Bacteria ; genetics ; growth & development ; Biofilms ; Genetic Heterogeneity

OBJECTIVETo observe the surface of Enterococcus faecalis and the dynamic forming process of those biofilms using atomic force microscopy (AFM) in air condition.

METHODSThe surface of Enterococcus faecalis which were dried in air were observed with AFM. We used the cellulose nitrate film to construct the Enterococcus faecalis biofilms model in vitro, and then placed the biofilms under AFM to observe the surface changes of biofilms' development.

RESULTSThe cell surfaces of strain Enterococcus faecalis were not regular because of the presence of the amorphous substance on the colony surface, which congregated globular, fibrous structure. Gradually determined that at 6 h the initial biofilm formed and at 24 h the biofilms maintained the steady-state. AFM height images showed topographical changes due to biofilms' development, which were used to characterize several aspects of the bacterial surface, such as the presence of extracellular polymeric substance, and the biofilms' development stage.

CONCLUSIONApplication of AFM in physiological conditions could be useful in observing Enterococcus faecalis surface ultrastructure and dynamic process of biofilms formation.

Bacterial Adhesion ; Biofilms ; Enterococcus faecalis ; growth & development ; Microscopy, Atomic Force

OBJECTIVETo investigate the biological function of mscL gene in S. epidermidis.

METHODSA plasmid pMAD-δmscL including the upstream and downstream homologous regions of mscL and spectinomycin resistance gene (spc) was constructed and transformed into S. epidermidis 1457 by electroporation with continuous subculture at 42 degrees celsius with shaking. The mscL knockout mutant (SE1457-δmscL) was selected by blue-white colony screening and antibiotic resistance. The D600 and numbers of viable cells were measured in the mutant and parent strains before and after an osmotic downshift of 0.9 M. The effect of the mscL knockout on biofilm formation was assessed using a semi-quantitative microtiter plate assay.

RESULTSThe plasmid pMAD-δmscL was constructed and mscL was deleted from the genome of S. epidermidis 1457. The mscL mutant was verified by PCR of the genomic DNA, direct sequencing and RT-PCR. During the exponential growth phase, the mutant showed significantly reduced ability to survive osmotic downshock in comparison with the wild-type strain but their capacity to form biofilm remained similar.

CONCLUSIONThe mscL gene may be involved in osmoregulation during the logarithmic growth of S. epidermidis, but it dose not affect biofilm formation of the bacterium.

Biofilms ; growth & development ; Gene Knockout Techniques ; Genes, Bacterial ; Plasmids ; Staphylococcus epidermidis ; genetics ; growth & development

OBJECTIVETo study dynamic relation between periodontal pathogens and cariogenic bacteria under analogous oral environment.

METHODSEight periodontopathic and cariogenic bacteria of Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), Provotella intermedium (Pi), Streptococcus mutans (Sm), Streptococcus sanguis (Ss), Actinomyces viscosus (Av) and Lactobacillus acidophilus (La) were used. These eight strains were cultured in modified chemostat under analogous oral environment which contained 600 ml modified BM medium supplemented with 2.5 g/L porcine gastric mucin, respectively. After 1, 24, 48 and 96 h, optical sectioning of plaque biofilms on removable and replaceable hydroxyapatite disks was analyzed by the combination of live bacterial Gram fluorescence staining and confocal laser scanning microscopy. Biofilm thickness and reconstruction of the three-dimensional architecture of plaque biofilms were made.

RESULTSBiofilm thickness increased significantly with time (P < 0.001). Biofilms of Aa were thinner than those of Ss and eight-specie biofilms were thicker than those formed by Ss and Aa per time point. Three-dimensional images showed periodontal pathogens mainly occurred in cariogenic bacterial complex or on the biofilm surface.

CONCLUSIONSGram-positive cariogenic species initially predominated in artificial plaque, followed by the increasing proportions of Gram-negative periodontal pathogens. The relation between microecological balance among bacteria and diseases is worthy of further studies.

Bacteria ; growth & development ; Biofilms ; Dental Caries ; microbiology ; Ecology ; Humans ; Microscopy, Confocal ; Periodontal Diseases ; microbiology

OBJECTIVETo study the effect of DNase I on biofilm formation of Staphylococcus aureus.

METHODSThe growth curve of S. aureus was detected using a spectrophotometer. The adhesion of S. aureus was analyzed using flat colony counting method, and the biofilm formation was assayed using the 96-well crystal violet staining method.

RESULTSExposure to different concentrations of DNase I did not obviously affect the growth of S. aureus but significantly inhibit the formation of bacterial biofilms in a dose-dependent manner. DNase I inhibited the adhesion of S. aureus at different growth stages. When combined with antibiotics, DNase I resulted in a signi?cant decrease in the established bio?lm biomass compared to antibiotics or DNase I used alone.

CONCLUSIONDNase I can effectively inhibit biofilm formation of S. aureus and enhance the inhibitory effect of antibiotics against S. aureus biofilms.

Anti-Bacterial Agents ; Biofilms ; drug effects ; Deoxyribonuclease I ; chemistry ; Staphylococcus aureus ; growth & development

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

Altered bowel flora is currently thought to play a role in a variety of disease conditions, and the use of Bifidobacterium spp. and Lactobacillus spp. as probiotics has been demonstrated to be health-promoting, even if the success of their administration depends on the applied bacterial strain(s) and the targeted disease. In the last few decades, specific probiotics have been shown to be effective in the treatment or the prevention of acute viral gastroenteritis, pediatric post-antibiotic-associated diarrhea, some pediatric allergic disorders, necrotizing enterocolitis in preterm infants, inflammatory bowel diseases and postsurgical pouchitis. The potential application of probiotics is continuously widening, with new evidence accumulating to support their effect on the prevention and treatment of other disease conditions, including several oral diseases, such as dental caries, periodontal diseases and oral malodor, as well as genitourinary and wound infections. Considering the increasingly widespread ability of pathogens to generate persistent biofilm-related infections, an even more attractive proposal is to administer probiotics to prevent or counteract biofilm development. The response of biofilm-based oral, intestinal, vaginal and wound infections to probiotics treatment will be reviewed here in light of the most recent results obtained in this field.
Antibiosis ; physiology ; Bacterial Infections ; prevention & control ; Bifidobacterium ; physiology ; Biofilms ; growth & development ; Humans ; Lactobacillus ; physiology ; Mucous Membrane ; microbiology ; Probiotics ; therapeutic use

OBJECTIVE: To establish the rat model of chronic suppurative otitis media and observe the formation of bacterial biofilm in middle ear mucosa of animal models and discuss the role of bacterial biofilm in the pathogenesis of chronic suppurative otitis media. METHOD: Twenty-eight rats were divided into six experimental groups and a control group evenly. All rats in experimental groups were infected with pseudomonas aeruginosa solution in 1 X 10(6) cfu/ml concentration through the tympanic membrane puncture approach to bilateral middle ear cavity. On the first, sixth, tenth, fifteenth and twenty-first st day after inoculation respectively, four rats in one experimental group were narcotized, then two-sided tympanic membrane of each rat were observed by using electric otoscope. We rated on the severity of the inflammation from the general pathology level (0 for normal, four for the most serious). After the execution, the two-sided otocysts were obtained. The left was made to SEM specimen and the shape of bacterial biofilm in middle ear mucous was observed. The right was observed by CLSM. Control group were executed at the beginning of the experiment. RESULT: (1) Bacterial biofilm in line with their respective criteria were found at the six days after the inoculation, and were more typical in shape after ten days. Then the states maintained stably within three weeks. (2) By observing tympanic membrane under electric otoscope, it can be seen that the inflammation severity of otitis media aggravated gradually in the first ten days and achieved the peak, then the state continued to the third week. The differences of tympanic membrane rating between one day group and six day group, six day group and ten day group were statistically significant (P < 0.05, P < 0.01). CONCLUSION (1) In this experiment, the process of bacterial biofilm development in rats model is: from beginning to the five days, the bacteria adhere and accumulates. After six days, the 3D structure of bacterial biofilm preliminary formatted. After ten days, the bacterial biofilm achieves the mature and steady state. (2) With the growth and maturity of bacterial biofilm in middle ear mucosa, the inflammation of otitis media is gradually increasing, which suggests that the inflammation severity of otitis media and the maturation of bacterial biofilm in middle ear mucosa are closely related.
Animals ; Biofilms ; growth & development ; Disease Models, Animal ; Mucous Membrane ; microbiology ; Otitis Media, Suppurative ; microbiology ; Pseudomonas aeruginosa ; Rats ; Rats, Wistar

OBJECTIVE: To observe the effect of biyuanshu oral liquid on the formation of pseudomonas aeruginosa biofilms in vitro. METHOD: Pseudomonas aeruginosa biofilm was established by plate culture and detected by Scanning electron microscopy and AgNO3 staining. After treated with different dosages of biyuanshu oral liquid and erythromycin, the pseudomonas aeruginosa biofilms were observed by AgNO3 staining and the number of viable bacteria were measured by serial dilution. RESULT: The pseudomonas aeruginosa biofilms could be detected by SEM at the seventh culture day and it was consistent with the detection of AgNO3 staining. The biyuanshu oral liquid and erythromycin have the effect on inhibiting the formation of pseudomonas aeruginosa biofilms. But with the already formed pseudomonas aeruginosa biofilms the inhibition was not significant. The serial dilution method showed that the viable counts of bacteria of biyuanshu oral liquid and erythromycin treated groups were significantly lower than those untreated groups (P < 0.05). CONCLUSION The biyuanshu oral liquid and erythromycin can inhibit the formation of pseudomonas aeruginosa biofilms in vitro.
Anti-Bacterial Agents ; pharmacology ; Biofilms ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Erythromycin ; pharmacology ; Pseudomonas aeruginosa ; drug effects ; growth & development

AIMThe purpose of this study was to develop a mathematical model to quantitatively describe the passive transport of macromolecules within dental biofilms.

METHODOLOGYFluorescently labeled dextrans with different molecular mass (3 kD, 10 kD, 40 kD, 70 kD, 2000 kD) were used as a series of diffusion probes. Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum were used as inocula for biofilm formation. The diffusion processes of different probes through the in vitro biofilm were recorded with a confocal laser microscope.

RESULTSMathematical function of biofilm penetration was constructed on the basis of the inverse problem method. Based on this function, not only the relationship between average concentration of steady-state and molecule weights can be analyzed, but also that between penetrative time and molecule weights.

CONCLUSIONThis can be used to predict the effective concentration and the penetrative time of anti-biofilm medicines that can diffuse through oral biofilm. Furthermore, an improved model for large molecule is proposed by considering the exchange time at the upper boundary of the dental biofilm.

Actinomyces ; growth & development ; Algorithms ; Biofilms ; growth & development ; Biological Transport ; Dental Plaque ; microbiology ; Dextrans ; pharmacokinetics ; Diffusion ; Fluorescent Dyes ; pharmacokinetics ; Fusobacterium nucleatum ; growth & development ; Macromolecular Substances ; pharmacokinetics ; Microscopy, Confocal ; Models, Biological ; Molecular Probe Techniques ; Streptococcus mutans ; growth & development ; Streptococcus sanguis ; growth & development