Adaptation of Bacillus species to dairy associated environment facilitates their biofilm forming ability.

Biofilm-forming Bacillus species are often involved in contamination of dairy products and therefore present a major microbiological challenge in the field of food quality and safety. In this study, we sequenced and analyzed the genomes of milk- and non-milk-derived Bacillus strains, and evaluated their biofilm-formation potential in milk. Unlike non-dairy Bacillus isolates, the dairy-associated Bacillus strains were characterized by formation of robust submerged and air-liquid interface biofilm (pellicle) during growth in milk. Moreover, genome comparison analysis revealed notable differences in putative biofilm-associated determinants between the dairy and non-dairy Bacillus isolates, which correlated with biofilm phenotype. These results suggest that biofilm formation by Bacillus species might represent a presumable adaptation strategy to the dairy environment.

In situ effect of enamel salivary exposure time and type of intraoral appliance before an erosive challenge.

OBJECTIVES: This study tested the effect of enamel salivary exposure time prior to an acid challenge (30 min, 1, 2, or 12 h) and type of intraoral appliance (palatal or mandibular) on initial erosion. METHODS: After initial surface hardness evaluation, enamel blocks (n = 340) were randomly divided into groups and volunteers (n = 20). The control group was not exposed to saliva previously to the erosive challenge. The volunteers wore palatal and mandibular appliances simultaneously. After salivary exposure, the blocks were subjected to acid exposure by immersion in hydrochloric acid (0.01 M, pH 2.3) for 30 s. Then, the enamel surface hardness was evaluated. Data were analyzed using ANOVA, Kruskal-Wallis and Tukey's test (p < 0.05). RESULTS: No difference was observed on percent surface hardness change (% SHC) in the enamel blocks between the types of intraoral appliances. Exposure to saliva for 30 min and 1 h promoted similar enamel resistance to the erosive attack, which was similar to the control group for both appliances. Blocks exposed to saliva for 2 h showed less hardness loss when compared to 30 min. Keeping the blocks in saliva during 12-h overnight resulted in similar percentage of enamel hardness loss compared to 2 h. CONCLUSIONS: A 2-hour in situ exposure to saliva is adequate to promote partial protection against initial erosive lesions, independently of the type of intraoral appliance used. CLINICAL SIGNIFICANCE: This finding will help researchers in the development of erosion studies, which will provide information for dentists to offer a better treatment for erosion.

Forced expression of FLO11 confers pellicle-forming ability and furfural tolerance on Saccharomyces cerevisiae in ethanol production.

We constructed a plasmid that expresses FLO11 encoding a cell surface glycoprotein of Saccharomyces cerevisiae under the control of a constitutive promoter. This plasmid conferred pellicle-forming ability on the non-pellicle-forming industrial strain of S. cerevisiae at the air-liquid interface of the glucose-containing liquid medium. The induced pellicle-forming cells exhibited tolerance to furfural, which is a key toxin in lignocellulosic hydrolysates, in ethanol production.

Biofouling on titanium implants: a novel formulation of poloxamer and peroxide for in situ removal of pellicle and multi-species oral biofilm.

Eradicating biofouling from implant surfaces is essential in treating peri-implant infections, as it directly addresses the microbial source for infection and inflammation around dental implants. This controlled laboratory study examines the effectiveness of the four commercially available debridement solutions '(EDTA (Prefgel®), NaOCl (Perisolv®), H2O2 (Sigma-Aldrich) and Chlorhexidine (GUM® Paroex®))' in removing the acquired pellicle, preventing pellicle re-formation and removing of a multi-species oral biofilm growing on a titanium implant surface, and compare the results with the effect of a novel formulation of a peroxide-activated 'Poloxamer gel (Nubone® Clean)'. Evaluation of pellicle removal and re-formation was conducted using scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy to assess the surface morphology, elemental composition and chemical surface composition. Hydrophilicity was assessed through contact angle measurements. The multi-species biofilm model included Streptococcus oralis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, reflecting the natural oral microbiome's complexity. Biofilm biomass was quantified using safranin staining, biofilm viability was evaluated using confocal laser scanning microscopy, and SEM was used for morphological analyses of the biofilm. Results indicated that while no single agent completely eradicated the biofilm, the 'Poloxamer gel' activated with 'H2O2' exhibited promising results. It minimized re-contamination of the pellicle by significantly lowering the contact angle, indicating enhanced hydrophilicity. This combination also showed a notable reduction in carbon contaminants, suggesting the effective removal of organic residues from the titanium surface, in addition to effectively reducing viable bacterial counts. In conclusion, the 'Poloxamer gel + H2O2' combination emerged as a promising chemical decontamination strategy for peri-implant diseases. It underlines the importance of tailoring treatment methods to the unique microbial challenges in peri-implant diseases and the necessity of combining chemical decontaminating strategies with established mechanical cleaning procedures for optimal management of peri-implant diseases.

Profiling the composition and metabolic functions of microbial community in pellicle-forming radish paocai.

Pellicle formation is an obvious indicator of spoilage and is followed by a loss of flavor in a variety of fermented vegetables. In this study, the pellicle-forming microorganisms were isolated using culture-dependent approaches, then a comparative analysis between the pellicle-forming (PF) radish paocai and normal fermented paocai in the diversity and function of microbial community was conducted by metagenome sequencing. Based on a pairwise t-test and OPLS-DA analysis, diallyl sulfide, (z)-1-allyl-2-(prop-1-en-1-yl) disulfane, and terpineol were considered to be the main components responsible for the unpleasant flavor of PF paocai. Yarrowia spp., Enterobacter spp., and Pichia spp. were the main pellicle-forming microorganisms. All 17 isolated Enterobacter strains showed pectinase-producing and cellulase-producing abilities, and 3 isolated Pichia strains showed gas-producing capacity. According to LEfSe analysis based on metagenomes, unclassified_g__Citrobacter and Yarrowia lipolytica were the uppermost biomarkers that distinguished the PF paocai from normal paocai. Unclassified_g__Lactobacillus and Lactobacillus plantarum were found to be actively engaged in starch and sucrose metabolism, cysteine and methionine metabolism, galactose metabolism, fructose and mannose metabolism, lysine biosynthesis, fatty acid biosynthesis, and arginine biosynthesis, all of which contributed to the flavor formation of paocai. Combining the results of metagenome sequencing with the data obtained based on the culture-dependent method, we could deduce that the growth of Yarrowia lipolytica first promoted the increase of pH and the formation of pellicle, which provided a suitable niche for the growth of some harmful bacteria such as Enterobacter, Citrobacter, and Serratia. These hazardous bacteria then worked in concert to induce the odorous stench and texture softening of paocai, as well as more pellicle formation.

Inhibition of Perilla frutescens Essential Oil on Pellicle Formation of Candida tropicalis and Pichia kluyveri and Its Effect on Volatile Compounds in Sichuan Pickles.

Pellicle formation is the most typical characteristic of deteriorating fermented vegetable products. Perilla frutescens essential oil (PEO) is widely used as a useful natural preservative. However, few studies have addressed the antifungal activity and mechanism of PEO in pellicle formation microorganisms, and it is still unclear whether it can inhibit pellicle formation and affect its volatile compounds in Sichuan pickles. The current study showed that PEO can inhibit pellicle formation during fermentation of Sichuan pickles as it had significant antifungal activity against the pellicle formation microorganisms Candida tropicalis SH1 and Pichia kluyveri SH2. The minimum inhibitory concentration (MIC) of PEO against C. tropicalis SH1 and P. kluyveri SH2 was determined to be 0.4 µL/mL, and the minimum fungicidal concentrations (MFCs) were 1.6 µL/mL and 0.8 µL/mL, respectively. The antifungal mechanism was activated as a result of damage to the cell membrane, an increase in the cell permeability, a decrease in the mitochondrial membrane potential, and the inhibition of ATPase activity. Meanwhile, the addition of PEO to Sichuan pickles can enrich the profiles of volatile compounds during fermentation, including limonene, myrcene, 1,8-cineole, linalool, perilla ketone, heptanal, hexanal, α-thujone and ß-terpineol and thus improve the overall sensory acceptability. These results indicated that PEO has the potential to be used as a novel food preservative to control pellicle formation in fermented vegetables.

Identification of pellicle formation related microorganisms in traditional Sichuan paocai through metagenomic sequence and the effects of Baijiu/Salt on pellicle and volatile components.

The occurrence of pellicle on the surface of paocai brine is a common undesirable phenomenon during the multi-rounds of paocai fermentation, which is mainly caused by the growth of microorganisms related to pellicle formation. But the detailed information on these microorganisms and volatile components produced by them, as well as the changes of the microorganisms during the process of paocai recovery, are still rare in the literature. Therefore, the purpose of this study was (1) to analyze the pellicle formation related microorganisms by comparing the differential microorganisms in initial brine and the brine when pellicle occurred through metagenomic sequencing technology, (2) to explore the changes of microorganisms in the fermentation system after addition Baijiu and/or salt, and (3) to further detect the VOCs in paocai samples by gas chromatography-mass spectrometry (GC-MS). The relationship between VOCs and the selected marker microorganisms was also determined. The results showed that the diversity of fungi was increased when pellicle formed, the pellicle formation related microorganisms mainly belonged to six genus, including Kazachstania, Lactobacillus, Pichia, Candida, Lachancea, and Saccharomyces. Apart from the unknown function and basic life activities of microorganisms, the metabolic activities of these pellicle formation related microorganisms were mainly carbohydrate transport and metabolism, and amino acid transport and metabolism. The growth of pellicle formation related microorganisms could be inhibited by adding Baijiu (1.5% v/v), but the addition of salt (7% salt (w/v) did not promote this inhibitory effect. Through PCA analysis, it was found that the VOCs of paocai were significantly affected by adding Baijiu and Baijiu and salt. The undesirable smell at the beginning of pellicle formation may be related to Propanoic acid, hexyl ester, 1,3-Dimethyl-1-cyclohexene, Oxime-, methoxy-phenyl- and Phenylethyl Alcohol.

Resistance of biofilm- and pellicle-embedded strains of Escherichia coli encoding the transmissible locus of stress tolerance (tLST) to oxidative sanitation chemicals.

Biofilm formation in food processing plants reduces the efficacy of sanitation. The presence of transmissible locus of stress tolerance (tLST) also enhances resistance of planktonic cells of Escherichia coli to sanitation chemicals but the role of tLST in resistance of biofilm-embedded cells remains unclear. This study investigated the link of tLST to biofilm formation and its contribution to resistance of biofilm-embedded E. coli to sanitation. Biofilms were formed as single-strain and as dual-strain biofilms in association with E. coli, Aeromonas australensis or Carnobacterium maltaromaticum. Biofilms on stainless steel were compared to floating biofilms formed at the air-liquid interface (pellicles). The resistance of biofilm-embedded tLST positive strains of E. coli to chlorine, hydrogen peroxide, and peroxyacetic acid was higher than the resistance of tLST negative strains. Higher biofilm density as measured by crystal violet staining was observed in tLST-positive strains of E. coli when compared to tLST negative strains. Biofilm density positively correlated to resistance to disinfectants. The use of confocal laser scanning microscopy detected more compact structure of pellicles compared to solid surface-attached biofilms, resulting in higher chlorine resistance despite the absence of tLST in strains of E. coli. Collectively, the findings of this study elucidated the impact of tLST in strains of E. coli on biofilm formation and sanitizer resistance. These findings may inform the development of improved sanitization protocols for food facilities.

Biofilm Formation and Heat Stress Induce Pyomelanin Production in Deep-Sea Pseudoalteromonas sp. SM9913.

Pseudoalteromonas is an important bacterial genus present in various marine habitats. Many strains of this genus are found to be surface colonizers on marine eukaryotes and produce a wide range of pigments. However, the exact physiological role and mechanism of pigmentation were less studied. Pseudoalteromonas sp. SM9913 (SM9913), an non-pigmented strain isolated from the deep-sea sediment, formed attached biofilm at the solid-liquid interface and pellicles at the liquid-air interface at a wide range of temperatures. Lower temperatures and lower nutrient levels promoted the formation of attached biofilm, while higher nutrient levels promoted pellicle formation of SM9913. Notably, after prolonged incubation at higher temperatures growing planktonically or at the later stage of the biofilm formation, we found that SM9913 released a brownish pigment. By comparing the protein profile at different temperatures followed by qRT-PCR, we found that the production of pigment at higher temperatures was due to the induction of melA gene which is responsible for the synthesis of homogentisic acid (HGA). The auto-oxidation of HGA can lead to the formation of pyomelanin, which has been shown in other bacteria. Fourier Transform Infrared Spectrometer analysis confirmed that the pigment produced in SM9913 was pyomelanin-like compound. Furthermore, we demonstrated that, during heat stress and during biofilm formation, the induction level of melA gene was significantly higher than that of the hmgA gene which is responsible for the degradation of HGA in the L-tyrosine catabolism pathway. Collectively, our results suggest that the production of pyomelanin of SM9913 at elevated temperatures or during biofilm formation might be one of the adaptive responses of marine bacteria to environmental cues.

Pellicle of thermotolerant Acetobacter pasteurianus strains: characterization of the polysaccharides and of the induction patterns.

Acetobacter species are well known to have the ability to grow floating on the surface of the medium by producing pellicle, which consists of cells and a self-produced matrix of cell-attached polysaccharide. We previously isolated three thermotolerant strains (SL13E-2, SL13E-3, and SL13E-4) from Sri Lankan coconut vinegar and identified all these strains as Acetobacter pasteurianus. The pellicle polysaccharides of these three strains and of A. pasteurianus SKU1108, which was originally isolated from Thailand, were characterized. The monosaccharide composition of the pellicle polysaccharides of these A. pasteurianus strains was found to be varied. For example, the pellicle polysaccharide of SL13E-2 is composed of rhamnose and glucose in the ratio 1:8, and that of SL13E-4 and mesophilic A. pasteurianus NBRC3191 consists of rhamnose, glucose and xylose in the ratio 1:5:2 and 1:4:2, respectively. On the other hand, the pellicle polysaccharides of SL13E-3 and SKU1108 strains are composed of rhamnose, glucose and galactose in the ratio 2:2:1 and 1:5:2.5, respectively. The pellicle formation of thermotolerant SL13E-2, SL13E-3, and SL13E-4 was found to be significantly induced by the addition of ethanol, while poor induction was observed with SKU1108. The size and sugar composition of the polysaccharides obtained from cells induced by ethanol and by uninduced cells were the same, indicating that the number of molecules of the polysaccharides had increased but the polysaccharide molecule remained unchanged. The addition of a sugar source such as glucose, sucrose or fructose slightly induced pellicle formation in SKU1108, especially at 40°C.