Salicylic acid-based poly(anhydride esters) for control of biofilm formation in Salmonella enterica serovar Typhimurium

Aims: Bacterial biofilms generally are more resistant to stresses as compared with free planktonic cells. Therefore, the discovery of antimicrobial stress factors that have strong inhibitory effects on bacterial biofilm formation would have great impact on the food, personal care, and medical industries. Methods and Results: Salicylate‐based poly(anhydride esters) (PAE) have previously been shown to inhibit biofilm formation, possibly by affecting surface attachment. Our research evaluated the effect of salicylate‐based PAE on biofilm‐forming Salmonella enterica serovar Typhimurium. To remove factors associated with surface physical and chemical parameters, we utilized a strain that forms biofilms at the air–liquid interface. Surface properties can influence biofilm characteristics, so the lack of attachment to a solid surface eliminates those constraints. The results indicate that the salicylic acid‐based polymers do interfere with biofilm formation, as a clear difference was seen between bacterial strains that form biofilms at the air–liquid interface (top‐forming) and those that form at the surface–liquid interface (bottom‐forming). Conclusion: These results lead to the conclusion that the polymers may not interfere with attachment; rather, the polymers likely affect another mechanism essential for biofilm formation in Salmonella. Significance and Impact of the study: Biofilm formation can be prevented through controlled release of nature‐derived antimicrobials formulated into polymer systems.     

vpsA- and luxO-independent biofilms of Vibrio cholerae

The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.     

The Role of Exopolymers Produced by Sphingomonas paucimobilis in Biofilm Formation and Composition

Exopolymers have been associated with the initial adhesion of bacteria, which is the primary step for biofilm formation. Moreover, the polymeric matrix of biofilms has a considerable influence on some of the most important physical and physiological properties of biofilms. The role of extracellular polymers in biofilm formation was studied using three mutants of Sphingomonas paucimobilis with increasing capabilities for exopolymer production. The physical, biochemical and physiological properties of three different layers of each biofilm were determined. The layers were detached by submitting the biofilm to increasing shear stress. The results revealed that the presence of exopolymers in the growth medium was essential for biofilm formation. The mutant producing the highest amount of exopolymer formed very thick biofilms, while the biofilms formed by the medium exopolymer producer were on average 8 times thinner. The lowest exopolymer producer did not form biofilm. In both types of biofilms, exopolymer density increased with depth, although this tendency was more significant in thinner biofilms. Cell distribution was also more heterogeneous in thinner biofilms, exhibiting a greater accumulation of cells in the inner layers. The thicker biofilms had very low activity in the inner layer. This was related to a high accumulation of proteins and DNA in this layer due to cell lysis and hydrolytic activity. Activity in the thin biofilm was constant throughout its depth, suggesting that there was no nutrient limitation. The production of exopolymers by each cell was constant throughout the depth of the biofilms, although it was greater in the case of the higher producer.     

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO₂ sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.     

Mini-review: convection around biofilms

Water that flows around a biofilm influences the transport of solutes into and out of the biofilm and applies forces to the biofilm that can cause it to deform and detach. Engineering approaches to quantifying and understanding these phenomena are reviewed in the context of biofilm systems. The slow-moving fluid adjacent to the biofilm acts as an insulator for diffusive exchange. External mass transfer resistance is important because it can exacerbate oxygen or nutrient limitation in biofilms, worsen product inhibition, affect quorum sensing, and contribute to the development of tall, fingerlike biofilm clusters. Measurements of fluid motion around biofilms by particle velocimetry and magnetic resonance imaging indicate that water flows around, but not through biofilm cell clusters. Moving fluid applies forces to biofilms resulting in diverse outcomes including viscoelastic deformation, rolling, development of streamers, oscillatory movement, and material failure or detachment. The primary force applied to the biofilm is a shear force in the main direction of fluid flow, but complex hydrodynamics including eddies, vortex streets, turbulent wakes, and turbulent bursts result in additional force components.     

Environmental factors that shape biofilm formation

Cells respond to the environment and alter gene expression. Recent studies have revealed the social aspects of bacterial life, such as biofilm formation. Biofilm formation is largely affected by the environment, and the mechanisms by which the gene expression of individual cells affects biofilm development have attracted interest. Environmental factors determine the cell’s decision to form or leave a biofilm. In addition, the biofilm structure largely depends on the environment, implying that biofilms are shaped to adapt to local conditions. Second messengers such as cAMP and c-di-GMP are key factors that link environmental factors with gene regulation. Cell-to-cell communication is also an important factor in shaping the biofilm. In this short review, we will introduce the basics of biofilm formation and further discuss environmental factors that shape biofilm formation. Finally, the state-of-the-art tools that allow us investigate biofilms under various conditions are discussed.     

环境因子对水霉菌生物膜形成的影响

【目的】体外构建水霉菌(Saprolegnia)生物膜(Biofilm,BF),研究环境因子对其生物膜形成的影响。【方法】采用改良的微孔板法研究静置培养条件下寄生水霉(Saprolegnia parasitica)ATCC200013在96孔酶标板上的成膜情况,CCK-8法(Cell Counting Kit-8)定量检测生物膜中水霉菌的活力。【结果】水霉菌的生物膜的OD450值在培养24 h达到峰值,48 h后趋于稳定。随着初始孢子浓度升高,水霉菌生物膜OD450值升高,差异显著(P0.05)。20-25°C生物膜形成量最多,OD450值显著高于其他温度组(P0.05)。在起始pH值为4-11的沙氏葡萄糖液体培养基中,水霉菌均能形成生物膜。在培养基中加入0.12 mmol/L以上CaCl2,能促进生物膜形成;添加0.03-2.00 mmol/L MgCl2,水霉菌生物膜形成量与未添加MgCl2对照组无显著性差异;Cu2+对水霉菌生物膜的形成有显著影响,0.5 mmol/L以上添加处理几乎不形成生物膜;NaCl能明显抑制水霉菌生物膜的形成,当NaCl质量分数低于0.12%时,对生物膜形成的影响较小(P0.05)。水霉菌在鲫皮和肌肉提取液包被后生物膜形成量与对照组相比无显著性差异,而鲫表皮黏液、鳃黏液包被后生物膜的形成量明显减少。【结论】研究首次采用微孔板法体外构建水霉菌生物膜,发现其生物膜的形成与多种环境因素有着密切的关系。这为了解水霉菌生物膜的形成规律提供了一定参考,为水霉菌生物膜的进一步研究奠定了基础。     

Impacts of hydrodynamic conditions and microscale surface roughness on the critical shear stress to develop and thickness of early-stage Pseudomonas putida biofilms

Biofilms can increase pathogenic contamination of drinking water, cause biofilm-related diseases, alter the sediment erosion rate, and degrade contaminants in wastewater. Compared with mature biofilms, biofilms in the early-stage have been shown to be more susceptible to antimicrobials and easier to remove. Mechanistic understanding of physical factors controlling early-stage biofilm growth is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and microscale surface roughness on the development of early-stage Pseudomonas putida biofilm through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that early-stage biofilm growth is suppressed under high flow conditions and that the local velocity for early-stage P. putida biofilms (growth time < 14 h) to develop is about 50 μm/s, which is similar to P. putida's swimming speed. We further illustrate that microscale surface roughness promotes the growth of early-stage biofilms by increasing the area of the low-flow region. Furthermore, we show that the critical average shear stress, above which early-stage biofilms cease to form, is 0.9 Pa for rough surfaces, three times as large as the value for flat or smooth surfaces (0.3 Pa). The important control of flow conditions and microscale surface roughness on early-stage biofilm development, characterized in this study, will facilitate future predictions and managements of early-stage P. putida biofilm development on the surfaces of drinking water pipelines, bioreactors, and sediments in aquatic environments.     

Protists with different feeding modes change biofilm morphology

The effect of Dexiostoma (filter feeder), Vannella, Chilodonella (raptorial feeders), Spumella , and Neobodo (direct interception feeders) on the morphology of multispecies bacterial biofilms was investigated in small flow cells. The filter feeder Dexiostoma campylum did not alter biofilm volume and porosity but stimulated the formation of larger microcolonies compared with ungrazed biofilms. In contrast, the raptorial feeder Vannella sp. efficiently grazed bacteria from the biofilm surface, leading to smaller microcolonies and lower maximal and basal layer thickness compared with ungrazed biofilms. Microcolony formation was not stimulated in the presence of the sessile Spumella sp. Chilodonella uncinata rasped bacteria from the outer surface leading to mushroom-shaped microcolonies. In the presence of C. uncinata and Spumella sp., the biofilm volume was 2.5–6.3 times lower compared with ungrazed biofilms. However, the biofilm porosity and the ratio of biofilm surface area to biofilm volume were 1.5–3.7 and 1.2–1.8 times higher, respectively. Thus, exchange of nutrients and gases between the biofilm and its surrounding fluid should also be improved in deeper biofilm layers, hence accelerating microbial growth.     

Mycoplasma biofilms ex vivo and in vivo

Biofilms are communities of microorganisms that are encased in polymeric matrixes and grow attached to biotic or abiotic surfaces. Despite their enhanced ability to resist antimicrobials and components of the immune system in vitro , few studies have addressed the interactions of biofilms with the host at the organ level. Although mycoplasmas have been shown to form biofilms on glass and plastic surfaces, it has not been determined whether they form biofilms on the tracheal epithelium. We developed a tracheal organ-mounting system that allowed the entire surface of the tracheal lumen to be scanned using fluorescence microscopy. We observed the biofilms formed by the murine respiratory pathogen Mycoplasma pulmonis on the epithelium of trachea in tracheal organ culture and in experimentally infected mice and found similar structure and biological characteristics as biofilms formed in vitro . This tracheal organ-mounting system can be used to study interactions between biofilms formed by respiratory pathogens and the host epithelium and to identify the factors that contribute to biofilm formation in vivo .     

Influence of Hydrodynamics and Cell Signaling on the Structure and Behavior of Pseudomonas aeruginosa Biofilms

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.     

Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.     

Spermidine regulates Vibrio cholerae biofilm formation via transport and signaling pathways

Vibrio cholerae , the causative agent of the devastating diarrheal disease cholera, can form biofilms on diverse biotic and abiotic surfaces. Biofilm formation is important for the survival of this organism both in its natural environment and in the human host. Development of V. cholerae biofilms are regulated by complex regulatory networks that respond to environmental signals. One of these signals, norspermidine, is a polyamine that enhances biofilm formation via the NspS/MbaA signaling system. In this work, we have investigated the role of the polyamine spermidine in regulating biofilm formation in V. cholerae . We show that spermidine import requires PotD1, an ortholog of the periplasmic substrate-binding protein of the spermidine transport system in Escherichia coli . We also show that deletion of the potD1 gene results in a significant increase in biofilm formation. We hypothesize that spermidine imported into the cell hinders biofilm formation. Exogenous spermidine further reduces biofilm formation in a PotD1-independent, but NspS/MbaA-dependent, manner. Our results suggest that polyamines affect biofilm formation in V. cholerae via multiple pathways involving both transport and signaling networks.     

Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB

The force-deflection and removal characteristics of bacterial biofilm were measured by two different techniques before and after chemical, or enzymatic, treatment. The first technique involved time lapse imaging of a biofilm grown in a capillary flow cell and subjected to a brief shear stress challenge imparted through increased fluid flow. Biofilm removal was determined by calculating the reduction in biofilm area from quantitative analysis of transmission images. The second technique was based on micro-indentation using an atomic force microscope. In both cases, biofilms formed by Staphylococcus epidermidis were exposed to buffer (untreated control), urea, chlorhexidine, iron chloride, or DispersinB. In control experiments, the biofilm exhibited force-deflection responses that were similar before and after the same treatment. The biofilm structure was stable during the post-treatment shear challenge (1% loss). Biofilms treated with chlorhexidine became less deformable after treatment and no increase in biomass removal was seen during the post-treatment shear challenge (2% loss). In contrast, biofilms treated with urea or DispersinB became more deformable and exhibited significant biofilm loss during the post-treatment flow challenge (71% and 40%, respectively). During the treatment soak phase, biofilms exposed to urea swelled. Biofilms exposed to iron chloride showed little difference from the control other than slight contraction during the treatment soak. These observations suggest the following interpretations: (1) chemical or enzymatic treatments, including those that are not frankly antimicrobial, can alter the cohesion of bacterial biofilm; (2) biocidal treatments (e.g., chlorhexidine) do not necessarily weaken the biofilm; and (3) biofilm removal following treatment with agents that make the biofilm more deformable (e.g., urea, DispersinB) depend on interaction between the moving fluid and the biofilm structure. Measurements such as those reported here open the door to development of new technologies for controlling detrimental biofilms by targeting biofilm cohesion rather than killing microorganisms.     

Biofilm formation by avian Escherichia coli in relation to media, source and phylogeny

AIMS: To assess the abilities of 105 avian pathogenic Escherichia coli (APEC) and 103 avian faecal commensal E. coli (AFEC) to form biofilms on a plastic surface and to investigate the possible association of biofilm formation with the phylotype of these isolates. METHODS AND RESULTS: Biofilm production was assessed in 96-well microtitre plates using three different media, namely, M63 minimal medium supplemented with glucose and casamino acids, brain-heart infusion broth, and diluted tryptic soy broth. Avian E. coli are highly variable in their ability to form biofilms. In fact, no strain produced a strong biofilm in all three types of media; however, most (75.7% AFEC and 55.2% APEC) were able to form a moderate or strong biofilm in at least one medium. Biofilm formation in APEC seems to be mostly limited to nutrient deplete media; whereas, AFEC are able to form biofilms in both nutrient deplete and replete media. Also, biofilm formation in E. coli from phylogenetic groups B2, D and B1 was induced by nutrient deplete conditions; whereas, biofilm formation by members of phylogenetic group A was strongest in a rich medium. CONCLUSIONS: Biofilm formation by APEC and phylotypes B2, D and B1 is induced by nutrient deplete conditions, while AFEC are able to form biofilms in both nutrient rich and deplete media. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to investigate biofilm formation by a large sample of avian E. coli isolates, and it provides insight into the conditions that induce biofilm formation in relation to the source (APEC or AFEC) and phylogenetic group (A, B1, B2 and D) of an isolate.     

In vitro effect of clarithromycin and alginate lyase against helicobacter pylori biofilm

It is now established that the gastric pathogen Helicobacter pylori has the ability to form biofilms in vitro as well as on the human gastric mucosa. The aim of this study is to evaluate the antimicrobial effects of Clarithromycin on H. pylori biofilm and to enhance the effects of this antibiotic by combining it with Alginate Lyase, an enzyme degrading the polysaccharides present in the extracellular polymeric matrix forming the biofilm. We evaluated the Clarithromycin minimum inhibition concentration (MIC) on in vitro preformed biofilm of a H. pylori. Then the synergic effect of Clarithromycin and Alginate Lyase treatment has been quantified by using the Fractional Inhibitory Concentration index, measured by checkerboard microdilution assay. To clarify the mechanisms behind the effectiveness of this antibiofilm therapeutic combination, we used Atomic Force Microscopy to analyze modifications of bacterial morphology, percentage of bacillary or coccoid shaped bacteria cells and to quantify biofilm properties. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1584–1591, 2016     

Metabolic dynamics of Desulfovibrio vulgaris biofilm grown on a steel surface

In this study, a comparative metabolomics approach combining gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) was applied first between planktonic cells and biofilms and then between pure cultures and biofilms of Desulfovibrio vulgaris. The results revealed that the overall metabolic level of the biofilm cells was down-regulated, especially for metabolites related to the central carbon metabolism, compared to the planktonic cells and the pure culture of D. vulgaris. In addition, pathway enrichment analysis of the 58 metabolites identified by GC-MS showed that fatty acid biosynthesis in the biofilm cells was up-regulated, suggesting that fatty acids may be important for the formation, maintenance and function of D. vulgaris biofilm. This study offers a valuable perspective on the metabolic dynamics of the D. vulgaris biofilm.     

Biofilm formation by ica-positive and ica-negative strains of Staphylococcus epidermidis in vitro

Staphylococcus epidermidis is a clinically important opportunistic pathogen that forms biofilm infections on nearly all types of indwelling medical devices. The biofilm forming capability of S. epidermidis has been linked to the presence of the ica operon in the genome, and the amount of biofilm formation measured by the crystal violet (CV) adherence assay. Six S. epidermidis strains were characterized for their ica status using PCR, and their biofilm forming ability over 6 days, using the CV assay and a flow cell system. Ica-negative strains characterized as ‘negative for biofilm formation’ based on the CV assay were demonstrated to form strongly attached biofilms after 6 days. However, the biofilms were not as extensive as the ica-positive strains. It was concluded that ica is not required for biofilm formation, nor is the 24-h CV assay generalizable for predicting the 6-day biofilm-forming ability for all S. epidermidis strains.     

Spent media from cultures of environmental isolates of Escherichia coli can suppress the deficiency of biofilm formation under anoxic conditions of laboratory E. coli strains

The prevailing lifestyle of bacteria is sessile and they attach to surfaces in structures known as biofilms. In Escherichia coli, as in many other bacteria, biofilms are formed at the air-liquid interface, suggesting that oxygen has a critical role in the biofilm formation process. It has been reported that anaerobically growing E. coli laboratory strains are unable to form biofilms even after 96 h of incubation on Luria Bertani (LB) medium. After analyzing 22,000 transposon-induced and 26,000 chemically-induced mutants we failed to isolate an E. coli laboratory strain with the ability to form biofilm under anaerobic growth conditions. Notably, seven strains from a collection of E. coli isolated from different hosts and the environment had the ability to form biofilm in the absence of oxygen. Interestingly, spent medium from cultures of one strain, Souza298, can promote biofilm formation of E. coli laboratory strains growing under anaerobic conditions. Our results led us to propose that laboratory E. coli strains do not release (or synthesize) a molecule needed for biofilm formation under anoxic conditions but that they bear all the required machinery needed for this process.