4 degrees C rather than at -80 degrees C. Preculturing L. monocytogenes in a lactic acid-supplemented medium increased the affinity of microbial cells to solvents and the bacterial attachment to stainless steel (P < 0.05).

Bacterial swimmers that infiltrate and take over the biofilm matrix

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.

Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation.

Fluorescence correlation spectroscopy (FCS) under two‐photon excitation was applied successfully to characterize the penetration and diffusion capabilities of fluorescent probes (latex beads and fluorescein isothiocyanate–dextran) of different size and electrical charge in two models of monomicrobial biofilms with low (Lactococcus lactis biofilm) or high (Stenotrophonas maltophilia biofilm) contents of extracellular polymeric substance (EPS). FCS measurements performed on each biofilm can show deviation from Brownian diffusion, depending on the local structure of the biofilm and the fluorophore size. In this case, we fitted the data to an anomalous diffusion model and determined apparent diffusion coefficients, which can be 50 times smaller than the values in aqueous solutions. This result was interpreted as steric hindrance of the diffusion of the fluorescent particles within the biofilm that can lead to a total inhibition as observed particularly in the mushroom‐like structure of the S. maltophilia biofilm. Alternatively, mechanisms for the absence of FCS signal behavior were related to attractive electrostatic interactions between cationic particles and negatively charged bacteria or to specific interactions between dextrans and EPS of the biofilm matrix.

Positive role of peptidoglycan breaks in lactococcal biofilm formation

Bacterial attachment to solid matrices depends on adhesive molecules present on the cell surface. Here we establish a positive correlation between peptidoglycan (PG) breaks, rather than particular molecules, and biofilm‐forming capacity in the Gram‐positive bacterium Lactococcus lactis. The L. lactis acmA strain, which is defective in PG hydrolase, adhered less efficiently than the wild‐type (wt) strain to different solid surfaces and was unable to form biofilms. These phenotypes were abolished by addition of lysozyme, a PG hydrolytic enzyme. Thus, the presence of PG breaks introduced by PG hydrolase, and not the AcmA protein itself, appears to be responsible for biofilm formation. Two different genetic screens confirmed the importance of PG breaks in L. lactis biofilm formation. Using the chain‐forming ability of the acmA strain as a phenotypic indicator of PG integrity, we selected for insertional mutants generating short chains. Five independent mutants were all mapped to ponA, which encodes the PG synthesis enzyme PBP1A. Double acmA ponA mutants displayed increased adhesion and biofilm‐forming capacity. Direct selection for strains with increased biofilm‐forming capacity resulted in the isolation of another five mutations in ponA. Based on these results, we conclude that PG breaks are important for both adhesion and biofilm formation in L. lactis.