Days Of Biofilm Growth Research Articles

Effect of high-fluoride dentifrice on root dentine and bacterial composition in a multispecies biofilm model

The present study evaluated the effect of high-fluoride dentifrice on dentine demineralization and bacterial composition in a multispecies biofilm model in vitro. A seven-organism bacterial consortium was grown on bovine dentine discs in a high-throughput active attachment model. The biofilms were submitted twice per day to the following dentifrices treatments: 5,000 ppm F, 1,100 ppm F, with placebo as a negative control. After 5 days of biofilm growth, dentine samples were assessed by transversal microradiography, the biofilm was collected for bacterial counts and the pH of the media was determined. Lower integrated mineral loss values were observed when 5,000 ppm F-treatment was used compared to the other treatments. Overall microbiological counts decreased with increasing F-concentration as well the pH of the media throughout the experiment. The 5,000 ppm F-treatment caused a shift in microbial composition and reduced dentine demineralization in the in-vitro experimental model.

Modelling formation and removal of biofilms in secondary dairy raw materials

Introduction. Microorganisms of dairy raw materials tend to adhere to the surfaces of processing equipment and form sustainable biofilms, which is a serious issue in the dairy industry. The goal of the present work was to investigate formation of biofilms on a glass surface in static model conditions, and removal of such biofilms by cleaning.
 Study objects and methods. The study objects were the permeates of skim milk, sweet whey and acid whey, as well as the biofilms formed and washings from glass slides. Biofilms were removed from the glass with detergents used in the dairy industry. Standard methods of determining microbiological and physicochemical properties were used to characterize the permeates. The biofilm structure and morphology of microorganisms participating in biofilm formation were investigated with a light spectroscopy. The efficiency of biofilm removal in a cleaning process was quantified with optical density of washings.
 Results and discussion. Biofilms in whey permeates formed slower compared to those in skimmed milk permeate during the first 24 h. Yeasts contributed significantly to the biofilm microflora in acid whey permeate throughout 5 days of biofilm growth. Well adhered biofilm layers were the most stable in skimmed milk permeate. The highest growth of both well and poorly adhered biofilm layers was observed in sweet whey permeate after 3–5 days. It was established that the primary attachment of microorganisms to a glass surface occurred within 8 h, mature multicultural biofilms formed within 48 h, and their partial destruction occurred within 72 h.
 Conclusion. The research results can be used to improve the cleaning equipment procedures in processing secondary dairy raw materials.

Distinct Temporal Succession of Bacterial Communities in Early Marine Biofilms in a Portuguese Atlantic Port.

Marine biofilms are known to influence the corrosion of metal surfaces in the marine environment. Despite some recent research, the succession of bacterial communities colonizing artificial surfaces remains uncharacterized in some temporal settings. More specifically, it is not fully known if bacterial colonizers of artificial surfaces are similar or distinct in the different seasons of the year. In particular the study of early biofilms, in which the bacterial cells communities first adhere to artificial surfaces, are crucial for the development of the subsequent biofilm communities. In this work, we used amplicon-based NGS (next-generation sequencing) and universal 16S rRNA bacterial primers to characterize the early biofilm bacterial communities growing on 316 L stainless steel surfaces in a Northern Portugal port. Sampling spanned 30-day periods in two distinct seasons (spring and winter). Biofilm communities growing in steel surfaces covered with an anti-corrosion paint and planktonic communities from the same location were also characterized. Our results demonstrated that distinct temporal patterns were observed in the sampled seasons. Specifically, a significantly higher abundance of Gammaproteobacteria and Mollicutes was found on the first days of biofilm growth in spring (day 1 to day 4) and a higher abundance of Alphaproteobacteria during the same days of biofilm growth in winter. In the last sampled day (day 30), the spring biofilms significantly shifted toward a dominance of photoautotrophic groups (mostly diatoms) and were also colonized by some macrofouling communities, something not observed during the winter sampling. Our results revealed that bacterial composition in the biofilms was particularly affected by the sampled day of the specific season, more so than the overall effect of the season or overall sampling day of both seasons. Additionally, the application of a non-fouling-release anti-corrosion paint in the steel plates resulted in a significantly lower diversity compared with plates without paint, but this was only observed during spring. We suggest that temporal succession of marine biofilm communities should be taken in consideration for future antifouling/anti-biofilm applications.

Spatio-temporal formation of biofilms and extracellular matrix analysis in Azospirillum brasilense.

Elucidation of biofilm structure formation in the plant growth-promoting rhizobacterium Azospirillum brasilense is necessary to gain a better understanding of the growth of cells within the extracellular matrix and its role in the colonization of plants of agronomic importance. We used immunofluorescence microscopy and confocal laser scanning microscopy to study spatio-temporal biofilm formation on an abiotic surface. Observations facilitated by fluorescence microscopy revealed the presence of polar flagellin, exopolysaccharides, outer major membrane protein (OmaA) and extracellular DNA in the Azospirillum biofilm matrix. In static culture conditions, the polar flagellum disaggregated after 3 days of biofilm growth, but exopolysaccharides were increasing. These findings suggest that the first step in biofilm formation may be attachment, in which the bacterium first makes contact with a surface through its polar flagellum. After attaching to the surface, the long flagella and OmaA intertwine the cells to form a network. These bacterial aggregates initiate biofilm development. The underlying mechanisms dictating how the biofilm matrix components of A. brasilense direct the overall morphology of the biofilm are not well known. The methods developed here might be useful in further studies that analyze the differential spatial regulation of genes encoding matrix components that drive biofilm construction.

Influence of the Inoculum Source on the Cariogenicity of in vitro Microcosm Biofilms

This study investigated the cariogenic potential of biofilms originating from different types of inoculum (saliva and dental plaque) from caries-active and caries-free individuals. Ten volunteers were selected from each caries condition for the paired collection of saliva and dental plaque. Microcosm biofilms were grown in triplicate from each inoculum on enamel specimens in 24-well plates under cariogenic challenge. After 10 days, the biofilms were collected for analysis of outcome variables: percentage of surface hardness change (%SHC) and microbiological composition of biofilms. Statistical analysis was performed using the t test, the linear multivariate analysis model and Pearson's correlation coefficients (α = 0.05). A comparative analysis between microbiological baseline data showed higher counts of mutans streptococci in plaque samples within caries-active individuals; a comparative analysis of colony-forming unit (CFU) counts between individuals with different caries status showed higher counts of acid-tolerant microorganisms and mutans streptococci in dental plaque and of acid-tolerant microorganisms in saliva. After 10 days of biofilm growth, the CFU values for total microorganisms, lactobacilli, mutans streptococci and acid-tolerant bacteria, as well as for SHC, were not statistically significant, considering the type of inoculum and caries condition (p > 0.05). A positive correlation was found for %SHC and CFU counts of acid-tolerant bacteria (r = 0.406) and lactobacilli (r = 0.379). Under the limits of this study, the cariogenic potential of biofilms, formed under identical conditions in vitro, is similar, regardless of baseline differences between the source and type of inoculum.

Biostabilization and Transport of Cohesive Sediment Deposits in the Three Gorges Reservoir.

Cohesive sediment deposits in the Three Gorges Reservoir, China, were used to investigate physical and geochemical properties, biofilm mass, and erosion and deposition characteristics. Biofilm cultivation was performed in a recirculating flume for three different periods (5, 10 and 15 days) under ambient temperature and with sufficient nutrients supply. Three groups of size-fractionated sediment were sequentially used, including 0–0.02 mm, 0.02–0.05 mm and 0.05–0.10 mm. Desired conditions for erosion and deposition were designed by managing high bed shear stress at the narrow part of upstream flume and low shear stress at the wide part of downstream flume. Biostabilization and transport characteristics of the biofilm coated sediment (bio-sediment) were strongly influenced by the cultivation period, and the results were compared with clean sediment. The bio-sediment was more resistant to erosion, and the mean shear stress was increased by factors of 2.65, 2.73 and 5.01 for sediment with 5, 10 and 15 days of biofilm growth compared with clean sediment, resulting in less sediment being eroded from the bed. Simultaneously, the settling velocity was smaller for bio-sediment due to higher organic content and porosity (i.e., lower density). Additionally, there was a smaller probability of deposition for sediment with a longer cultivation period after erosion, resulting in more retention time in aquatic systems. These results will benefit water management in natural rivers.

Potency and penetration of telavancin in staphylococcal biofilms

Due to the emergence of staphylococcal biofilm infections, the need for advanced antibiotics is crucial. The aim of this investigation was to evaluate the potency and penetration of telavancin against staphylococcal biofilms using two different biofilm models. Multiple staphylococcal strains, including meticillin-sensitive Staphylococcus aureus ATCC 29213, vancomycin-intermediate S. aureus ATCC 700787, heterogeneously vancomycin-intermediate S. aureus ATCC 700698 and meticillin-sensitive Staphylococcus epidermidis ATCC 12228, were grown and treated in drip-flow reactors to determine log reductions due to telavancin treatment. After 3 days of biofilm growth and 24h of treatment, mean log reductions for telavancin ranged from 1.65 to 2.17 depending on the bacterial strain tested. Penetration was evaluated qualitatively using confocal scanning laser microscopy to image the infiltration of fluorescently labelled antibiotic into a staphylococcal biofilm grown in a flow cell. Fluorescently labelled telavancin rapidly penetrated the biofilms with no alteration in the biofilm structure.

Gas chromatography-mass spectrometry-based metabolite profiling of Salmonella enterica serovar Typhimurium differentiates between biofilm and planktonic phenotypes.

The aim of this study was to utilize gas chromatography coupled with mass spectrometry (GC-MS) to compare and identify patterns of biochemical change between Salmonella cells grown in planktonic and biofilm phases and Salmonella biofilms of different ages. Our results showed a clear separation between planktonic and biofilm modes of growth. The majority of metabolites contributing to variance between planktonic and biofilm supernatants were identified as amino acids, including alanine, glutamic acid, glycine, and ornithine. Metabolites contributing to variance in intracellular profiles were identified as succinic acid, putrescine, pyroglutamic acid, and N-acetylglutamic acid. Principal-component analysis revealed no significant differences between the various ages of intracellular profiles, which would otherwise allow differentiation of biofilm cells on the basis of age. A shifting pattern across the score plot was illustrated when analyzing extracellular metabolites sampled from different days of biofilm growth, and amino acids were again identified as the metabolites contributing most to variance. An understanding of biofilm-specific metabolic responses to perturbations, especially antibiotics, can lead to the identification of novel drug targets and potential therapies for combating biofilm-associated diseases. We concluded that under the conditions of this study, GC-MS can be successfully applied as a high-throughput technique for "bottom-up" metabolomic biofilm research.

Broad-spectrum anti-biofilm peptide that targets a cellular stress response.

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.

Effect of cleansing of biofilm formed on titanium discs

To study the combined effect of mechanical and chemical cleansing on a 4-day biofilm grown intra-orally on titanium discs with different surface characteristics. Twenty subjects used a splint with two metal plates in the upper jaw. Each plate was placed in the premolar-molar region and carried four titanium discs with four different surface characteristics (OsseoSpeed(™), TiOblast(™), experimental and turned surface). After 4 days of biofilm growth, the discs were cleaned mechanically and chemically with saline or chlorhexidine. Following cleansing, microbial samples were obtained and analysed by culture. The titanium discs were processed for scanning electron microscope (SEM) analysis. The experiment was repeated 3 days later using delmopinol or a mixture of essential oils during cleansing. The combination of mechanical and chemical cleansing was ineffective in complete biofilm removal from all four titanium discs. The microbiological analysis did not reveal any statistically significant differences between surface types or between cleaning agents regarding logarithmic mean counts of CFU for specific bacteria, aerobes, anaerobes or the TVC. Aerobes were more numerous than anaerobes on all surface types. The SEM analysis disclosed that the remaining biofilm on moderately rough surfaces (OsseoSpeed(™), TiOblast(™) and experimental) was complex and firmly attached, while the biofilm on turned surface had a pattern of spread bacteria forming less clusters. Cleansing may call for prolonged time of chemomechanical debridement and/or more effective disinfectants to suppress biofilms on dental implant surfaces.

Biodegradable nanofibrous drug delivery systems: effects of metronidazole and ciprofloxacin on periodontopathogens and commensal oral bacteria

The purposes of this study were to fabricate biodegradable polydioxanone (PDS II®) electrospun periodontal drug delivery systems (hereafter referred to as matrices) containing either metronidazole (MET) or ciprofloxacin (CIP) and to investigate the effects of antibiotic incorporation on both periodontopathogens and commensal oral bacteria. Fibrous matrices were processed from PDS polymer solution by electrospinning. Antibiotic-containing PDS solutions were prepared to obtain four distinct groups: 5wt.% MET, 25wt.% MET, 5wt.% CIP, and 25wt.% CIP. Pure PDS was used as a control. High-performance liquid chromatography (HPLC) was done to evaluate MET and CIP release. Dual-species biofilms formed by Lactobacillus casei (Lc) and Streptococcus salivarius (Ss) were grown on the surface of all electrospun matrices. After 4days of biofilm growth, the viability of bacteria on biofilms was assessed. Additionally, antimicrobial properties were evaluated against periodontopathogens Fusobacterium nucleatum (Fn) and Aggregatibacter actinomycetemcomitans (Aa) using agar diffusion assay. A three-dimensional interconnected porous network was observed in the different fabricated matrices. Pure PDS showed the highest fiber diameter mean (1,158 ± 402nm) followed in a descending order by groups 5wt.% MET (1,108 ± 383nm), 25wt.% MET (944 ± 392nm), 5wt.% CIP (871 ± 309nm), and 25wt.% CIP (765 ± 288nm). HPLC demonstrated that groups containing higher amounts (25wt.%) of incorporated drugs released more over time, while those with lower levels (5wt.%) the least. No inhibitory effect of the tested antibiotics was detected on biofilm formation by the tested commensal oral bacteria. Meanwhile, CIP-containing matrices inhibited growth of Fn and Aa. CIP-containing matrices led to a significant inhibition of periodontopathogens without negatively impairing the growth of periodontal beneficial bacteria. Based on the proven in vitro inhibition of periodontitis-related bacteria, future in vivo research using relevant animal models is needed to confirm the effectiveness of these drug delivery systems.

An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies

Microcosm biofilms formed in microplates have demonstrated complex community dynamics similar to natural dental biofilm. No simplified microcosm models to evaluate enamel demineralization and dose-response effect to anticariogenic therapies have yet been established, thus this study was designed to develop a pre-clinical model fulfilling this purpose. Experiments were carried out to establish the time of biofilm formation and the sucrose concentration and exposure regimen. Biofilms were initiated from saliva and grown for up to 10 days on bovine enamel discs in 24-well plates, with a saliva analogue medium. Data were collected as pH readings and thepercentage enamel surface hardness change. A dose-response evaluation was performed with chlorhexidine, which significantly affected the pH and mineral loss. Overall, the established model parameters, 5 days of biofilm growth with intermittent 1% sucrose exposure of 6 h per day, was suitable as a pre-clinical model for enamel demineralization and dose-response studies.

Efficacy of Using a Hydrodebrider and of Citric Acid/Zwitterionic Surfactant on a Staphylococcus Aureus Bacterial Biofilm in the Sheep Model of Rhinosinusitis

Biofilms in chronic rhinosinusitis (CRS) patients are associated with recalcitrant disease patterns. Citric acid/zwitterionic surfactant (CAZS) has been shown to have significant efficacy at in vitro biofilm eradication. Unique hydrodebrider systems have been shown to have effective antibiofilm efficacy. The aim of this study was to evaluate the efficacy of the CAZS/hydrodebrider treatment on Staphylococcus aureus biofilms in the sheep model of CRS. Forty-two sheep frontal sinuses were inoculated with S. aureus, followed by 7 days of biofilm growth. Sinuses were randomized to one of nine treatment groups: control, hydrodebrider with saline or CAZS (killed on day 0 or day 7 posttreatment), CAZS, or saline flush (killed on day 0 or day 7 posttreatment). Confocal scanning laser microscopy (CSLM) was used to confirm extent of biofilm reduction. Samples of each sinus were taken, assessing cilia morphology using electron microscopy. A mean of 37.22 ± 9.65% of control (no treatment) CSLM images taken showed biofilms. Both CAZS flush and CAZS/hydrodebrider treatments showed initial improvements in biofilms but biofilm regrowth followed treatment, 14.74 ± 9.58% to 18.91 ± 12.14% and 15.60 ± 13.92% to 24.70 ± 3.66%, respectively (p > 0.05). Saline/hydrodebrider treatments showed a nonsignificant improvement in biofilm treatment, which was maintained, 18.34 ± 11.85% to 12.04 ± 10.28% (p > 0.05). Cilial morphology grade was significantly worse in the CAZS treatment groups (p < 0.05). CAZS solution (with and without a hydrodebrider system) can significantly adversely affect cilia morphology. The hydrodebrider system, when combined with saline, may be a useful treatment adjunct for mucosal biofilms.

Phenotypic Characterization ofStreptococcus pneumoniaeBiofilm Development

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching approximately 8 x 10(8) cells and approximately 15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.

Nutrient dynamics and successional changes in a lentic freshwater biofilm

SUMMARY 1. Colonisation, species composition, succession of microalgae and nutrient dynamics in biofilms grown under light and dark conditions were examined during the initial phases of biofilm development in a lentic freshwater environment.2. Biofilms were developed on inert (perspex) panels under natural illuminated and experimental dark conditions and the panels were retrieved for analysis after different incubation periods. Analysed parameters included biofilm thickness, algal density, biomass, chlorophyll a, species composition, total bacterial density and nutrients such as nitrite, nitrate, phosphate and silicate.3. Biofilm thickness, algal density, biomass, chlorophyll a and species richness were significantly higher in light‐grown biofilms, compared with dark‐grown biofilms. The light‐grown biofilms showed a three‐phased succession pattern, with an initial domination of Chlorophyceae followed by diatoms (Bacillariophyceae) and finally by cyanobacteria. Dark‐grown biofilms were mostly dominated by diatoms.4. Nutrients were invariably more concentrated in biofilms than in ambient water. Nutrient concentrations were generally higher in dark‐grown biofilms except in the case of phosphate, which was more concentrated in light‐grown biofilms. Significant correlations between nutrients and biofilm parameters were observed only in light‐grown biofilms.5. The N : P ratio in the biofilm matrix decreased sharply in the initial 4 days of biofilm growth; ensuing N‐limitation status seemed to influence biofilm community structure. The N : P ratios showed significant positive correlations with the chlorophycean fraction in both light and dark‐grown biofilms, and low N : P ratio in the older biofilms favoured cyanobacteria. Our data indicate that nutrient chemistry of biofilm matrix shapes community structure in microalgal biofilms.