OBJECTIVE: To investigate the regulatory effects of two traditional mineral medicines (TMMs), Gypsum Fibrosum (Shigao, GF) and Terra Flava Usta (Zaoxintu, TFU), on gut-beneficial bacteria in mice, and preliminarily explore their mechanisms of action. METHODS: Mice were randomly divided into 3 groups (n=10 per group): the control group (standard diet), the GF group (diet supplemented with 2% GF), and the TFU group (diet supplemented with 2% TFU). After 4-week intervention, 16S rRNA gene sequencing was used to analyze the changes in the gut microbiota (GM). Scanning electron microscopy, in combination with coumarin A tetramethyl rhodamine conjugate and Hoechst stainings, was used to observe the bacteria and biofilm formation. RESULTS: Principal coordinate analysis revealed that GF and TFU significantly altered the GM composition in mice. Further analysis revealed that GF and TFU affected different types of gut bacteria, suggesting that different TMMs may selectively modulate specific bacterial populations. For certain bacteria, such as Faecalibaculum and Ileibacterium, both GF and TFU exhibited growth-promoting effects, implying that they may be sensitive to TMMs and that different TMMs can increase their abundance through their respective mechanisms. Notably, Lactobacillus reuteri, a widely recognized and used probiotic, was significantly enriched in the GF group. Random forest analysis identified Ileibacterium valens as a potential indicator bacterium for TMMs' impact on GM. Further mechanistic studies showed that gut bacteria formed biofilm structures on the TFU surface. CONCLUSIONS This study provides new insights into the interaction between TMMs and GM. As safe and effective natural clays, GF and TFU hold promise as potential candidates for prebiotic development.
Animals ; Gastrointestinal Microbiome/drug effects* ; Bacteria/growth & development* ; Mice ; Biofilms/drug effects* ; Male ; RNA, Ribosomal, 16S/genetics*

OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity. METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants. RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes. CONCLUSION The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.
Legionella pneumophila/pathogenicity* ; CRISPR-Cas Systems ; Biofilms/growth & development* ; Phenotype ; Bacterial Proteins/metabolism* ; Gene Deletion

LysR-type transcriptional regulators are involved in the regulation of numerous cellular metabolic processes in Klebsiella pneumoniae, leading to severe infection. Earlier, we found a novel LysR family gene, named kp05372, in a strain of K. pneumoniae (designated GPKP) isolated from forest musk deer. To study the function of this gene in relation to the biological characteristics of GPKP, we used the suicide plasmid and conjugative transfer methods to construct deletion mutant strain GPKP-Δkp05372; moreover, we also constructed the GPKP-Δkp05372+ complemented strain. The role of this gene was determined by comparing the following characteristics of three strains: growth curves, biofilm formation, drug resistance, stress resistance, median lethal dose (LD₅₀), organ colonization ability, and the histopathology of GPKP. Real-time polymerase chain reaction (RT-PCR) was used to test the expression level of seven genes upstream of kp05372. There was no significant difference in the growth rates when comparing the three bacterial strains, and no significant difference was recorded at different osmotic pressures, temperatures, salt contents, or hydrogen peroxide concentrations. The GPKP-Δkp05372 mutant formed a weak biofilm, and the other two strains formed medium biofilm. The drug resistance of the GPKP-Δkp05372 mutant toward cephalothin, cotrimoxazole, and polymyxin B was changed. The acid tolerance of the deletion strain was stronger than that of the other two strains. The LD₅₀ values of the wild-type and complemented strains were 174-fold and 77-fold higher than that of the GPKP-Δkp05372 mutant, respectively. The colonization ability of the GPKP-Δkp05372 mutant in the heart, liver, spleen, kidney, and intestine was the weakest. The three strains caused different histopathological changes in the liver and lungs. In the GPKP-Δkp05372 mutant, the relative expression levels of kp05374 and kp05379 were increased to 1.32-fold and 1.42-fold, respectively, while the level of kp05378 was decreased by 42%. Overall, the deletion of kp05372 gene leads to changes in the following: drug resistance and acid tolerance; decreases in virulence, biofilm formation, and colonization ability of GPKP; and regulation of the upstream region of adjacent genes.
Animals ; Bacterial Proteins/physiology* ; Biofilms ; Deer/microbiology* ; Drug Resistance, Bacterial ; Female ; Klebsiella Infections/pathology* ; Klebsiella pneumoniae/growth & development* ; Male ; Mice ; Transcription Factors/physiology*

BACKGROUND AND OBJECTIVES: Manuka honey has anti-microbial, anti-inflammatory, and anti-proliferative action with a high concentration of methylglyoxal compound. It is also effective in killing Staphylococcus aureus biofilm and effective for the acute exacerbation of chronic rhinosinusitis. The aim of this study was to determine the anti-fibrotic effect of manuka honey in nasal polyp fibroblasts. MATERIALS AND METHOD: Primary nasal fibroblasts were isolated from nasal polyps and treated with transforming growth factor-beta 1 (TGF-β1). To determine the anti-fibrotic effect of manuka honey, fibroblasts were pre-treated with various concentration of the honey. Reverse transcription-polymerase chain reaction and western blot analysis were then performed to determine α-smooth muscle actin (α-SMA), collagen type I, and matrix metalloproteinase-9 (MMP-9) messenger ribonucleic acid (mRNA) expression and protein production in nasal polyp fibroblasts. Phosphorylated Smad (pSmad) 2/3 and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were then determined by western blotting. RESULTS: TGF-β1 stimulation increased α-SMA, collagen type I, and MMP-9 mRNA expression and protein production in nasal polyp fibroblasts. Manuka honey effectively suppressed α-SMA, collagen type I, and MMP-9 mRNA expression and protein production. Its inhibitory role on TGF-β1 induced myofibroblast differentiation and its extracellular matrix production was associated with Smad2/3 and AMPK pathway. CONCLUSION: Manuka honey can inhibit TGF-β1 induced myofibroblast differentiation, collagen type I, and MMP-9 production in nasal fibroblasts. These results suggest that manuka honey might be a useful candidate for the inhibition of nasal polyp formation if further studies in vivo were accompanied.
Actins ; Adenosine ; AMP-Activated Protein Kinases ; Biofilms ; Blotting, Western ; Collagen Type I ; Extracellular Matrix ; Fibroblasts ; Homicide ; Honey ; Matrix Metalloproteinase 9 ; Methods ; Myofibroblasts ; Nasal Polyps ; Protein Kinases ; Pyruvaldehyde ; RNA ; RNA, Messenger ; Staphylococcus aureus ; Transforming Growth Factor beta ; Transforming Growth Factors

Bacterial Proteins ; genetics ; metabolism ; Biofilms ; DNA-Binding Proteins ; genetics ; metabolism ; Flagella ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Transcription Factors ; genetics ; metabolism ; Vibrio parahaemolyticus ; cytology ; genetics ; growth & development ; physiology

Three new alkyl substituted anthraquinone derivatives, trivially named as symploquinones A-C (Compounds 1-3) were isolated from Symplocos racemosa. The structures of these compounds were determined on the basis of extensive spectroscopic analyses (UV, IR, Mass, H- and C-NMR, and two-dimensional (2D) NMR techniques). The resulting data were also compared with the reported literature. These compounds were then subjected to antibacterial or antibiofilm testing. Compounds 1 and 3 exhibited good antibacterial activity in the concentration range of 160-83 μg·mL against Streptococcus mutans, methicillin resistant Staphylococcus aureus and Proteus mirabilis. Both compounds were further screened for anti-biofilm activity, which revealed promising activities at sub-MIC concentrations. None of the compounds were found to be active against Klebsiella pneumoniae.
Anthraquinones ; chemistry ; isolation & purification ; pharmacology ; Anti-Bacterial Agents ; chemistry ; isolation & purification ; pharmacology ; Biofilms ; drug effects ; growth & development ; Ericales ; chemistry ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; physiology ; Microbial Sensitivity Tests ; Proteus mirabilis ; drug effects ; physiology ; Spectrophotometry, Infrared ; Streptococcus mutans ; drug effects ; physiology

OBJECTIVETo evaluate the effect of 2-methacryloyloxyethyl phosphorylcholine (MPC) and nanoparticles of amorphous calcium phosphate (NACP) on the protein-repellent property of dental adhesive.

METHODSMPC and NACP were incorporated into SBMP as the test group. Scotchbond Multi-Purpose (SBMP) was used as control group. Human dentin shear bond strengths were measured. Protein adsorption onto samples was determined by micro bicinchoninic acid (BCA) method. A dental plaque microcosm biofilm model with human saliva as inoculum was used to investigate biofilm viability.

RESULTSThe dentin bond strength of modified group was (28.7±2.2) MPa, which was not significantly different from that of the SBMP control group. The amount of protein adsorption in the modified group and the SBMP control group were (0.21±0.02) µg/cm(2) and (4.17±0.45) µg/cm(2) respectively. Lactic acid production of biofilms in modified group and SBMP control were (7.71 ± 1.01) mmol/L and (19.18 ± 2.34) mmol/L repectively.

CONCLUSIONSMPC-NACP based dental adhesive greatly reduce the protein adsorption and bacterial adhesion, without compromising dentin shear bond strength. This novel bonding agent may have wide application.

Adsorption ; Biofilms ; drug effects ; growth & development ; Calcium Phosphates ; pharmacology ; Dental Cements ; pharmacology ; Dental Plaque ; Dentin ; chemistry ; Humans ; Lactic Acid ; biosynthesis ; Methacrylates ; pharmacology ; Nanoparticles ; Phosphorylcholine ; analogs & derivatives ; pharmacology ; Resin Cements ; pharmacology ; Saliva ; Tensile Strength

Tooth bleaching agents contain powerful oxidizing agents, which serve as the main part of bleaching agents because of its release of effective bleaching component. It has been a hot topic whether tooth bleaching agents exert negative influence on oral health. In order to provide train of thoughts and reference for further clinical researches and treatments, this review paper focuses on bleaching agents' effects on the growth of oral microbes and the formation of biofilms.
Bacteria ; drug effects ; growth & development ; Biofilms ; drug effects ; growth & development ; Humans ; Hydrogen Peroxide ; Mouth ; microbiology ; Oral Health ; Oxidants ; pharmacology ; Tooth Bleaching ; Tooth Bleaching Agents ; pharmacology

OBJECTIVETo study the effect of extracellular DNA(eDNA) on the formation of Streptococcus mutans(Sm) biofilms during different growth periods in sucrose environment.

METHODSSm biofilms were established on smooth glass surfaces under the environment of 1% sucrose and cultured in the condition of 37 ℃, 5% O2, 85% N2 and 10% CO2. Samples were randomly divided into four groups based on fourculture time(6,12, 24 and 48 h), respectively. Each group was further divided into two subgroups: control group(without deoxyribonuclease Ⅰ[DNaseⅠ] treatment) and test group(with DNaseⅠtreatment). DNaseⅠ was added 1 h advance in the treatment group to a final concentration of 100 U/ml. Each sample was stained with mixed SYTO-9/PI fluorescent dye. Confocal laser scanning microscopy was used for biofilm observation and scanning. The total biomass, the thickness and the volume of red fluorescence of each biofilm sample were measured following three-dimensional reconstruction using the softwear of Imaris 7.2.3.

RESULTSUnder the environment of 1% sucrose, the Sm bacterial adhesion and distribution density increased over time, the quantity of eDNA and membrane-damaged bacteria which were indicated by red fluorescence also increased within 24 h but dropped later. The biofilm biomasses of Sm biofilm in 6, 12, 24 and 48 h DNaseⅠ treatment group reduced significantly(P<0.05) compared to those in the corresponding control groups by 81.3%, 85.0%, 90.1% and 12.4%, respectively. The biofilm thicknesses in each DNase Ⅰ treatment group (except 6 h group) also reduced significantly(P<0.05) compared to those in the corresponding control group by 34.4%, 45.6% and 23.6%, respectively. The quantities of eDNA and membrane-damaged bacteria reduced in each treatment group except 48 h group compared to that in the corresponding control group.

CONCLUSIONSUnder the environment of 1% sucrose, eDNA plays an important role in promoting the formation of Sm biofilm.

Bacterial Adhesion ; drug effects ; Biofilms ; growth & development ; DNA ; physiology ; Deoxyribonuclease I ; pharmacology ; Microscopy, Confocal ; Streptococcus mutans ; physiology ; Sucrose ; Sweetening Agents ; Temperature

OBJECTIVETo study the effects of inhibitory peptide of Staphylococcus epidermidis (SE) biofilm (briefly referred to as inhibitory peptide) on adhesion and biofilm formation of SE at early stage.

METHODSBy using peptide synthesizer, the inhibitory peptide was synthesized with purity of 96.8% and relative molecular mass of 874.4. (1) Solution of SE ATCC 35984 (the same below) was cultivated with inhibitory peptide in the final concentrations of 1-256 µg/mL, and the M-H broth without bacteria solution was used as blank control. The MIC of the inhibitory peptide against SE was determined (n=3). (2) Solution of SE was cultivated with trypticase soy broth (TSB) culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 µg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Growth of SE was observed every one hour from immediately after cultivation (denoted as absorbance value), and the growth curve of SE during the 24 hours of cultivation was drawn, with 3 samples in each group at each time point. (3) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentrations of 16, 32, 64, 128, and 256 µg/mL (set as inhibitory peptide groups in corresponding concentration), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE was observed after cultivation for 4 hours (denoted as absorbance value, n=10); biofilm formation of SE was observed after cultivation for 20 hours (denoted as absorbance value, n=10). (4) Solution of SE was cultivated with TSB culture solution containing inhibitory peptide in the final concentration of 128 µg/mL (set as 128 µg/mL inhibitory peptide group), and solution of SE being cultivated with TSB culture medium was used as negative control group. Adhesive property of SE and its biofilm formation were observed with confocal laser scanning microscope (CLSM), and the sample numbers were both 3. Data were processed with one-way analysis of variance, LSD test, and Dunnett T3 test.

RESULTS(1) The MIC of inhibitory peptide against SE exceeded 256 µg/mL. (2) There was no significant difference in the growth curve of SE between inhibitory peptide groups in different concentrations and negative control group. (3) After 4 hours of cultivation, the absorbance values of adhesive property of SE in 256, 128, 64, and 32 µg/mL inhibitory peptide groups were respectively 0.20 ± 0.04, 0.27 ± 0.03, 0.35 ± 0.04, and 0.40 ± 0.04, which were significantly lower than the absorbance value in negative control group (0.53 ± 0.10, P<0.05 or P<0.01); the absorbance value of adhesive property of SE in 16 µg/mL inhibitory peptide group was 0.47 ± 0.09, which was close to the absorbance value in negative control group (P>0.05). After 20 hours of cultivation, the absorbance values of biofilm formation of SE in 256, 128, and 64 µg/mL inhibitory peptide groups were respectively 0.49 ± 0.10, 0.68 ± 0.06, and 0.93 ± 0.13, which were significantly less than the absorbance value in negative control group (1.21 ± 0.18, P<0.05 or P<0.01); the absorbance values of biofilm formation in 32 and 16 µg/mL inhibitory peptide groups were respectively 1.18 ± 0.22 and 1.15 ± 0.26, which were close to the absorbance value in negative control group (with P values above 0.05). (4) CLSM showed that more adhering bacteria and compact structure of biofilm were observed in negative control group, but less adhering bacteria and loose structure of biofilm were observed in 128 µg/mL inhibitory peptide group.

CONCLUSIONSThe inhibitory peptide can inhibit adhesion and biofilm formation of SE at early stage, but its structure still needs to be further modified.

Bacterial Adhesion ; Biofilms ; growth & development ; Humans ; Microscopy, Confocal ; Peptides ; Staphylococcus epidermidis ; genetics ; metabolism ; physiology