Diversity in biofilm formation and production of curli fimbriae and cellulose of Salmonella Typhimurium strains of different origin in high and low nutrient medium

The biofilm forming behavior of 51 Salmonella Typhimurium strains was determined in Tryptone Soya Broth (TSB) and 20 times diluted TSB (1/20TSB) at 25°C and 37°C. The results indicated that biofilm forming behavior is influenced by environmental conditions and associated with the origin of the strains. Clinical, outbreak-associated and retail product isolates showed dense biofilm formation in both media at 25°C, and in TSB also at 37°C. However, industrial isolates only showed dense biofilm formation in 1/20TSB at 25°C. By enumeration of biofilm cells, LIVE/DEAD staining and SEM analysis of biofilms it was found that the ratio of cells and extracellular matrix is affected by environmental conditions. Indeed, the genes involved in curli fimbriae and cellulose production are highly induced during biofilm formation at 25°C in 1/20TSB. This indicates that these are important matrix components during biofilm formation in 1/20TSB at 25°C and that other factors contribute to biofilm formation of clinical, outbreak-associated and retail product isolates at 37°C and/or nutrient-rich conditions.     

Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties

Bacterial biofilms are communities of bacteria entangled in a self‐produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid‐polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum‐of‐the‐parts ¹³C solid‐state nuclear magnetic resonance (NMR) analysis to define the curli‐to‐pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well‐studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid‐air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic‐associated) and the strain itself.     

In vitro effect of subinhibitory concentrations of antibiotics on biofilm formation by clinical strains of Salmonella enterica serovar Typhimurium isolated in Slovakia

Aims: In this study, we examined the biofilm formation of 75 Salmonella enterica serovar Typhimurium (Salm. Typhimurium) human clinical isolates and the effect of subinhibitory concentrations (sub-MICs) of gentamicin, ciprofloxacin and cefotaxime on biofilm formation and exopolysaccharides (EPS) production. Methods and Results: Quantification of biofilm formation and EPS production were carried out using a modified microtitre plate assay and spectrophotometric method, respectively. The results indicate that 38 isolates (50·7%), which are predominantly of DT104 phage type, presented as the strong biofilm producers in vitro on plastic surface. When strains with the highest biofilm-forming capacity were grown in the presence of sub-MICs of gentamicin and ciprofloxacin, the inhibition of biofilm formation and EPS production was observed. In contrast, cefotaxime at 1/2 MIC (0·039 μg ml⁻¹) was able to significantly induce the production of biofilm as well as EPS in three isolates with nontypable and DT104 phage type, respectively. Conclusions: These results clearly indicate that all the three antibiotics tested are able to interfere with biofilm formation and EPS production by Salm. Typhimurium isolates. Significance and Impact of the Study: The current study demonstrated that cefotaxime at sub-MIC can be beneficial for the behaviour of pathogen Salm. Typhimurium in vitro.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

aroA and aroD mutations influence biofilm formation in Salmonella Enteritidis

aro mutants of Salmonella enterica are frequently used as live vaccines for the oral vaccination of domestic animals. Interestingly, besides their auxotrophy, they appear to be of reduced resistance to the components of innate immune response due to a defect in outer membrane and/or cell wall integrity. Because different extracellular structures associated with the cell wall or outer membrane are involved in biofilm formation, we were interested in the ability of aroA and aroD mutants of S . Enteritidis to adhere to solid surfaces. We found that aroA and aroD mutants did not adhere to solid surfaces although they bind Congo red and produced d -mannose and d -glucose capsular polysaccharides in the same amounts as the wild-type strain. However, the aro mutants exhibited a decreased production of cellulose, N -acetyl- d -glucosamine or N -acetylneuraminic acid containing capsular polysaccharide and fimbriae which explains their inability to form biofilms. aroA and aroD containing plasmids complemented all the defects of the aro mutants. Beside its attenuation for different hosts, the loss of ability to form biofilm is an additional interesting characteristic of aro mutants.     

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.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

Effect of cinnamaldehyde on biofilm formation and sarA expression by methicillin‐resistant Staphylococcus aureus

Aims: To investigate the antibiofilm effect of cinnamaldehyde on methicillin‐resistant Staphylococcus aureus (MRSA) and analyse the effect of subminimum inhibitory concentrations (MICs) of cinnamaldehyde on the expression of the biofilm‐related gene sarA. Methods and Results: The MICs and minimum bactericidal concentrations (MBCs) were determined using a microtitre broth dilution method. Biofilm susceptibility was determined using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) staining and colony forming unit (CFU) counting assays. Antibiofilm effects were studied with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). SarA expression was assessed by real‐time PCR. MICs and MBCs were in the range 0·0625–0·5% (v/v). The killing effects were concentration dependent. At a concentration of 5× MIC, all strains in biofilm were decreased to lower than 20% of the control groups. SEM and CLSM images indicated that a 5× MIC concentration of cinnamaldehyde was able to detach and kill existing biofilms. Apart from strain JB‐06, real‐time PCR showed that the expression of sarA of all other strains was decreased upon exposure to sub‐MICs of cinnamaldehyde. Conclusions: These data showed the strong killing effect of cinnamaldehyde against MRSA within biofilms. Significance and Impact of the Study: This study indicated the potential of cinnamaldehyde as an inhibitory agent for use in MRSA biofilm‐related infections.     

Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524

Aims:  To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced.
Methods and Results:  Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga . xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3·83 and 3·75 g l⁻¹ respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2·5 g l⁻¹. Solid state ¹³C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles.
Conclusions:  The production rate of bacterial cellulose by Ga . xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features.
Significance and Impact of the Study:  Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga . xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.     

高盐环境下鼠伤寒沙门氏菌差异糖蛋白富集及组学分析

【背景】沙门氏菌是一种革兰阴性肠道病原菌,主要依靠III型分泌系统(typeIIIsecretion systems,T3SSs)来产生与致病性相关的效应蛋白。其中沙门氏菌致病岛(Salmonellapathogenicity island,SPI)区域是关键的基因区域。高盐浓度条件可以诱导SPI-1上效应蛋白的表达。【目的】探究在高盐浓度条件下鼠伤寒沙门氏菌(SalmonellaentericaserovarTyphimurium)糖蛋白的差异表达情况,寻找有意义的效应糖蛋白。【方法】将鼠伤寒沙门氏菌在普通培养基和高盐培养基中培养,收集菌体并超声裂解,提取蛋白后,用肼偶联法富集糖蛋白并用胰酶酶解,通过二甲基标记定量及LC/MS定量蛋白质组学方法进行糖蛋白的定量,用Thermo Proteome Discoverer 2.2软件对标记蛋白进行定性及定量分析。【结果】质谱结果显示,高盐环境中,沙门氏菌有19个糖蛋白的表达发生显著改变,其中,上调蛋白10个,最为显著的是ompC基因编码的外膜孔蛋白;下调表达糖蛋白9个,最为显著的是yjgF基因编码的翻译起始抑制因子。【结论】根据定量蛋白质组...     

HSP90 and eNOS partially co-localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes

Background information. Cultivation techniques promoting three‐dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel‐based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co‐cultures to evaluate, on both two‐ and three‐dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat‐shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase). Results. Freshly isolated rat cardiomyocytes were cultured on fibronectin, Matrigel gel or laminin, or in co‐culture with cardiac fibroblasts, and tested for both integrity and viability. As validation criteria, integrity of both plasma membrane and mitochondria was evaluated by transmission electron microscopy. Cell sensitivity to microenvironmental stimuli was monitored by immunofluorescence and confocal microscopy. We found that HSP90 and eNOS expression and localization are affected by changes in ECM composition. Elaboration of the images revealed, on Matrigel‐cultured cardiomyocytes, areas of high co‐localization between HSP90 and eNOS and co‐localization coefficients, which indicated the highest correlation with respect to the other substrates. Conclusions. Our three‐dimensional adult cardiomyocyte cultures are suitable for both analysing cell—ECM interactions at electron and confocal microscopy levels and monitoring micro‐environment impact on cardiomyocyte phenotype.     

Functional Amyloid and Other Protein Fibers in the Biofilm Matrix

Biofilms are ubiquitous in the natural and man-made environment. They are defined as microbes that are encapsulated in an extracellular, self-produced, biofilm matrix. Growing evidence from the genetic and biochemical analysis of single species biofilms has linked the presence of fibrous proteins to a functional biofilm matrix. Some of these fibers have been described as functional amyloid or amyloid-like fibers. Here we provide an overview of the biophysical and biological data for a wide range of protein fibers found in the biofilm matrix of Gram-positive and Gram-negative bacteria.     

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens,Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48–72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N_{21 days} of Serratia to be ca 3 Log₁₀(CFU cm^?2) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.     

The Role of Functional Amyloids in Bacterial Virulence

Amyloid fibrils are best known as a product of human and animal protein misfolding disorders, where amyloid formation is associated with cytotoxicity and disease. It is now evident that for some proteins, the amyloid state constitutes the native structure and serves a functional role. These functional amyloids are proving widespread in bacteria and fungi, fulfilling diverse functions as structural components in biofilms or spore coats, as toxins and surface-active fibers, as epigenetic material, peptide reservoirs or adhesins mediating binding to and internalization into host cells. In this review, we will focus on the role of functional amyloids in bacterial pathogenesis. The role of functional amyloids as virulence factor is diverse but mostly indirect. Nevertheless, functional amyloid pathways deserve consideration for the acute and long-term effects of the infectious disease process and may form valid antimicrobial targets.     

Multifaceted applications of nanomaterials in cell engineering and therapy

Nanomaterials with superior physiochemical properties have been rapidly developed and integrated in every aspect of cell engineering and therapy for translating their great promise to clinical success. Here we demonstrate the multifaceted roles played by innovatively-designed nanomaterials in addressing key challenges in cell engineering and therapy such as cell isolation from heterogeneous cell population, cell instruction in vitro to enable desired functionalities, and targeted cell delivery to therapeutic sites for prompting tissue repair. The emerging trends in this interdisciplinary and dynamic field are also highlighted, where the nanomaterial-engineered cells constitute the basis for establishing in vitro disease model; and nanomaterial-based in situ cell engineering are accomplished directly within the native tissue in vivo. We will witness the increasing importance of nanomaterials in revolutionizing the concept and toolset of cell engineering and therapy which will enrich our scientific understanding of diseases and ultimately fulfill the therapeutic demand in clinical medicine.