Microglia extracellular traps in Oreochromis niloticus infected with Weissella cibaria.

The mechanism of extracellular traps (ETs) is important in the cellular response against bacteria. Thus, in the present study, we describe for the first time the capacity of the Nile tilapia (Oreochromis niloticus) microglia in the formation of ETs in Weissella cibaria in vitro infection. Thus, we evaluated the ultrastructure of the microglia culture and observed the formation of ETs 6 h after stimulation with lipopolysaccharide (LPS) and during the course of infection. Our results shed light on the mechanism of formation of ETs in the microglia of teleost fish and the ability of W. cibaria to infect these cells.

Safety demonstration of a microbial species for use in the food chain: Weissella confusa.

Due to their traditional use in food fermentation process for centuries, microbial food cultures are considered to have a safe history of use. A specific microbial risk assessment is therefore rarely conducted for fermented foods and their food cultures, inoculated or naturally present. Some of those food cultures have been also considered for their potential health effect as probiotic strain candidates, for which a specific safety demonstration process has been proposed by a joint expert report of FAO and WHO. The European Food Safety Authority (EFSA) Biohazard panel also provides an approach for evaluating the safety of a strain to be added in the food chain, the Qualified Presumption of Safety (QPS). Weissella confusa, former taxon Lactobacillus confusus, is a food culture characterized in the fermentation process of sourdough. Some strains have been recently proposed for their probiotic potential. The species is also documented in recent infection case reports. It is considered nevertheless to be opportunistic as underlying factors have been suggested to explain the infection. We report here the microbial risk assessment of the species, by studying a collection of 26 food and 17 clinical isolates of Weissella confusa. The phenotypic study, genomic characterization and bibliographical survey will allow us to conclude about the safety of the species and confirm its use for food fermentation and consider specific strains for demonstration of their respective health effects as probiotic candidates.

Weissella cibaria CMU exerts an anti­inflammatory effect by inhibiting Aggregatibacter actinomycetemcomitans­induced NF­κB activation in macrophages.

Periodontitis is a chronic inflammatory disease caused by various periodontal pathogens. Weissella cibaria CMU (oraCMU) is a probiotic that promotes oral health. However, its anti­inflammatory effects against periodontal pathogens have not yet been investigated. The present study evaluated the anti­inflammatory effects of live oraCMU against stimulation with the formalin­inactivated periodontal pathogen Aggregatibacter actinomycetemcomitans in RAW 264.7 macrophages. Cell viability was analyzed by the MTS assay in a dose­dependent manner (at multiplicities of infection of 0.1, 1, 10, 100 and 1,000). Nitric oxide (NO) was monitored using the Griess test. The mRNA expression of proinflammatory cytokines such as interleukin (IL)1ß and IL6 was assessed by reverse transcription­quantitative PCR. Western blotting was used to examine the effects of oraCMU on the phosphorylation of NF­κB inhibitor α (IκBα) and IκBα kinase (IKK), the nuclear translocation of the NF­κB subunit p65 and the expression of inducible NO synthase (iNOS). Live oraCMU had no cytotoxic effects on RAW 264.7 macrophages. In A. actinomycetemcomitans­stimulated RAW 264.7 macrophages, oraCMU reduced NO production by suppressing iNOS expression and downregulating the mRNA expression of proinflammatory cytokines in a dose­dependent manner. IKK phosphorylation and IκBα degradation were dose­dependently inhibited by oraCMU and the nuclear translocation of p65 via the canonical NF­κB pathway was simultaneously reduced. The results indicated that oraCMU possessed anti­inflammatory activity associated with the inhibition of NF­κB signal activation in response to periodontal pathogens. This suggests that oraCMU is a beneficial anti­inflammatory probiotic that can aid in the maintenance of oral health.

Probiotic and technological features of Enterococcus and Weissella isolates from camel milk characterised by an Argane feeding regimen.

The objectives of this study were to isolate lactic acid bacteria (LAB) from a raw Moroccan camel milk collected after the incorporation of a specific Argane by-products diet, and to investigate their technological properties as well as their probiotic features. The molecular identification of the isolates indicated that they belong to Weissella confusa, Weissella cibaria or Enterococcus durans species. Our results revealed that the tested isolates have a fast acidifying ability (values ranging between 0.045 ± 0.01 to 0.93 ± 0.01 after only 4 h incubation), important proteolysis, autolysis, lipolytic activities and an important diacetyl and exopolysaccharides production. All these isolates demonstrated a high tolerance to gastrointestinal conditions, namely to gastric simulated juice (survival rate ranged between 75.05 ± 7.88 and 85.55 ± 1.77%) and to bile salts (survival rate between 42.79 ± 1.11 and 82.75 ± 1.01%). The autoaggregation, hydrophobicity and antioxidant activity mean values of the isolates were 13.26-41.16%, 13.23-54.47% and 47.57-63.31%, respectively. Importantly, LAB cultures exhibited antibacterial activity against Gram-negative and Gram-positive pathogenic bacteria and none of the tested isolates presented antibiotic resistance, haemolytic or DNase activities. This study revealed interesting properties for LAB isolated and supported their utilization as autochthone starters for camel milk fermentation that represent a challenge process. These results presented as well the probiotic potential for a possible human consumption.

Weissella paramesenteroides WpK4 ameliorate the experimental amoebic colitis by increasing the expression of MUC-2 and the intestinal epithelial regeneration.

AIMS: This study evaluates the action of Weissella paramesenteroides WpK4 on amoebic colitis. METHODS AND RESULTS: Weissella paramesenteroides WpK4 was administered in Entamoeba dispar infected and noninfected mice and clinical parameters were evaluated. Following 7 days, the caeca were collected for histopathology, morphometry and immunohistochemical staining of MUC-2, CDC-47 and IgA. The treatment reduced diarrhoea and the presence of blood in the faeces and diminished the area of necrosis, also causing weight gain. Also, the addition of this bacterium enhanced the expression of the mucin (MUC-2). The reduction in necrosis and increased CDC-47 expression indicates significant epithelial regeneration. The negative correlation between CDC-47 and the necrosis area reveals that the bacterium favoured the recovery of the necrotic regions and the positive correlation found between the expression of MUC-2 and CDC-47 indicates that the epithelial regeneration also supports the synthesis of MUC-2. CONCLUSIONS: Weissella paramesenteroides WpK4 was able to increase the protection of the intestinal mucosa against experimental amoebic colitis through the increase of MUC-2 and epithelial regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: Weissella paramesenteroides WpK4 presents the potential to become a complementary tool in the treatment of amoebic colitis.

In vitro assessment of potential probiotic characteristics of indigenous Lactococcus lactis and Weissella oryzae isolates from rainbow trout (Oncorhynchus mykiss Walbaum).

AIM: The objective of this study was to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from the intestinal ecosystem of rainbow trout. METHODS AND RESULTS: Among LAB isolates, 10 of them were selected and screened for resistance to acid and bile salts, pancreatin, sodium chloride and temperature, hydrophobicity, growth profile and antimicrobial activity against fish pathogens. Then, biosafety assessments were investigated. Selected LAB tolerated to gastrointestinal physiological conditions, pancreatin and a range of sodium chloride and temperature. They also exhibited hydrophobicity and showed antagonistic activity against Streptococcus iniae and Yersinia ruckeri. Results of 16S rRNA gene sequencing showed that selected LAB belonged to the Lactococcus lactis (n = 5) and Weissella oryzae (n = 5) species. They exhibited no ß-haemolytic activity, while six selected LAB were resistant to some antibiotics. None of them harboured virulence factors. CONCLUSIONS: This study revealed probiotic characteristics of indigenous LAB isolated from the intestinal ecosystem of rainbow trout. However, further studies are required to confirm the effectiveness of these isolates as probiotics in aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, for the first time, the presence of probiotic candidates belonging to W. Oryzae was confirmed in fish intestinal microbiota.

IL-10 Expression-Inducing Gut Bacteria Alleviate High-Fat Diet-Induced Obesity and Hyperlipidemia in Mice.

In the present study, we examined the effects of interleukin (IL)-10 expression-inducing bacteria Bifidobacterium adolescentis HP1, Lactobacillus mucosae HP2, and Weissella cibaria HP3 on high-fat diet (HFD)-induced obesity and liver steatosis in mice. Oral gavage of HP1, HP2, and HP3 reduced HFD-induced bodyweight gain, triglycerides, and total cholesterol levels in the blood and liver. They also suppressed HFD-induced colitis and the fecal δ,γ-Proteobacteria population. Of the tested bacteria, HP2, which most potently inhibited IL-10 expression, also suppressed HFD-induced bodyweight gain, liver steatosis, and colitis most effectively. These findings suggest that IL-10 expression-inducing gut bacteria can suppress obesity and liver steatosis.

Anti-Inflammatory Potential of Probiotic Strain Weissella cibaria JW15 Isolated from Kimchi through Regulation of NF-κB and MAPKs Pathways in LPS-Induced RAW 264.7 Cells.

Probiotics are known to provide the host with immune-modulatory effects and are therefore of remarkable interest for therapeutic and prophylactic applications against various disorders, including inflammatory diseases. Weissella cibaria JW15 (JW15) has been reported to possess probiotic and antioxidant properties. However, the effect of JW15 on inflammatory responses has not yet been reported. Therefore, the objective of the current study was to evaluate the anti-inflammatory potential of JW15 against lipopolysaccharide (LPS) stimulation. The production of pro-inflammatory factors and the cellular signaling pathways following treatment with heat-killed JW15 was examined in LPS-induced RAW 264.7 cells. Treatment with heat-killed JW15 decreased nitric oxide and prostaglandin E2 production via downregulation of the inducible nitric oxide synthase and cyclooxygenase-2. In addition, treatment with heat-killed JW15 suppressed the expression of pro-inflammatory cytokines, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α. The anti-inflammatory properties of treating with heat-killed JW15 were associated with mitogen-activated protein kinase signaling pathwaymediated suppression of nuclear factor-κB. These results indicated that JW15 possesses antiinflammatory potential and provide a molecular basis regarding the development of functional probiotic products.

Safety Evaluation of Oral Care Probiotics Weissella cibaria CMU and CMS1 by Phenotypic |and Genotypic Analysis.

Weissella cibaria CMU and CMS1 are known to exert beneficial effects on the oral cavity but have not yet been determined to be generally recognized as safe (GRAS), although they are used as commercial strains in Korea. We aimed to verify the safety of W. cibaria CMU and CMS1 strains through phenotypic and genotypic analyses. Their safety was evaluated by a minimum inhibitory concentration assay for 14 antibiotics, DNA analysis for 28 antibiotic resistance genes (ARGs) and one conjugative element, antibiotic resistance gene transferability, virulence gene analysis, hemolysis, mucin degradation, toxic metabolite production, and platelet aggregation reaction. W. cibaria CMU showed higher kanamycin resistance than the European Food Safety Authority (EFSA) cut-off, but this resistance was not transferred to the recipient strain. W. cibaria CMU and CMS1 lacked ARGs in chromosomes and plasmids, and genetic analysis confirmed that antibiotic resistance of kanamycin was an intrinsic characteristic of W. cibaria. Additionally, these strains did not harbor virulence genes associated with pathogenic bacteria and lacked toxic metabolite production, ß-hemolysis, mucin degradation, bile salt deconjugation, ß-glucuronidase, nitroreductase activity, gelatin liquefaction, phenylalanine degradation, and platelet aggregation. Our findings demonstrate that W. cibaria CMU and CMS1 can achieve the GRAS status in future.

Probiotic potential of Weissella strains isolated from horse feces.

In this study, we isolated four Weissella confusa strains from the healthy horse feces to test their potential as equine probiotics. The identification and characteristics of these isolates were determined as per standard methods. Resistance and susceptibility of the isolated strains were tested to low pHs, different heat treatments, commonly used antibiotics and against the pathogenic strains of Salmonella, Pasteurella, Staphylococcus aureus, and Escherichia coli. After 3 h cultural in different pH medium, the 4 strains still had a certain amount of survival above pH 3.0. WH2 and WH4 were still viable at pH2.5. All the isolated strains showed proper growth at 60 °C while no strain survived at 80 °C. The inhibition of α-amylase, the scavenging ability of free radical DPPH· and hydroxyl free radical HO·were also investigated. The results showed that WH4 had highest inhibition rate of α-amylase activity and DPPH· free radical scavenging rate, and the inhibition rate of α-amylase activity was 24.09% and the DPPH· free radical scavenging rate was 35.78%. The inhibition rate ofα-amylase activity and DPPH· scavenging rate of free radicals in the other three strains were about 10%. The clearance rate of hydroxyl radical (HO·) in 4 strains was between 12% and 15%. The antibiotic susceptibilities varied for these four Weisella strains but all of them showed resistance against the frequently used equine antibiotics. All the four strains successfully suppressed the growth of standard strains in in vitro bacteriostasis experiment, which included Salmonella enteritidis (NTNC13349), Escherichia coli (C83902) and Staphylococcus aureus (BNCC186335). they also successfully suppressed the growth of state key laboratory isolating pathogens, which are Pasterurella multocida and Salmonella. Our findings suggest that the isolated strains of Weissella confusa can act as potential equine probiotics and should be explored further.

Isolation and Preliminary Screening of a Weissella confusa Strain from Giant Panda (Ailuropoda melanoleuca).

Weissella confusa has recently received attention for its probiotic potential. Some W. confusa and Weissella cibaria strains isolated from fermented foods show favorable probiotic effects. However, the probiotic properties of W. confusa isolated from giant panda remain unreported to date. Thus, this study isolated a W. confusa strain from giant panda feces and then investigated its characteristics and probiotic properties. A lactic acid bacteria strain was isolated from giant panda fecal samples. The isolated strain was screened by in vitro probiotic property tests, including in vitro antimicrobial test, antioxidant test, surface hydrophobicity, and stress resistance. On the basis of biochemical identification and 16S rDNA sequencing, the W. confusa strain was identified as BSP201703. This Weissella confusa strain can survive at pH 2 and 0.3% (w/v) concentration of bile salt environment and inhibit common intestinal pathogens. It also possesses an in vitro antioxidant capacity, a high auto-aggregation ability, and a high surface hydrophobicity. BSP201703 might serve as a probiotic to giant pandas.

Adhesion Capacity of Weissella cibaria to Bovine Mammary Tissue and the Effect of Bio-Sealant Topical Application on Physicochemical Properties of Milk.

The ability of a probiotic strain (Weissella cibaria) to adhere on tissue and the effect of its topical application in nipples of lactating cows on physicochemical characteristics of milk were evaluated. An ex vivo model was used to demonstrate the adhesion capacity of W. cibaria. Tissue samples were randomly distributed in three different solutions corresponding to three treatments (a nipple bio-sealant formulation, sterile PBS solution and biomass of W. cibaria, sterile PBS solution without microorganism addition). The number of bacteria adhered in tissue was quantified and observed using electron microscopy. Additionally, a bio-sealant prepared with W. cibaria was topically applied to nipples of dairy cows. Milk samples were taken every 7 days for 60 days. Two controls were used. California mastitis test (CMT), somatic cell count, electrical conductivity, pH, density, and acidity were evaluated. The adhesion capacity of W. cibaria strain to epithelial cells of bovine teat tissue samples was demonstrated. When the strain was added as a bio-sealant, the adhesion capacity of W. cibaria was 80.44%. The response variables did not show significant differences among treatments; these results indicate the safety of the topical application of W. cibaria on the bovine mammary gland. In this study, a new safe way of administering probiotic microorganisms in nipples of lactating cows was demonstrated. W. cibaria adheres to the bovine mammary tissue and can be topically applied in nipples of lactating cows without affecting the physicochemical characteristics of milk.

Functional Probiotic Assessment and In Vivo Cholesterol-Lowering Efficacy of Weissella sp. Associated with Arid Lands Living-Hosts.

The research and the selection of novel probiotic strains from novel niches are receiving increased attention on their proclaimed health benefits to both humans and animals. This study aimed to evaluate the functional properties of Weissella strains from arid land living-hosts and to select strains with cholesterol-lowering property in vitro and in vivo, for use as probiotics. They were assessed for acid and bile tolerance, antibiotic susceptibility, membrane properties, antibacterial activity, antiadhesive effect against pathogens to host cell lines, and cholesterol assimilation in vitro. Our results showed that the majority of strains revealed resistance to gastrointestinal conditions. All the strains were nonhemolytic and sensitive to most of the tested antibiotics. They also exhibited high rates of autoaggregation and some of them showed high coaggregation with selected pathogens and high adhesion ability to two different cell lines (Caco-2 and MIM/PPk). Particularly, W. halotolerans F99, from camel feces, presented a broad antibacterial spectrum against pathogens, reduced Enterococcus faecalis and Escherichia coli adhesion to Caco-2 cells, and was found to reduce, in vitro, the cholesterol level by 49 %. Moreover, W. halotolerans F99 was evaluated for the carbohydrate utilization as well as the serum lipid metabolism effect in Wistar rats fed a high-cholesterol diet. W. halotolerans F99 showed an interesting growth on different plant-derivative oligosaccharides as sole carbon sources. Compared with rats fed a high-fat (HF) diet without Weissella administration, total serum cholesterol, low-density lipoprotein cholesterol, and triglycerides levels were significantly (p<0.001) reduced in W. halotolerans F99-treated HF rats, with no significant change in high-density lipoprotein cholesterol HDL-C levels. On the basis of these results, this is the first study to report that W. halotolerans F99, from camel feces, can be developed as cholesterol-reducing probiotic strain. Further studies may reveal their potential and possible biotechnological and probiotic applications.

Probiotic attributes and prevention of LPS-induced pro-inflammatory stress in RAW264.7 macrophages and human intestinal epithelial cell line (Caco-2) by newly isolated Weissella cibaria strains.

Probiotic lactic acid bacteria are known to modulate gut associated immune responses. Not many studies have reported on the role of Weissella species in preventing lipopolysaccharide (LPS) induced proinflammatory stress in murine macrophages as well as in human intestinal epithelial cells (Caco-2). Therefore, the present study was taken up to evaluate the probiotic attributes of four newly isolated Weissella strains (two each from fermented dosa batter and a human infant faecal sample); these attributes are cholesterol reduction, adhesion to Caco-2 cells and mucin and their ability to prevent LPS-induced nitric oxide and proinflammatory cytokine (IL-6, IL-1ß and TNFα) production by the murine macrophages and IL-8 production by the human epithelial cells. Reduction in LPS induced pro-inflammatory stress was compared with a well-studied probiotic bacterium Lactobacillus rhamnosus GG. The results suggested that the strains were tolerant to gastric conditions (pH 3.0) and bile salts. In addition, the strains exhibited moderate cell surface hydrophobicity, cholesterol reduction and adhesion to Caco-2 cells and gastric mucin. All the strains could prevent LPS-induced nitric oxide and IL-6 production in murine macrophages, while strain 28 alone prevented IL-1ß production. All the strains could prevent IL-8 production by the human epithelial cells. The present study led to the first line selection of W. cibaria 28 as a putative strain for future studies as it showed adhesion to Caco-2 cells and gastric mucin and cholesterol reduction besides preventing LPS-induced pro-inflammatory stress in macrophages and in human colonic epithelial cells.

A fibronectin-binding protein (FbpA) of Weissella cibaria inhibits colonization and infection of Staphylococcus aureus in mammary glands.

Staphylococcus aureus (S. aureus) is a frequent cause of infections in both humans and animals. Probiotics are known to inhibit colonization of pathogens on host tissues. However, mechanisms for the inhibition are still elusive due to complex host-microbe and microbe-microbe interactions. Here, we show that reduced abilities of S. aureus to infect mammary glands in the presence of Weissella cibaria (W. cibaria) were correlated with its poor adherence to mammary epithelial cells. Such inhibition by W. cibaria isolates was at least partially attributed to a fibronectin-binding protein (FbpA) on this lactic acid bacterium. Three W. cibaria isolates containing fbpA had higher inhibitory abilities than other three LAB isolates without the gene. The fbpA-deficient mutant of W. cibaria isolate LW1, LW1ΔfbpA, lost the inhibitory activity to reduce the adhesion of S. aureus to mammary epithelial cells and was less able to reduce the colonization of S. aureus in mammary glands. Expression of FbpA to the surface of LW1ΔfbpA reversed its inhibitory activities. Furthermore, addition of purified FbpA inhibited S. aureus biofilm formation. Our results suggest that W. cibaria FbpA hinders S. aureus colonization and infection through interfering with the S. aureus invasion pathway mediated by fibronectin-binding proteins and inhibiting biofilm formation of S. aureus.

Community dynamics and metabolite target analysis of spontaneous, backslopped barley sourdough fermentations under laboratory and bakery conditions.

Barley flour is not commonly used for baking because of its negative effects on bread dough rheology and loaf volume. However, barley sourdoughs are promising ingredients to produce improved barley-based breads. Spontaneous barley sourdough fermentations were performed through backslopping (every 24h, 10days) under laboratory (fermentors, controlled temperature of 30°C, high dough yield of 400) and bakery conditions (open vessels, ambient temperature of 17-22°C, low dough yield of 200), making use of the same batch of flour. They differed in pH evolution, microbial community dynamics, and lactic acid bacteria (LAB) species composition. After ten backsloppings, the barley sourdoughs were characterized by the presence of the LAB species Lactobacillus fermentum, Lactobacillus plantarum, and Lactobacillus brevis in the case of the laboratory productions (fast pH decrease, pH<4.0 after two backslopping steps), and of Leuconostoc citreum, Leuconostoc mesenteroides, Weissella confusa and Weissella cibaria in the case of the bakery productions (slow pH decrease, pH4.0 after eight backslopping steps). In both sourdough productions, Saccharomyces cerevisiae was the sole yeast species. Breads made with wheat flour supplemented with 20% (on flour basis) barley sourdough displayed a firmer texture, a smaller volume, and an acceptable flavour compared with all wheat-based reference breads. Hence, representative strains of the LAB species mentioned above, adapted to the environmental conditions they will be confronted with, may be selected as starter cultures for the production of stable barley sourdoughs and flavourful breads.

Elucidating the Mechanism of Weissella-dependent Lifespan Extension in Caenorhabditis elegans.

The mechanism whereby lactic acid bacteria extend the lifespan of Caenorhabditis elegans has previously been elucidated. However, the role of Weissella species has yet not been studied. We show that Weissella koreensis and Weissella cibaria significantly (p < 0.05) extend the lifespan of C. elegans compared with Escherichia coli OP50 and induce the expression of several genes related to lifespan extension (daf-16, aak-2, jnk-1, sod-3 and hif-1). Oral administration of Weissella altered reactive oxygen species (ROS) production and lowered the accumulation of lipofuscin and increased locomotor activity (which translates to a delay in ageing). Moreover, Weissella-fed C. elegans had decreased body sizes, brood sizes, ATP levels and pharyngeal pumping rates compared with E. coli OP50-fed worms. Furthermore, mutations in sod-3, hif-1 or skn-1 did not alter lifespan extension compared with wild-type C. elegans. However, C. elegans failed to display lifespan extension in loss-of-function mutants of daf-16, aak-2 and jnk-1, which highlights the potential role of these genes in Weissella-induced longevity in C. elegans. Weissella species extend C. elegans lifespan by activating DAF-16 via the c-Jun N-terminal kinase (JNK) pathway, which is related to stress response, and the AMP-activated protein kinase (AMPK)-pathway that is activated by dietary restriction.

Evaluation of Time-Temperature Integrators (TTIs) with Microorganism-Entrapped Microbeads Produced Using Homogenization and SPG Membrane Emulsification Techniques.

A comparative study was conducted to evaluate precision and accuracy in controlling the temperature dependence of encapsulated microbial time-temperature integrators (TTIs) developed using two different emulsification techniques. Weissela cibaria CIFP 009 cells, immobilized within 2% Na-alginate gel microbeads using homogenization (5,000, 7,000, and 10,000 rpm) and Shirasu porous glass (SPG) membrane technologies (10 µm), were applied to microbial TTIs. The prepared micobeads were characterized with respect to their size, size distribution, shape and morphology, entrapment efficiency, and bead production yield. Additionally, fermentation process parameters including growth rate were investigated. The TTI responses (changes in pH and titratable acidity (TA)) were evaluated as a function of temperature (20°C, 25°C, and 30°C). In comparison with conventional methods, SPG membrane technology was able not only to produce highly uniform, small-sized beads with the narrowest size distribution, but also the bead production yield was found to be nearly 3.0 to 4.5 times higher. However, among the TTIs produced using the homogenization technique, poor linearity (R(2)) in terms of TA was observed for the 5,000 and 7,000 rpm treatments. Consequently, microbeads produced by the SPG membrane and by homogenization at 10,000 rpm were selected for adjusting the temperature dependence. The Ea values of TTIs containing 0.5, 1.0, and 1.5 g microbeads, prepared by SPG membrane and conventional methods, were estimated to be 86.0, 83.5, and 76.6 kJ/mol, and 85.5, 73.5, and 62.2 kJ/mol, respectively. Therefore, microbial TTIs developed using SPG membrane technology are much more efficient in controlling temperature dependence.

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms.

Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 µg to as high as 50 µg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 µg ml(-1)) showed little or no sugar interference up to 2000 µg ml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1 µg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms.