Individual growth detection of bacterial species in an in vitro oral polymicrobial biofilm model

Most in vitro studies on the antibacterial effects of antiseptics have used planktonic bacteria in monocultures. However, this study design does not reflect the in vivo situation in oral cavities harboring different bacterial species that live in symbiotic relationships in biofilms. The aim of this study was to establish a simple in vitro polymicrobial model consisting of only three bacterial strains of different phases of oral biofilm formation to simulate in vivo oral conditions. Therefore, we studied the biofilm formation of Actinomyces naeslundii (An), Fusobacterium nucleatum (Fn), and Enterococcus faecalis (Ef) on 96-well tissue culture plates under static anaerobic conditions using artificial saliva according to the method established by Pratten et al. that was supplemented with 1 g l^?1 sucrose. Growth was separately determined for each bacterial strain after incubation periods of up to 72 h by means of quantitative real-time polymerase chain reaction and live/dead staining. Presence of an extracellular polymeric substance (EPS) was visualized by Concanavalin A staining. Increasing incubation times of up to 72 h showed adhesion and propagation of the bacterial strains with artificial saliva formulation. An and Ef had significantly higher growth rates than Fn. Live/dead staining showed a median of 49.9 % (range 46.0–53.0 %) of living bacteria after 72 h of incubation, and 3D fluorescence microscopy showed a three-dimensional structure containing EPS. An in vitro oral polymicrobial biofilm model was established to better simulate oral conditions and had the advantage of providing the well-controlled experimental conditions of in vitro testing.     

Spatial associative memory: a possible species difference in rats and pigeons

Previous research has shown that pigeons can remember which of four spatially distinct responses was last reinforced, for at least 72 h. The present study sought to replicate this finding using rats. Rats were tested in an operant chamber containing four spatially distinct levers. In each session one lever was randomly selected to provide reinforcement for 15 min. This reinforced period was preceded by a non-reinforced period that was 30 s long, on average. During the non-reinforced period the amount the rat pressed on the previously reinforced lever was compared to responding on the other three levers, and was taken as a measure of memory. Sessions were separated either by 17 min, 24 or 72 h. Unlike pigeons, rats responded at chance levels following each of these retention intervals. This finding adds to previous research suggesting differences in cognitive processes in rats and pigeons.     

骨科内植物念珠菌生物被膜体外模型的构建及药物敏感性

利用24孔板在骨科内植物表面构建骨科内植物念珠菌生物被膜模型,并使用荧光镜检和菌落计数法(colony forming unit,CFU)检测醋酸氯己定(以下简称氯己定)与氟康唑对骨科内植物念珠菌生物被膜的联合抗菌效应.本研究成功在体外构建出骨科内植物念珠菌生物被膜模型,并发现无论是白念珠菌(SC5314)、近平滑念珠...     

Use of an in vitro flat-bed biofilm model to measure biologically active anti-odour compounds

The objective of this study was to demonstrate the utility of a modified flat-bed perfusion biofilm matrix system for testing toothpaste formulations directly, without dilution, as a layer in direct contact with the biofilm matrix surface. Final biofilm yields and volatile sulphur compounds (VSC) biogenesis were measured to show the relative efficacy of toothpaste formulations. Diffusion characteristics of the flat-bed system to exposure with Meridol® tooth and tongue gel (TTG; 1,400 ppm F^? from amine fluoride/stannous fluoride, 0.5 % zinc lactate, oral malodour counteractives) was assessed using a bioluminescent target species Escherichia coli Nissle 1917/pGLITE coupled with a low-light photon camera to visualise the kill kinetics. Tongue-flora derived, mixed culture biofilms (n?=?4) received 5, 15 and 30 min treatment with TTG, respectively, to determine the optimum time of exposure. VSC biogenesis was measured from headspace samples by gas chromatography prior to and following treatment of two daily applications for 4 days of treatment (TTG), positive control (CHX gel) and negative controls (placebo and sham treatment). Viable counts were performed at the end of experiments by destructive sampling of the biofilms and plating onto selective and non-selective agar. Following a single treatment with TTG, the E. coli biofilm with lux target gave >50 % reduction of luminescence within 2 to 3 h before recovering to a steady state over 10 h, suggesting biofilm cidal activity rather biostasis. For mixed culture biofilms, 15- and 30-min treatment exposure with TTG gave almost identical reductions in final biofilm yields. For comparing efficacy of treatments, biofilms treated with TTG gave greatest reductions in both pre–post levels of H₂S (P?<?0.01) and CH₃SH (P?<?0.05) and population yields at the end of the experiments (P?<?0.001) compared to placebo and positive control. The in vitro flat-bed perfusion model may be used to replicate many of the activities and reactions believed to be occurring by the tongue biofilm microflora within a real mouth, including VSC biogenesis and its inhibition by exposure to active agents as components of toothpastes and gels applied in direct contact with the biofilm.     

Detachment characteristics and oxacillin resistance of Staphyloccocus aureus biofilm emboli in an in vitro catheter infection model

Catheter-related bloodstream infections due to Staphylococcus aureus are of increasing clinical importance. The pathophysiological steps leading to colonization and infection, however, are still incompletely defined. We observed growth and detachment of S. aureus biofilms in an in vitro catheter-infection model by using time-lapse microscopy. Biofilm emboli were characterized by their size and their susceptibility for oxacillin. Biofilm dispersal was found to be a dynamic process in which clumps of a wide range of diameters detach. Large detached clumps were highly tolerant to oxacillin compared with exponential-phase planktonic cultures. Interestingly, the degree of antibiotic tolerance in stationary-phase planktonic cultures was equal to that in the large clumps. The mechanical disruption of large clumps reduced the minimal bactericidal concentration (MBC) by more than 1,000 times. The MBC for whole biofilm effluent, consisting of particles with an average number of 20 bacteria was 3.5 times higher than the MBC for planktonic cultures. We conclude that the antibiotic resistance of detached biofilm particles depends on the embolus size and could be attributed to nutrient-limited stationary-phase physiology of cells within the clumps. We hypothesize that the detachment of multicellular clumps may explain the high rate of symptomatic metastatic infections seen with S. aureus.     

口腔静态生物膜模型研究进展北大核心CSCD

口腔静态生物膜模型是体外模拟口腔微生态环境的重要手段,因其成本低、通量高、可靠性好、操作容易等优点,已成为研究各种口腔疾病的发病机制,测试各种药物、口腔护理用品、食品的重要工具。建立口腔静态生物膜模型,可根据研究目的,选择不同的装置、接种源、培养基、基质和培养条件,并通过测定生物量、代谢活性、群落结构以及进行可视化分析等多种方法评价生物膜的生长情况。本文汇总了近年来报道的口腔静态生物膜模型建立和评价的方法学要素,并分析讨论了各种方法的适用范围,希望有助于相关领域研究和生产实践的开展。     

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P?<?0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20–50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.     

An in vitro biofilm model system to facilitate study of microbial communities of the human oral cavity

The human oral cavity is host to a diverse microbiota. Much of what is known about the behaviour of oral microbes derives from studies of individual or several cultivated species, situations which do not totally reflect the function of organisms within more complex microbiota or multispecies biofilms. The number of validated models that allow examination of the role that biofilms play during oral cavity colonization is also limited. The CDC biofilm reactor is a standard method that has been deployed to study interactions between members of human microbiotas allowing studies to be completed during an extended period under conditions where nutrient availability, and washout of waste products are controlled. The objective of this work was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum to study the development, reproducibility and stability of the oral microbiota. By employing deep sequencing of the variable regions of the 16S rRNA gene, we found that the CDC biofilm reactor could be used to efficiently cultivate microbiota containing all six major phyla previously identified as the core saliva microbiota. After an acclimatisation period, communities in each reactor stabilised. Replicate reactors were predominately populated by a shared core microbiota; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units. We conclude that the CDC biofilm reactor can be used to cultivate communities that replicate key features of the human oral cavity and is a useful tool to facilitate studies of the dynamics of these communities.     

基于微流控的生物被膜研究进展及口腔微生态系统应用展望

口腔是人体最重要的微生物储藏库之一,这些微生物通常以生物被膜的形式稳定地黏附于口腔内粘膜及牙齿表面,在病理条件下则可以深入髓腔及牙槽骨内,成为龋病、根尖周炎、牙周病等常见口腔疾病的主要病因,甚至与许多全身性疾病密切相关.在人类口腔微生态系统中,微生物种类繁多,生物被膜的构成及相互作用关系复杂,目前对其认识还十分有限;此...     

In vitro biofilm model for studying tongue flora and malodour

AIMS: To develop a perfusion biofilm system to model tongue biofilm microflora and their physiological response to sulfur-containing substrates (S-substrates) in terms of volatile sulfide compound (VSC) production. METHODS AND RESULTS: Tongue-scrape inocula were used to establish in vitro perfusion biofilms which were examined in terms of ecological composition using culture-dependent and independent (PCR-DGGE) approaches. VSC-specific activity of cells was measured by a cell suspension assay, using a portable industrial sulfide monitor which was also used to monitor VSC production from biofilms in situ. Quasi steady states were achieved by 48 h and continued to 96 h. The mean (+/-SEM) growth rate for 72-h biofilms (n=4) was micro=0.014 h(-1) (+/-0.005 h(-1)). Comparison of biofilms, perfusate and original inoculum showed their ecological composition to be similar (Pearson coefficient>0.64). Perfusate and biofilm cells derived from the same condition (co-sampled) were equivalent with regard to VSC-specific activities which were up-regulated in the presence of S-substrates. CONCLUSIONS: The model maintained a stable tongue microcosm suitable for studying VSC production; biofilm growth in the presence of S-substrates up-regulated VSC activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is apt for studying ecological and physiological aspects of oral biofilms and could be useful for screening inhibitory agents.     

肺炎克雷伯菌生物被膜的体外模型建立

目的研究临床分离的肺炎克雷伯菌在体外形成生物被膜的情况,为进一步研究生物被膜肺炎克雷伯菌的耐药机制奠定基础。方法采用改良平板法建立肺炎克雷伯菌生物被膜模型,用喷金法和扫描电镜观察鉴定,并对生物被膜的形成进行定量分析。结果23株临床分离的肺炎克雷伯菌菌株生物被膜形成能力不同,以强阳性生物被膜形成能力者为最多数。结论绝大多数临床分离的肺炎克雷伯菌菌株具有较强的生物被膜形成能力。应用改良平板法能够较好的在体外建立其生物被膜模型。     

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.     

伊犁黑蜂蜂胶对口腔主要致龋细菌及生物膜生长的实验研究

目的研究伊犁黑蜂蜂胶对口腔常见致龋细菌及其生物膜生长的影响,观察其防龋效果。方法 (1)通过液体稀释法测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小抑菌浓度(MIC)及最小杀菌浓度(MBC);(2)生物膜形成抑制试验测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小生物膜形成抑制浓度(MBIC50);(3)通过结晶紫染色法测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小生物膜清除浓度(MBEC);结果(1)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、嗜酸乳杆菌、血链球菌、粘性放线菌和内氏放线菌的最低抑菌浓度分别是0.78、0.39、1.56、0.39、0.20和0.20 mg/mL,最低杀菌浓度分别为1.56、0.78、3.125、0.78、0.39和0.39mg/mL;(2)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌的MBIC50分别是0.39、0.39、0.39、0.05和0.10mg/mL;(3)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌的MBEC分别是6.25、1.56、3.1256、0.78和0.78mg/mL。结论伊犁黑蜂蜂胶对口腔主要致龋细菌的生长具有抑制作用,能够抑制主要致龋细菌单菌生物膜的形成,并具有一定清除作用,是具有一定防龋效果的天然药物。     

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 the percentage 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.     

Spatial variability in nutrient concentration and biofilm nutrient limitation in an urban watershed

Nutrient enrichment threatens river ecosystem health in urban watersheds, but the influence of urbanization on spatial variation in nutrient concentrations and nutrient limitation of biofilm activity are infrequently measured simultaneously. In summer 2009, we used synoptic sampling to measure spatial patterns of nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), and soluble reactive phosphorus (SRP) concentration, flux, and instantaneous yield throughout the Bronx River watershed within New York City and adjacent suburbs. We also quantified biofilm response to addition of NO₃ ⁻, phosphate (PO₄ ³⁻), and NO₃ ⁻ + PO₄ ³⁻ on organic and inorganic surfaces in the river mainstem and tributaries. Longitudinal variation in NO₃ ⁻ was low and related to impervious surface cover across sub-watersheds, but spatial variation in NH₄ ⁺ and SRP was higher and unrelated to sub-watershed land-use. Biofilm respiration on organic surfaces was frequently limited by PO₄ ³⁻ or NO₃ ⁻ + PO₄ ³⁻, while primary production on organic and inorganic surfaces was nutrient-limited at just one site. Infrequent NO₃ ⁻ limitation and low spatial variability of NO₃ ⁻ throughout the watershed suggested saturation of biological N demand. For P, both higher biological demand and point-sources contributed to greater spatial variability. Finally, a comparison of our data to synoptic studies of forested, temperate watersheds showed lower spatial variation of N and P in urban watersheds. Reduced spatial variation in nutrients as a result of biological saturation may represent an overlooked effect of urbanization on watershed ecology, and may influence urban stream biota and downstream environments.     

An algebraic model of an associative noise-like coding memory

A mathematical model of an associative memory is presented, sharing with the optical holography memory systems the properties which establish an analogy with biological memory. This memory system-developed from Gabor's model of memoryis based on a noise-like coding of the information by which it realizes a distributed, damage-tolerant, equipotential storage through simultaneous state changes of discrete substratum elements. Each two associated items being stored are coded by each other by means of two noise-like patterns obtained from them through a randomizing preprocessing. The algebraic braic transformations operating the information storage and retrieval are matrix-vector products involving Toeplitz type matrices. Several noise-like coded memory traces are superimposed on a common substratum without crosstalk interference; moreover, extraneous noise added to these memory traces does not injure the stored information. The main performances shown by this memory model are: i) the selective, complete recovering of stored information from incomplete keys, both mixed with extraneous information and translated from the position learnt; ii) a dynamic recollection where the information just recovered acts as a new key for a sequential retrieval process; iii) context-dependent responses. The hypothesis that the information is stored in the nervous system through a noise-like coding is suggested. The model has been simulated on a digital computer using bidimensional images.     

Development and application of a polymicrobial, in vitro, wound biofilm model

Aims: The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements. Methods and Results: The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip‐flow reactor (DFR), and a three‐species biofilm model was established using methicillin‐resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Cl. perfringens in the colony‐drip‐flow reactor model. Plate counts revealed that MRSA, Ps. aeruginosa and Cl. perfringens grew to 7·39 ± 0·45, 10·22 ± 0·22 and 7·13 ± 0·77 log CFU per membrane, respectively. The three‐species model was employed to evaluate the efficacy of two antimicrobial dressings, Curity? AMD and Acticoat?, compared to sterile gauze controls. Microbial growth on Curity? AMD and gauze was not significantly different, for any species, whereas Acticoat? was found to significantly reduce growth for all three species. Conclusions: Using the colony‐DFR, a three‐species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species. Significance and Impact of the Study: The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment.     

Transfer of a conjugative transposon, Tn5397 in a model oral biofilm

A tetracycline resistance profile was established from a microcosm dental plaque in a constant depth film fermenter. The fermenter was inoculated with a Bacillus subtilis strain which contained the conjugative transposon, Tn5397, which confers tetracycline resistance upon its host. After 6 hour and 24 hour the tetracycline resistance profile of the biofilm was redetermined and a tetracycline resistant Streptococcus species was isolated. A molecular analysis of this strain confirmed that Tn5397 was present in the genomic DNA of the isolate. These data represent the first report, to our knowledge, of intergeneric transfer of a conjugative transposon in a mixed species biofilm and demonstrates the ability of conjugative transposons to disseminate antibiotic resistance genes in a mixed species environment.     

Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners

doi:10.1111/j.1741‐2358.2009.00329.x
Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners Objectives: Microorganisms may colonise polysiloxane soft liners leading to bio‐deterioration. The aim of this study was to investigate in vitro adhesion and in vivo biofilm formation of Candida species on polysiloxane surfaces. Methods: The materials used in this study were Molloplast B, GC Reline soft, Mollosil Plus, Silagum Comfort and Palapress Vario. The in vitro retention of clinical isolates of Candida albicans to the relining and denture‐base materials by microscopic (scanning electron microscopy, SEM), conventional culturing methods and antimicrobial properties of these materials were studied. Candida found on materials and mucosa following long‐term use were identified and quantified, and biofilms covering the surfaces were investigated by SEM. Results: There was a significant decrease in the number of cells attached in vitro to saliva‐coated surfaces compared with non‐treated surfaces. An oral Candida carriage of 78% was found. Candida albicans, C. glabrata, C. intermedia and C. tropicalis were identified. In vivo biofilm formation on the liners appeared as massive colonisation by microorganisms. Conclusions: The results of the in vitro experiments suggest that salivary film influences early colonisation of different C. albicans strains. The film layer also minimises the differences among different strains. The Candida carriage of these patients was similar to denture‐wearing patients without soft liners.