Plant-based oral care product exhibits antibacterial effects on different stages of oral multispecies biofilm development in vitro

Abstract Background Excessive biofilm formation on surfaces in the oral cavity is amongst the main reasons for severe infection development like periodontitis and peri-implantitis. Mechanical biofilm removal as well as the use of adjuvant antiseptics supports the prevention of pathogenic biofilm formation. Recently, the antibacterial effect of the oral care product REPHA-OS®, based on medicinal plant extracts and essential oils, has been demonstrated on oral pathogens grown on agar plates. In the present study, the effectiveness of the product on medical relevant oral biofilm development should be demonstrated for the first time. Methods An established in vitro oral multispecies biofilm, composed of Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, was used to analyze the antibacterial effect of different REPHA-OS® concentrations on planktonic bacteria, biofilm formation and mature biofilms. It was quantified using metabolic activity assays and live/dead fluorescence staining combined with three-dimensional confocal laser-scanning microscopy. Additionally, effects on species distribution inside the biofilm were assessed by means of quantitative real-time PCR. Results REPHA-OS® showed statistically significant antimicrobial effects on all stages of biofilm development: a minimal inhibitory concentration of 5% could be detected for both, for planktonic bacteria and for biofilm formation. Interestingly, only a slightly higher concentration of 10% was necessary to completely kill all bacteria in mature biofilms also. In contrast, an influence on the biofilm matrix or the species distribution could not be observed. The effect could be attributed to the herbal ingredients, not to the contained ethanol. Conclusion The strong antibacterial effect of REPHA-OS® on different stages of oral biofilm development strengthens its application as an alternative adjuvant in oral care therapies.

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Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species

Journal of Medical Microbiology ◽

10.1099/jmm.0.021832-0 ◽

2010 ◽

Vol 59 (10) ◽

pp. 1225-1234 ◽

Cited By ~ 51

Author(s):

H. M. H. N. Bandara ◽

O. L. T. Lam ◽

R. M. Watt ◽

L. J. Jin ◽

L. P. Samaranayake

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Significant Inhibition ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Dependent Manner ◽

Bacterial Lipopolysaccharides ◽

Species Specific ◽

Biofilm Development

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

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Effects of CO2laser irradiation on matrix-rich biofilm development formation–an in vitro study

PeerJ ◽

10.7717/peerj.2458 ◽

2016 ◽

Vol 4 ◽

pp. e2458 ◽

Cited By ~ 6

Author(s):

Bruna Raquel Zancopé ◽

Vanessa B. Dainezi ◽

Marinês Nobre-dos-Santos ◽

Sillas Duarte ◽

Vanessa Pardi ◽

...

Keyword(s):

Contact Angle ◽

Biofilm Formation ◽

Dental Enamel ◽

In Vitro Study ◽

Dry Weight ◽

Vitro Study ◽

Enamel Surface ◽

Control Group ◽

Biofilm Development

BackgroundA carbon dioxide (CO2) laser has been used to morphologically and chemically modify the dental enamel surface as well as to make it more resistant to demineralization. Despite a variety of experiments demonstrating the inhibitory effect of a CO2laser in reduce enamel demineralization, little is known about the effect of surface irradiated on bacterial growth. Thus, this in vitro study was preformed to evaluate the biofilm formation on enamel previously irradiated with a CO2laser (λ = 10.6 µM).MethodsFor this in vitro study, 96 specimens of bovine enamel were employed, which were divided into two groups (n = 48): 1) Control-non-irradiated surface and 2) Irradiated enamel surface. Biofilms were grown on the enamel specimens by one, three and five days under intermittent cariogenic condition in the irradiated and non-irradiated surface. In each assessment time, the biofilm were evaluated by dry weigh, counting the number of viable colonies and, in fifth day, were evaluated by polysaccharides analysis, quantitative real time Polymerase Chain Reaction (PCR) as well as by contact angle. In addition, the morphology of biofilms was characterized by fluorescence microscopy and field emission scanning electron microscopy (FESEM). Initially, the assumptions of equal variances and normal distribution of errors were conferred and the results are analyzed statistically by t-test and Mann Whitney test.ResultsThe mean of log CFU/mL obtained for the one-day biofilm evaluation showed that there is statistical difference between the experimental groups. When biofilms were exposed to the CO2laser, CFU/mL and CFU/dry weight in three day was reduced significantly compared with control group. The difference in the genes expression (Glucosyltransferases (gtfB) and Glucan-binding protein (gbpB)) and polysaccharides was not statically significant. Contact angle was increased relative to control when the surface was irradiated with the CO2laser. Similar morphology was also visible with both treatments; however, the irradiated group revealed evidence of melting and fusion in the specimens.ConclusionIn conclusion, CO2laser irradiation modifies the energy surface and disrupts the initial biofilm formation.

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Role of the Streptococcus mutans irvA Gene in GbpC-Independent, Dextran-Dependent Aggregation and Biofilm Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01015-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 7037-7043 ◽

Cited By ~ 15

Author(s):

Min Zhu ◽

Dragana Ajdić ◽

Yuan Liu ◽

David Lynch ◽

Justin Merritt ◽

...

Keyword(s):

Streptococcus Mutans ◽

Biofilm Formation ◽

Growth Conditions ◽

Parental Background ◽

A Cell ◽

Major Surface ◽

Biofilm Development ◽

Small Aggregation

ABSTRACT Dextran-dependent aggregation (DDAG) of Streptococcus mutans is an in vitro phenomenon that is believed to represent a property of the organism that is beneficial for sucrose-dependent biofilm development. GbpC, a cell surface glucan-binding protein, is responsible for DDAG in S. mutans when cultured under defined stressful conditions. Recent reports have described a putative transcriptional regulator gene, irvA, located just upstream of gbpC, that is normally repressed by the product of an adjacent gene, irvR. When repression of irvA is relieved, there is a resulting increase in the expression of GbpC and decreases in competence and synthesis of the antibiotic mutacin I. This study examined the role of irvA in DDAG and biofilm formation by engineering strains that overexpressed irvA (IrvA+) on an extrachromosomal plasmid. The IrvA+ strain displayed large aggregation particles that did not require stressful growth conditions. A novel finding was that overexpression of irvA in a gbpC mutant background retained a measure of DDAG, albeit very small aggregation particles. Biofilms formed by the IrvA+ strain in the parental background possessed larger-than-normal microcolonies. In a gbpC mutant background, the overexpression of irvA reversed the fragile biofilm phenotype normally associated with loss of GbpC. Real-time PCR and Northern blot analyses found that expression of gbpC did not change significantly in the IrvA+ strain but expression of spaP, encoding the major surface adhesin P1, increased significantly. Inactivation of spaP eliminated the small-particle DDAG. The results suggest that IrvA promotes DDAG not only by GbpC, but also via an increase in P1.

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Searching Hit Potential Antimicrobials in Natural Compounds Space against Biofilm Formation

Molecules ◽

10.3390/molecules25225334 ◽

2020 ◽

Vol 25 (22) ◽

pp. 5334

Author(s):

Roberto Pestana-Nobles ◽

Jorge A. Leyva-Rojas ◽

Juvenal Yosa

Keyword(s):

Natural Products ◽

Biofilm Formation ◽

Caulobacter Crescentus ◽

Resistant Bacteria ◽

Chronic Infections ◽

Aconitic Acid ◽

Starting Point ◽

In Silico Studies ◽

Biofilm Development

Biofilms are communities of microorganisms that can colonize biotic and abiotic surfaces and thus play a significant role in the persistence of bacterial infection and resistance to antimicrobial. About 65% and 80% of microbial and chronic infections are associated with biofilm formation, respectively. The increase in infections by multi-resistant bacteria instigates the need for the discovery of novel natural-based drugs that act as inhibitory molecules. The inhibition of diguanylate cyclases (DGCs), the enzyme implicated in the synthesis of the second messenger, cyclic diguanylate (c-di-GMP), involved in the biofilm formation, represents a potential approach for preventing the biofilm development. It has been extensively studied using PleD protein as a model of DGC for in silico studies as virtual screening and as a model for in vitro studies in biofilms formation. This study aimed to search for natural products capable of inhibiting the Caulobacter crescentus enzyme PleD. For this purpose, 224,205 molecules from the natural products ZINC15 database, have been evaluated through molecular docking and molecular dynamic simulation. Our results suggest trans-Aconitic acid (TAA) as a possible starting point for hit-to-lead methodologies to obtain new inhibitors of the PleD protein and hence blocking the biofilm formation.

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Early Biofilm Formation on UV Light Activated Nanoporous TiO2SurfacesIn Vivo

International Journal of Biomaterials ◽

10.1155/2018/7275617 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Nagat Areid ◽

Eva Söderling ◽

Johanna Tanner ◽

Ilkka Kangasniemi ◽

Timo O. Närhi

Keyword(s):

Titanium Alloy ◽

Biofilm Formation ◽

Uv Light ◽

Antibacterial Properties ◽

Mutans Streptococci ◽

Human Gingival Fibroblast ◽

Gingival Fibroblast ◽

Biofilm Development

Purpose. To explore earlyS. mutansbiofilm formation on hydrothermally induced nanoporous TiO2surfacesin vivoand to examine the effect of UV light activation on the biofilm development.Materials and Methods. Ti-6Al-4V titanium alloy discs (n = 40) were divided into four groups with different surface treatments: noncoated titanium alloy (NC); UV treated noncoated titanium alloy (UVNC); hydrothermally induced TiO2coating (HT); and UV treated titanium alloy with hydrothermally induced TiO2coating (UVHT).In vivoplaque formation was studied in 10 healthy, nonsmoking adult volunteers. Titanium discs were randomly distributed among the maxillary first and second molars. UV treatment was administered for 60 min immediately before attaching the discs in subjects’ molars. Plaque samples were collected 24h after the attachment of the specimens. Mutans streptococci (MS), non-mutans streptococci, and total facultative bacteria were cultured, and colonies were counted.Results. The plaque samples of NC (NC + UVNC) surfaces showed over 2 times more oftenS. mutanswhen compared to TiO2surfaces (HT + UVHT), with the number of colonized surfaces equal to 7 and 3, respectively.Conclusion. Thisin vivostudy suggested that HT TiO2surfaces, which we earlier showed to improve blood coagulation and encourage human gingival fibroblast attachmentin vitro, do not enhance salivary microbial (mostly mutans streptococci) adhesion and initial biofilm formation when compared with noncoated titanium alloy. UV light treatment provided Ti-6Al-4V surfaces with antibacterial properties and showed a trend towards less biofilm formation when compared with non-UV treated titanium surfaces.

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Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae

mBio ◽

10.1128/mbio.01466-17 ◽

2017 ◽

Vol 8 (6) ◽

Cited By ~ 16

Author(s):

Jayajit Das ◽

Elaine Mokrzan ◽

Vinal Lakhani ◽

Lucia Rosas ◽

Joseph A. Jurcisek ◽

...

Keyword(s):

Middle Ear ◽

In Silico ◽

Biofilm Formation ◽

Rational Design ◽

Bacterial Biofilm ◽

Confocal Imaging ◽

Extracellular Dna ◽

Fractal Structures ◽

Biofilm Development

ABSTRACT Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.

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Characterization and application of a flow system for in vitro multispecies oral biofilm formation

Journal of Periodontal Research ◽

10.1111/jre.12110 ◽

2013 ◽

Vol 49 (3) ◽

pp. 323-332 ◽

Cited By ~ 27

Author(s):

V. Blanc ◽

S. Isabal ◽

M. C. Sánchez ◽

A. Llama-Palacios ◽

D. Herrera ◽

...

Keyword(s):

Biofilm Formation ◽

Flow System ◽

Oral Biofilm

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Biofilm Formation by Candida Species on Silicone Surfaces and Latex Pacifier Nipples: An in vitro Study

Journal of Clinical Pediatric Dentistry ◽

10.17796/jcpd.33.3.7572960tn46837k4 ◽

2009 ◽

Vol 33 (3) ◽

pp. 235-240 ◽

Cited By ~ 15

Author(s):

Luiz Cezar da Silveira ◽

Senda Charone ◽

Lucianne Cople Maia ◽

Rosangela Maria de Araújo Soares ◽

Maristela Barbosa Portela

Keyword(s):

Candida Species ◽

Biofilm Formation ◽

In Vitro Study ◽

Reduction Reaction ◽

Fungal Colonization ◽

Candida Spp ◽

Biofilm Growth ◽

Species Analysis ◽

Biofilm Development

The present study assessed the growth and development of biofilm formation by isolates of C. albicans, C. glabrata and C. parapsilosis on silicone and latex pacifier nipples. The silicone and latex surfaces were evaluated by scanning electronic microscopy (SEM). The plastic component of the nipple also seems to be an important factor regarding the biofilm formation by Candida spp. The biofilm growth was measured using the MTT reduction reaction. C. albicans was found to have a slightly greater capacity of forming biofilm compared to the other Candida species. Analysis of the pattern of biofilm development by C. albicans,C. glabrata and C. parapsilosis on latex and silicon pacifier shields showed an increased biofilm formation regarding the latter substrate. Silicone was shown to be more resistant to fungal colonization, particularly in the case of C. parapsilosis, despite the lack of any statistically significant differences (P > 0.05). In addition, silicone has a smoother surface compared to latex, whose surface was found to be rugose and irregular

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Next Science Wound Gel Technology, a Novel Agent That Inhibits Biofilm Development by Gram-Positive and Gram-Negative Wound Pathogens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00108-14 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3060-3072 ◽

Cited By ~ 12

Author(s):

Kyle G. Miller ◽

Phat L. Tran ◽

Cecily L. Haley ◽

Cassandra Kruzek ◽

Jane A. Colmer-Hamood ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Skin Barrier ◽

Host Responses ◽

Content Type ◽

Planktonic Bacteria ◽

Topical Antimicrobials ◽

Biofilm Development ◽

Biofilm Infection

ABSTRACTLoss of the skin barrier facilitates the colonization of underlying tissues with various bacteria, where they form biofilms that protect them from antibiotics and host responses. Such wounds then become chronically infected. Topical antimicrobials are a major component of chronic wound therapy, yet currently available topical antimicrobials vary in their effectiveness on biofilm-forming pathogens. In this study, we evaluated the efficacy of Next Science wound gel technology (NxtSc), a novel topical agent designed to kill planktonic bacteria, penetrate biofilms, and kill the bacteria within.In vitroquantitative analysis, using strains isolated from wounds, showed that NxtSc inhibited biofilm development byStaphylococcus aureus,Staphylococcus epidermidis,Pseudomonas aeruginosa,Acinetobacter baumannii, andKlebsiella pneumoniaeby inhibiting bacterial growth. The gel formulation NxtSc-G5, when applied to biofilms preformed by these pathogens, reduced the numbers of bacteria present by 7 to 8 log10CFU/disc or CFU/g.In vivo, NxtSc-G5 prevented biofilm formation for 72 h when applied at the time of wounding and infection and eliminated biofilm infection when applied 24 h after wounding and infection. Storage of NxtSc-G5 at room temperature for 9 months did not diminish its efficacy. These results establish that NxtSc is efficaciousin vitroandin vivoin preventing infection and biofilm development by different wound pathogens when applied immediately and in eliminating biofilm infection already established by these pathogens. This novel antimicrobial agent, which is nontoxic and has a usefully long shelf life, shows promise as an effective agent for the prevention and treatment of biofilm-related infections.

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Antibiotic susceptibility of Staphylococcus aureus with different degrees of biofilm formation

Journal of Analytical Science & Technology ◽

10.1186/s40543-021-00294-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hyo-Jung Shin ◽

Sungtae Yang ◽

Yong Lim

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Resistance Mechanisms ◽

Minimal Bactericidal Concentration ◽

Chronic Infections ◽

Planktonic Bacteria ◽

Growth Modes ◽

Biofilm Bacteria ◽

Biofilm Development

AbstractStaphylococcus aureus is one of the most common pathogens in biofilm-associated chronic infections. S. aureus living within biofilms evades the host immune response and is more resistant to antibiotics than planktonic bacteria. In this study, we generated S. aureus with low and high levels of biofilm formation using the rbf (regulator of biofilm formation) gene and performed a BioTimer assay to determine the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of various types of antibiotics. We showed that biofilm formation by S. aureus had a greater effect on MBC than MIC, probably due to the different growth modes between planktonic and biofilm bacteria. Importantly, we found that the MBC for biofilm S. aureus was much higher than that for planktonic cells, but there was little difference in MBC between low and high levels of biofilm formation. These results suggest that once the biofilm is formed, the bactericidal activity of antibiotics is significantly reduced, regardless of the degree of S. aureus biofilm formation. We propose that S. aureus strains with varying degrees of biofilm formation may be useful for evaluating the anti-biofilm activity of antimicrobial agents and understanding antibiotic resistance mechanisms by biofilm development.

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