• Journal of Periodontal and Implant Science
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• v.44 no.2
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• pp.79-84
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• 2014

Purpose: While single-species biofilms have been studied extensively, we know notably little regarding multispecies biofilms and their interactions. The purpose of this study was to develop and evaluate an in vitro multispecies dental biofilm model that aimed to mimic the environment of chronic periodontitis. Methods: Streptococcus gordonii KN1, Fusobacterium nucleatum ATCC23726, Aggregatibacter actinomycetemcomitans ATCC33384, and Porphyromonas gingivalis ATCC33277 were used for this experiment. The biofilms were grown on 12-well plates with a round glass slip (12 mm in diameter) with a supply of fresh medium. Four different single-species biofilms and multispecies biofilms with the four bacterial strains listed above were prepared. The biofilms were examined with a confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM). The minimum inhibitory concentrations (MIC) for four different planktonic single-species and multispecies bacteria were determined. The MICs of doxycycline and chlorhexidine for four different single-species biofilms and a multispecies biofilm were also determined. Results: The CLSM and SEM examination revealed that the growth pattern of the multispecies biofilm was similar to those of single-species biofilms. However, the multispecies biofilm became thicker than the single-species biofilms, and networks between bacteria were formed. The MICs of doxycycline and chlorhexidine were higher in the biofilm state than in the planktonic bacteria. The MIC of doxycycline for the multispecies biofilm was higher than were those for the single-species biofilms of P. gingivalis, F. nucleatum, or A. actinomycetemcomitans. The MIC of chlorhexidine for the multispecies biofilm was higher than were those for the single-species biofilms of P. gingivalis or F. nucleatum. Conclusions: To mimic the natural dental biofilm, a multispecies biofilm composed of four bacterial species was grown. The 24-hour multispecies biofilm may be useful as a laboratory dental biofilm model system.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.473-486
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• 2019

In the aquatic environment, microorganisms are predominantly organized as biofilms. Biofilms are formed by the aggregation of microbial cells and are surrounded by a matrix of extracellular polymeric substances (EPS) secreted by the microbial cells. Biofilms are attached to various surfaces, such as the living tissues, indwelling medical devices, and piping of the industrial potable water system. Biofilms formed from a single species has been extensively studied. However, there is an increased research focus on multispecies biofilms in recent years. It is important to assess the microbial mechanisms underlying the regulation of multispecies biofilm formation to determine the drinking water microbial composition. These mechanisms contribute to the predominance of the best-adapted species in an aquatic environment. This review focuses on the interactions in the multispecies biofilms, such as coaggregation, co-metabolism, cross-species protection, jamming of quorum sensing, lateral gene transfer, synergism, and antagonism. Further, this review explores the dynamics and the factors favoring biofilm formation and pathogen transmission within the drinking water distribution systems. The understanding of the physiology and biodiversity of microbial species in the biofilm may aid in the development of novel biofilm control and drinking water disinfection processes.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1053-1064
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• 2017

A biofilm is a community of microbes that typically inhabit on surfaces and are encased in an extracellular matrix. Biofilms display very dissimilar characteristics to their planktonic counterparts. Biofilms are ubiquitous in the environment and influence our lives tremendously in both positive and negative ways. Pseudomonas aeruginosa is a bacterium known to produce robust biofilms. P. aeruginosa biofilms cause severe problems in immunocompromised patients, including those with cystic fibrosis or wound infection. Moreover, the unique biofilm properties further complicate the eradication of the biofilm infection, leading to the development of chronic infections. In this review, we discuss the history of biofilm research and general characteristics of bacterial biofilms. Then, distinct features pertaining to each stage of P. aeruginosa biofilm development are highlighted. Furthermore, infections caused by biofilms on their own or in association with other bacterial species (i.e., multispecies biofilms) are discussed in detail.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.348-355
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• 2023

Epifluorescence microscopy with image analysis was evaluated as a biofilm quantification method (i.e., quantification of surface area colonized by biofilms), in comparison with crystal violet (CV) staining. We performed different experiments to generate multispecies biofilms with natural and artificial bacterial assemblages. First, four species were inoculated daily in 16 different sequences to form biofilms (surface colonization, 0.1%-56.6%). Second, a 9-species assemblage was allowed to form biofilms under 10 acylase treatment episodes (33.8%-55.6%). The two methods comparably measured the quantitative variation in biofilms, exhibiting a strong positive relationship (R² ≥ 0.7). Moreover, the two methods exhibited similar levels of variation coefficients. Finally, six synthetic and two natural consortia were allowed to form biofilms for 14 days, and their temporal dynamics were monitored. The two methods were comparable in quantifying four biofilms colonizing ≥18.7% (R² ≥ 0.64), but not for the other biofilms colonizing ≤ 3.7% (R² ≤ 0.25). In addition, the two methods exhibited comparable coefficients of variation in the four biofilms. Microscopy and CV staining comparably measured the quantitative variation of biofilms, exhibiting a strongly positive relationship, although microscopy cannot appropriately quantify the biofilms below the threshold colonization. Microscopy with image analysis is a promising approach for easily and rapidly estimating absolute quantity of multispecies biofilms.

• The Journal of Advanced Prosthodontics
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• v.15 no.2
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• pp.80-92
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• 2023

PURPOSE. This study incorporated the nanomaterial, nanostructured silver vanadate decorated with silver nanoparticles (AgVO₃), into heat-cured resin (HT) at concentrations of 2.5%, 5%, and 10% and compared the adhesion of multispecies biofilms, surface characteristics, and mechanical properties with conventional heat-cured (HT 0%) and printed resins. MATERIALS AND METHODS. AgVO₃ was incorporated in mass into HT powder. A denture base resin was used to obtain printed samples. Adhesion of a multispecies biofilm of Candida albicans, Candida glabrata, and Streptococcus mutans was evaluated by colony-forming units per milliliter (CFU/mL) and metabolic activity. Wettability, roughness, and scanning electron microscopy (SEM) were used to assess the physical characteristics of the surface. The mechanical properties of flexural strength and elastic modulus were tested. RESULTS. HT 10%-AgVO₃ showed efficacy against S. mutans; however, it favored C. albicans CFU/mL (P < .05). The printed resin showed a higher metabolically active biofilm than HT 0% (P < .05). There was no difference in wettability or roughness between groups (P > .05). Irregularities on the printed resin surface and pores in HT 5%-AgVO₃ were observed by SEM. HT 0% showed the highest flexural strength, and the resins incorporated with AgVO₃ had the highest elastic modulus (P < .05). CONCLUSION. The incorporation of 10% AgVO₃ into heat-cured resin provided antimicrobial activity against S. mutans in a multispecies biofilm did not affect the roughness or wettability but reduced flexural strength and increased elastic modulus. Printed resin showed higher irregularity, an active biofilm, and lower flexural strength and elastic modulus than heat-cured resin.

• Microbiology and Biotechnology Letters
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• v.50 no.4
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• pp.548-556
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• 2022

Acylases can have a significant effect on biofilm formation owing to their quorum quenching activity. In this study, we investigated the effects of acylase treatment episodes on multispecies biofilm development. A consortium composed of 9 species belonging to different genera was allowed to form biofilms for 5 days under various treatment episodes (different treatment periods, 1, 2, 3, or 4 days; and two application timings, beginning or later) at 1, 5, 10, 20 and 50 mg·l^-1 acylase concentrations. The acylase treatment for 5 days showed that acylase concentration was negative with biofilm development (linear regression, Y = -0.05·x + 2.37, p < 0.05, R² = 0.88). Acylase was more effective in reducing biofilm formation when it was applied in the beginning (vs. in later development stage) at all acylase concentrations (p < 0.05). ANOVA indicated that treatment period was significant on biofilm formation in both application timings at ≥ 10 mg·l^-1 (p < 0.05). Linearity test results showed that all slope values between period and biofilm were negative in both timings at ≥ 10 mg·l^-1 (p < 0.05, except for the later application at 20 mg·l^-1). When temporal biofilm dynamics were monitored at 20 mg·l^-1, biofilms gradually increased with time at all treatment episodes (p < 0.05), and slope values in linear regression between biofilm and time were lower when acylase was applied in the beginning (p < 0.05). Our findings suggest the importance of the acylase treatment period and application timing on biofilm control.

• Journal of the korean academy of Pediatric Dentistry
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• v.48 no.1
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• pp.21-30
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• 2021

The purpose of this study is to investigate the antibacterial effects of indocyanine green (ICG) and near-infrared diode lasers on multispecies biofilms. Multispecies biofilms of Streptococcus mutans, Lactobacillus casei and Candida albicans were treated with different irradiation time using photosensitizer ICG and 808 nm near-infrared diode laser. Colony forming unit (CFU) was measured, and qualitative evaluation of biofilm was performed with confocal laser scanning microscopy (CLSM). Temperature measurement was conducted to evaluate photothermal effect. In the groups using ICG and diode laser, reduction in CFU was statistically significant, but the difference in antibacterial effect on L. casei and C. albicans with irradiation time was not significant, and similar results were confirmed with CLSM. Groups with ICG and diode laser showed higher temperature elevation than groups without ICG, and results of measured temperature were similar to the range of hyperthermia. In conclusion, ICG and near-infrared diode laser showed antibacterial effects on multispecies biofilms, but studies on protocol are necessary for clinical application.

• Restorative Dentistry and Endodontics
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• v.46 no.2
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• pp.20.1-20.11
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• 2021

Objectives: The aim of this study was to evaluate bacterial nanocellulose (BNC) membranes incorporated with antimicrobial agents regarding cytotoxicity in fibroblasts of the periodontal ligament (PDLF), antimicrobial activity, and inhibition of multispecies biofilm formation. Materials and Methods: The tested BNC membranes were BNC + 1% clindamycin (BNC/CLI); BNC + 0.12% chlorhexidine (BNC/CHX); BNC + nitric oxide (BNC/NO); and conventional BNC (BNC; control). After PDLF culture, the BNC membranes were positioned in the wells and maintained for 24 hours. Cell viability was then evaluated using the MTS calorimetric test. Antimicrobial activity against Enterococcus faecalis, Actinomyces naeslundii, and Streptococcus sanguinis (S. sanguinis) was evaluated using the agar diffusion test. To assess the antibiofilm activity, BNC membranes were exposed for 24 hours to the mixed culture. After sonicating the BNC membranes to remove the remaining biofilm and plating the suspension on agar, the number of colony-forming units (CFU)/mL was determined. Data were analyzed by 1-way analysis of variance and the Tukey, Kruskal-Wallis, and Dunn tests (α = 5%). Results: PDLF metabolic activity after contact with BNC/CHX, BNC/CLI, and BNC/NO was 35%, 61% and 97%, respectively, compared to BNC. BNC/NO showed biocompatibility similar to that of BNC (p = 0.78). BNC/CLI showed the largest inhibition halos, and was superior to the other BNC membranes against S. sanguinis (p < 0.05). The experimental BNC membranes inhibited biofilm formation, with about a 3-fold log CFU reduction compared to BNC (p < 0.05). Conclusions: BNC/NO showed excellent biocompatibility and inhibited multispecies biofilm formation, similarly to BNC/CLI and BNC/CHX.

• The Journal of Advanced Prosthodontics
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• v.9 no.6
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• pp.482-485
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• 2017

PURPOSE. This laboratory study aimed to investigate the effect of doping an acrylic denture base resin material with nanoparticles of ZnO, CaO, and $TiO_2$ on biofilm formation. MATERIALS AND METHODS. Standardized specimens of a commercially available cold-curing acrylic denture base resin material were doped with 0.1, 0.2, 0.4, or 0.8 wt% commercially available ZnO, CaO, and $TiO_2$ nanopowder. Energy dispersive X-ray spectroscopy (EDX) was used to identify the availability of the nanoparticles on the surface of the modified specimens. Surface roughness was determined by employing a profilometric approach; biofilm formation was simulated using a monospecies Candida albicans biofilm model and a multispecies biofilm model including C. albicans, Actinomyces naeslundii, and Streptococcus gordonii. Relative viable biomass was determined after 20 hours and 44 hours using a MTT-based approach. RESULTS. No statistically significant disparities were identified among the various materials regarding surface roughness and relative viable biomass. CONCLUSION. The results indicate that doping denture base resin materials with commercially available ZnO, CaO, or $TiO_2$ nanopowders do not inhibit biofilm formation on their surface. Further studies might address the impact of varying particle sizes as well as increasing the fraction of nanoparticles mixed into the acrylic resin matrix.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1908-1919
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• 2015

This work investigated the potential of curcumin (CCM) and (-)-epigallocatechin gallate (EGCG) to inhibit N-acyl homoserine lactone (AHL)-mediated biofilm formation in gram-negative bacteria from membrane bioreactor (MBR) activated sludge. The minimum inhibitory concentrations (MICs) of CCM alone against all the tested bacteria were 200-350 μg/ml, whereas those for EGCG were 300-600 μg/ml. Biofilm formation at one-half MICs indicated that CCM and EGCG alone respectively inhibited 52-68% and 59-78% of biofilm formation among all the tested bacteria. However, their combination resulted in 95-99% of biofilm reduction. Quorum sensing inhibition (QSI) assay with known biosensor strains demonstrated that CCM inhibited the expression of C4 and C6 homoserine lactones (HSLs)-mediated phenotypes, whereas EGCG inhibited C4, C6, and C10 HSLs-based phenotypes. The Center for Disease Control biofilm reactor containing a multispecies culture of nine bacteria with one-half MIC of CCM (150 μg/ml) and EGCG (275 μg/ml) showed 17 and 14 μg/cm² of extracellular polymeric substances (EPS) on polyvinylidene fluoride membrane surface, whereas their combination (100 μg/ml of each) exhibited much lower EPS content (3 μg/cm²). Confocal laser scanning microscopy observations also illustrated that the combination of compounds tremendously reduced the biofilm thickness. The combined effect of CCM with EGCG clearly reveals for the first time the enhanced inhibition of AHL-mediated biofilm formation in bacteria from activated sludge. Thus, such combined natural QSI approach could be used for the inhibition of membrane biofouling in MBRs treating wastewaters.